AFCEC
 
TR­­
77­
7
BIOASSAY
OF
AIR
FORCE
FC­
206
FIRE­
FIGHTING
FOAM
Eric
H.
Wang
Civil
Engineering
Research
Facility
University
of
New
Mexico
Albuquerque,
New
Mexico
87131
April
1977
Final
Report:
November
1974
 
June
1976
Approved
for
public
release;
distribution
unlimited.

Prepared
for
AIR
FORCE
CIVIL
ENGINEERING
CENTER
Air
Force
Systems
Command
Tyndall
Air
Force
Base,
Florida
32401
ECJ~
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TR
 
77­
7
~
4.
TITLE
(
o~
dSuStiile)

~
BIOASSAYOF
AIR
FORCE
FC­
205
FIRE­
FIGHTING
FOAM
I
i~.
TVVE
.),~~`
O~
T
6
PERIOD
:
ovEP.~
D
Final
Technical
Report
~.
P~
3~\~
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cr~.
REPORT
N~
JM3ER
.

CERF
EE­
7
7.
AUTHOR(
s)
8.
CC,~~
1~.
CT
O'~
GRA~
iT
Y~
JMbER(~)

Harvey
Green
F29601
 
76
 
C­
0015
9.
PERFORMING
ORGANIZATION
NAME
AND
ADDRESS
~`~._
EJ.
iE~!
T.
~?.
3J~
Z7.
TASK
Eric
H.
Wang
Civil
Engineering
Research
Facility,
~
C~(
UfliT
NLMDERS
University
of
New
Mexico,
Box
25,
University
T.
D.
4.01
Station,
Albuquerque,
NM
87131
1Z.
REPORT
tisIE
11.
CONTROLLING
OFFICE
NAME
AND
ADDRESS
Air
Force
Civil
Engineering
Center
Air
Force
Systems
Command
Tyndall
Air
Force
Base,
FL
32401
~
i
r.
i]
1977
13.
NJ~)
3EPO~
PAGES
62
 
14.
MONITORING
AGENCY
NAME
b
ADDHESS(
If
difieren:
from
CottrolUr~
Office)
IS.
SECURITY
CLASS.
(
ot
this
r~
port~)

Unclassified
iSa.
DECLASSFICATION/
OOWN3RAOIP4D
.
SCHEDULE
~_____________________________________________________________
16.
O~
STRIDUTION
STATEMENT
(
of
thfs
Report)

Approved
for
public
release;
distribution
unlimited.

17.
D!
STRIBUTION
STATEMENT
(
of
the
abstract
entered
in
Block
20.
If
difleren!
from
Report)

18.
SUPPLEMENTARY
NOTES
Available
in
DDC
19.
KEY
WORD$
(
Continue
on
reverse
side
if
necessary
and
identify
by
block
number)

Bioassay
Eutrophication
Ecosystem
Acute
and
Chronic
Testing
Toxic
Lethality
Probit
Analysis
Conspecific
20.
ABSTRACT
(
Continue
on
ro"
er3e
side
Ii
necessary
end
Identify
by
block
number)

Tests
were
conducted
to
assess
the
effects
of
an
Air
Force
fire­
fighting
foam
(
FC­
206)
on
an
aquatic
system.
A
model
ecosystem
was
constructed
to
maintain
a
more
natural
setting
for
total
ecosystem
monitoring.
A
holding
facility
was
also
constructed
and
used
to
maintain
a
variety
of
test
organisms
for
bioassay
experiments.
Several
flowthrough
bioassay
systems
were
used
for
both
invertebrate
and
vertebrate
bioassay
testing;
data
were
collected
on
five
different
vertebrate
species
and
three
invertebrate
species.

r~
r­~
FORM
1473
EDITION
OF
1
NOV
65
IS
OBSOLETE
IJU
1
JAN
73
UNCLASSIFI
ED
S~
CURITV
CL~
S;
IFICATI~
OF
THIS
PAGE
r~
thenDe~
aEnter
PREFACE
This
report
documents
work
performed
during
the
period
November
1974
through
June
1976
by
the
University
of
New
Mexico
under
contract
F­
2960l­
76
 
C­
00l5
with
the
Air
Force
Civil
Engineering~
Center,
Air
Force
Systems
Command,
Tyndall
Air
Force
Base,
Florida
32401.
This
research
was
directed
by
Dr.
David
Nyquist
as
Principal
Investigator
from
November
1974
until
February
15,
1976
and
by
Mr.
Harvey
Green
from
February
16,
1976
to
June
1976.
Dr.
Kathryn
G.
Vogel
was
responsible
for
the
research
contained
in
the
appendix.
This
program
was
managed
at
Kirtland
Air
Force
Base,
New
Mexico
by
Maj.
Michael
G.
MacNaughton,
Commander
AFCEC/
OL
 
AA
and
at
the
Civil
Engineering
Center
by
Sgt.
Claude
Scott.

This
report
has
been
reviewed
by
the
Information
Office
(
UI)
and
is
releasable
to
the
National
Technical
Information
Service
(
NTIS).
At
NTIS
it
will
be
available
to
the
general
public,
including
foreign
nationals.

This
technical
report
has
been
reviewed
and
is
approved
for
publication.

1
(
The
reverse
of
this
page
is
blank.)
TABLE
OF
CONTENTS
Section
Page
I
INTRODUCTION
1
II
EXPERIMENTAL
FACILITIES
3
1.
Laboratory
3
2.
Hatchery
3
3.
Ecosystem
Model
6
III
TEST
PROCEDURES
9
1.
Ecosystem
Testing
9
2.
Bioassay
Experiments
9
a.
Introduction
b.
Statistical
Analysis
Techniques
10
c.
Diluter
13
d.
Chemical
Analysis
14
e.
Selection
of
Test
Organisms
23
IV
EXPERIt~
1ENTALRESULTS
24
1.
Ecosystem
24
2.
Bioassay
Tests
25
V
CONCLUSIONS
AND
RECOMMENDATIONS
35
REFERENCES
37
APPENDIX
A:
ADEOSINE
TRIPHOSPHATE
(
ATP)
ANALYSIS
TO
ASSESS
TOXICITY
OF
AQUATIC
POLLUTANTS
39
111
LIST
OF
ILLUSTRATIONS
2
Outdoor
Hatchery
3
Ecosystem
Model
4
Chemical
Analysis
Rainbow
Trout
5
Chemical
Analysis
Fathead
Minnow
6
Chemical
Analysis
7
Chemical
Analysis
8
Chemical
Analysis
9
Tolerance
of
Test
for
MBAS
of
Test
Water
for
for
MBAS
of
Test
Water
for
for
MBAS
of
Test
Water
for
for
MBAS
of
Test
Water
for
for
MBAS
of
Test
Water
for
Species
to
FC
 
2O6
Toxicant
Catfish
Bluegill
Flagfish
Figure
1
Environmental
Testing
Laboratory
Page
4
5
8
16
18
20
21
22
28
31
32
34
Page
10
Egg
Production
and
Hatchability
of
P~
imaphaiespromelus
and
Jordanella
floridae
11
Age
Versus
Lethality
to
FC­
206
for
Vertebrate
Species
12
Growth
Length
and
Weight
Data
for
Saimo
gairdneria
and
Jordanella
floridae
LIST
OF
TABLES
Table
1
Chemical
Analysis
of
Dilution
Water
15
2
LC­
50
Data
for
Test
Species
26
iv
SECTION
I
I
NTRODUCT
ION
1.
BACKGROUND
As
part
of
its
overall
environmental
quality
program,
the
Air
Force
is
required
to
assess
the
impact
of
its
operations
on
existing
ecosystems.
Because
of
the
unique
type
and
locations
of
many
Air
Force
facilities,
the
wastes
generated
by
the
many
varied
operations
are
not
common
to
the
civilian
sector.

Presently,
the
acute
(
short
 
term)
bioassay
is
being
effectively
used
to
assess
the
effects
of
toxic
materials
on
aquatic
systems.
However,
because
of
the
types
of
wastes
discharged
by
the
Air
Force,
a
chronic
(
long
 
term)
flowthrough
bioassay
was
deemed
necessary.

2.
OBJECTIVES
In
order
to
develop
sufficient
data
on
the
toxicity
of
Air
Force
wastes
to
aquatic
organisms,
the
University
of
New
Mexico
Civil
Engineering
Research
Facility
(
CERF)
was
contracted
to
construct
a
facility
in
which
to
conduct
bioassay
studies
of
Air
Force
wastes
and
to
develop
new
techniques
for
assessing
their
influence
on
natural
aquatic
systems.
The
specific
objectives
of
this
research
were
as
follows:

1.
To
establish
a
facility
to
accomplish
bioassay
and
total
ecosystem
monitoring.

2.
To
examine
the
effects
of
unique
Air
Force
wastes
on
aquatic
systems.

3.
To
establish
an
ATP
technique
to
assess
the
effects
of
wastes
on
aquatic
systems.

3.
SCOPE
Toxicity
studies
were
undertaken
on
a
new
fluorocarbon
fire­
fighting
foam
(
FC
 
206)
which
is
being
used
by
the
Air
Force.
Although
not
considered
a
hazard
1
during
emergency
fire­
fighting,
FC­
206
could
have
adverse
environmental
effects
when
used
in
large
volume
at
a
training
facility.
Continual
exposure
in
a
limited
area
makes
specific
knowledge
of
acceptable
levels
for
disposal
into
natural
waters
a
necessity.
Thus,
to
evaluate
the
effects
that
an
Air
Force
waste
could
have
on
a
total
ecosystem,
a
test
model
stream
was
constructed.

2
SECTION
II
EXPERIMENTAL
FACILITIES
1.
LABORATORY
The
main
laboratory
was
housed
in
a
3000­
ft2,
concrete­
block
building
(
Figure
1).
The
laboratory
was
divided
by
task
so
that
the
test
animals
would
be
disturbed
as
little
as
possible.
The
test
animals
were
subjected
to
a
16­

hour
photoperiod,
which
was
maintained
by
a
timer
that
was
not
equipped
to
simulate
dawn/
dusk
conditions;
however,
to
prevent
spontaneous
shock,
a
small
light
was
left
on.
Room
temperature
was
maintained
at
17
°
Cthroughout
the
year.
One
room
in
the
laboratory
was
maintained
for
the
culturing
of
tropical
fish
(
flagfish).
This
room
also
housed
the
refrigerating
unit
for
the
culturing
of
algae.

Acute
testing
was
performed
in
20.82­
liter
test
chambers.
The
cold­
water
species
(
rainbow
trout,
etc.)
were
placed
in
a
cold
 
water
bath
at
15
°
C.
The
chronic
flowthrough
experiments
were
conducted
in
five
banks
of
six
56.78­
liter
chambers,
each
divided
in
half
by
a
double­
strength
glass
panel.
The
temperature
of
the
water
in
these
chambers
was
maintained
at
15
°
Cby
a
gas­
fired
air
conditioner
adapted
with
a
heat
exchanger.
The
water
fed
to
the
Mount
and
Brungs
diluters
for
the
warm
 
water
organisms
was
heated
in
the
feeder
tank,

which
elevated
the
temperature
to
20
°
C.
The
tropical
fish
chambers
were
further
heated
by
individual
100
 
watt
heaters
on
each
side
of
the
test
chambers.

With
these
individual
heaters
the
temperature
in
the
test
chambers
could
be
maintained
at
any
level
between
200
and
27
°
C.

2.
HATCHERY
The
outdoor
facility
was
maintained
as
a
typical
hatchery
(
Figure
2).
Only
one
species
of
the
test
vertebrates
(
Jordanella
floridac)
was
actually
hatched
at
this
facility;
the
other
test
animals
came
from
a
variety
of
sources.
The
facility
has
six
18,927
 
liter
rearing
tanks
for
vertebrates,
six
refrigerated
(
minnow
cool)
473
 
liter
tanks,
twelve
1140­
liter
holding
tanks
for
algae
and
3
Storage
Wet
Chemistry
Figure
1.
EnvirOnmental
Testing
LaboratOrY
Proportional
DiluterS
for
Bioassay
and
~
jomonitOriflg
.­.
(
71
Solar
/
Heater~

/
Ecosystem
Biomoni
tori
ng
Ii
Biomonitoring
Test
Channel
Model
Stream
Rearing
and
Holding
­
Tanks
­
Laboratory
(
See
figure
1
for
details.)

Figure
2.
Outdoor
Hatchery
invertebrates,
and
ten
1136­
liter
circular
holding
tanks
for
maintaining
invertebrate
and
vertebrate
populations;
six
3028­
liter
holding
tanks
were
available
for
ecosystem
testing.
In
addition,
a
104­
meter­
long
model
stream
with
six
7.1
 
meter
 
long
substreams
and
holding
reservoirs
was
constructed.
Six
solar
collectors
were
employed
to
heat
water
to
facilitate
the
maintenance
of
the
warm­
water
test
organisms
during
the
colder
months
of
the
year.

3.
ECOSYSTEM
MODEL
During
the
past
several
decades
much
concern
has
developed
over
the
continual
deterioration
of
our
natural
waters.
Thus,
a
total
biological
ecosystem
polluted
with
FC­
206
was
examined.
The
term
ecosystem
in
its
broadest
sense
means
the
economics
of
a
system.
In
other
words,
it
encompasses
what
exactly
is
occurring
within
a
system
at
a
given
time
under
known
conditions.
The
idea
of
modeling
a
total
system
(
foodweb)
is
not
new,
but
relatively
little
work
has
been
accomplished
in
this
field.
Since
CERF
does
not
have
a
natural
body
of
water
to
work
with,
a
model
ecosystem
was
constructed.

Biologically,
the
significance
of
modeling
a
natural
environment
enhances
the
data
that
can
be
collected
in
more
conventional
laboratory
testing
(
static
and
flowthrough
bioassays).
The
effects
that
a
foreign
substance
can
have
on
a
total
system
can
be
greatly
varied
by
the
natural
system
itself.
What
occurs
in
the
form
of
recovery
is
also
helpful
in
the
total
assessment
of
the
effect
of
a
toxic
substance.
If
the
bottom
of
the
foodweb
is
destroyed
and
does
not
recover
quickly,
the
entire
system
might
be
considered
dead.
The
speed
of
recolonization
after
the
advent
of
a
polluted
condition
is
also
important.

Another
consideration
in
assessing
effects
after
introduction
of
a
foreign
substance
is
the
nature
of
the
recovery;
i.
e.,
does
the
normal
community
return
or
does
a
new
system
develop?
Combining
the
data
collected
from
laboratory
studies
~
iththat
from
simulated
natural
settings
can
greatly
enhance
knowledge
of
the
true
effects
that
a
particular
toxicant
will
have
in
a
natural
setting
(
Reference
1).

Reference
1.
Modeling
the
Eutrophication
Process,
Ann
Arbor
Science
Publishers,
Inc.,
Ann
Arbor,
Michigan,
1975.

6
To
establish
the
effects
that
a
foreign
substance
can
have
on
an
ecosystem,

total
ecosystem
monitoring
is
the
best
approach.
The
general
objective
behind
the
ecosystem
modeling
experiment
was
to
evaluate
the
effects
during
and
after
the
introduction
of
a
foreign
substance
to
a
littoral
body
of
water,
since
these
effects
can
be
reflected
in
the
initial
damage
done
to
the
total
ecosystem
and
the
speed
with
which
the
total
ecosystem
returns
to
normal.

The
main
stream
of
the
ecosystem
model
(
Figure
3)
was
constructed
of
76.2
 
cm
 
diameter
aluminum
culvert
cut
in
half.
The
unit
was
104
meters
long
with
a
vertical
drop
at
the
headwaters
of
1.22
meters
to
0.61
meter
at
the
catch
basin.

The
main
channel
substrate,
composed
of
clay
to
stones
as
large
as
5
cm
in
diameter
was
varied
to
best
approximate
a
natural
stream
bed.
The
flow
rate
was
established
at
127
liters/
mm.
The
depth
of
water
within
the
stream
varied
from
2.5
to
30.5
cm.
The
depth
in
each
section
was
maintained
by
a
series
of
wood
weirs.
The
entire
system
was
fed
by
domestic
water
which
had
been
passed
through
a
commercial
dechlorinator.

The
side
channels
were
6.1
 
meter­
long,
30.5
 
cm
 
diameter
aluminum
culverts
cut
in
half.
There
was
a
5.1
 
cm
drop
from
the
headwaters
to
the
catch
reservoir
The
catch
basins
at
the
end
of
the
side
channels
were
303­
liter,

galvanized
 
steel
tanks
coated
with
polypoxy
epoxy
marine
paint
to
stop
leaching
from
the
galvanized
metal.
1
The
substrate
in
the
side
channels
was
sand
and
small
stones
(
5
cm
or
less).

The
main
channel,
which
was
used
as
a
feeder
for
the
side
channels
during
testing
and
as
a
source
for
recolonization
after
testing,
was
established
to
act
as
a
positive
control;
the
actual
testing
was
accomplished
in
the
side
channels.

The
water
entering
the
side
channels
was
filtered
during
testing
to
prevent
recolonization
or
contamination
by
the
main
stream.
Monitoring
of
the
population
numbers
within
the
system
without
erroneous
introductions
from
the
main
channel
was
accomplished
in
this
way.
Although
there
is
no
effective
way
to
prevent
Footnote
1
Routine
analysis
of
the
water
from
this
reservoir
did
not
show
any
changes
in
water
quality
after
the
water
entered
the
system.

7
recolonization
from
airborne
spores,
random
seeding
occurred
in
both
the
polluted
section
and
the
control
section.
The
apparatus
allowed
an
escape
mechanism
for
the
motile
organisms.
The
toxicant
was
fed
into
one
channel
while
the
other
channel
was
fed
only
fresh
water
for
a
control.
Sampling
stations
were
placed
along
the
side
channels
to
monitor
the
pollutant
concentrations.
Note:
Circled
numbers
indicate
sampling
stations.

Figure
3.
Ecosystem
Model
8
SECTION
III
TEST
PROCEDURES
1.
ECOSYSTEM
TESTING
After
preliminary
testing
of
the
FC­
205
fire­
fighting
foam
in
the
laboratory
experiments
were
conducted
on
the
total
ecosystem.
The
side
channel
was
established
8
weeks
prior
to
testing,
and
the
bluegills
which
were
to
be
used
in
these
tests
were
allowed
a
30­
day
period
to
acclimate
themselves.
The
toxicant
was
gravity
fed
into
a
known
volume
of
water.
The
concentration
at
the
headwaters
was
9320
mg/.
e
(
volume/
volume)
of
concentrated
FC
 
2O6.
The
opposite
side
channel
acted
as
a
control
with
an
equal
volume
of
water
flow.
It
was
known
in
advance
that
a
concentration
in
excess
of
4000
mg/
Z
would
destroy
the
fish.
The
high
concentration
was
established
to
judge
the
effects
on
the
standing
algae
community
and
on
the
motile
organisms
within
the
test
area.
The
resident
fish
population
was
dispersed
uniformly
throughout
the
entire
system
before
testing'.

To
maintain
concentration
levels,
a
complete
chemical
backup
was
adminis
 
tered.
The
total
oxygen
content
and
1~
J~
S
~
eve~
swere
recorded
by
station.

Sanipies
were
taken
from
each
of
the
12
stations,
3
times
each
day.
Because
of
the
expense
and
time
to
process
samples,
only
a
random
spot­
check
analysis
was
performed
to
verify
that
the
`
toxicant
levels
during
the
test
remained
constant.

2.
BIOASSAY
EXPERIMENTS
a.
Introduction
Bioassay
involves
the
monitoring
of­
effects
on
living
organisms
produced
by
the
introduction
of
a
foreign
substance
to
the
environment.
The
techniques
for
this
monitoring
are
as
varied
as
the
effects
produced.
In
this
study,

the
most
commonly
accepted
procedures
for
biological
monitoring
were
used­­
acute
and
chronic
test
methods.

The
acute
test
is
normally
done
as
a
screening
test
for
96
hours
or
less.
Death
of
the
organisms
serves
as
the
means
of
evaluation
and
provides
9
answers
to
problems
quickly
and
economically.
The
chronic
test,
on
the
other
hand,
is
normally
run
over
a
longer
duration
of
time
(
24
to
2160
hour)
and
provides
monitoring
of
a
wider
variety
of
parameters
such
as
reproduction,

morphological
and
behavioral
changes,
etc.
This
test
also
provides
a
closer
approximation
of
what
is
actually
the
true
effect
of
a
toxicant
within
an
existing
aquatic
system.

Generally
the
toxicant
level
which
produces
death
in
50
percent
of
the
test
organisms
(
LC­
50)
is
used
as
a
measure
of
toxicity.
A
factor
of
10
is
used
in
some
cases
to
determine
the
level
of
the
material
which
is
acceptable
for
release
to
the
environment;
however,
this
must
be
determined
from
other
considerations
such
as
the
potential
harm
and
the
additional
cost
of
reducing
the
concentration
of
the
material
in
the
waste.

b.
Statistical
Analysis
Techniques
Interpretation
of
the
LC­
50
is
commonly
accomplished
by
the
graphic
technique
described
in
~
Reference
2.
Saiiilogarithmic
paper
with
the
percentage
 
of­
survival
plotted
on
the
X
 
axis
and
the
exposure
concentration
plotted
on
the
Y
 
axis
°
is
used.
The
upper
limit
is
that
point
at
which
more
than
50
percent
and
less
than
90
percent
of
the
organisms
die;
the
lower
limit
is
that
point
at
which
more
than
15
percent
and
less
than
40
percent
of
the
organisms
die.

The
LC­
50
is
that
point
at
which
the
line
connecting
these
two
points
(
high
and
low
limits)
intersects
the
50­
percent
line.
The
LC­
50
concentration
can
then
be
extrapolated
directly
from
the
curve.
This
graphic
expression
is
attractive
since
it
is
quick,
economical,
and
reliable.

Probit
analysis,
which
may
also
be
used
to
determine
the
LC­
50,

provides
a
somewhat
closer
approximation.
In
a
biological
assay
the
potency
of
a
stimulus
 
 
physical,
chemical,
biological,
or
physiological
 
 
is
tested
by
means
of
the
reaction
which
it
produces
in
living
matter.
In
so
doing,
a
quantal
(
all
or
nothing)
response
is
obtained.
In
the
flowthrough
bioassay
Reference
2.
Standard
Methods
for
the
Evaluation
of
Water
and
Wastewater,
13th
Edition,
American
Public
Health
Association,
Washington,
D.
C.,
1973.

10
experimental
technique,
a
group
of
subjects
on
which
a
stimulus
is
applied
to
the
environment,
resulting
in
a
response.
A
response
is
a
measure
of
the
magnitude
of
the
stimulus
and,
more
importantly,
the
intensity
required
to
produce
equal
responses.

Probit
analysis
is
one
accepted
method
for
handling
quantile
response
data
in
lieu
of
the
graphic
technique
described
above.
Graphic
methods
are
likely
to
be
misleading
if
the
curvature
of
the
dosage
response
curve
appreciably
exceeds
the
range
of
dosages
tested,
especially
if
the
distribution
of
the
dosages
tested
is
markedly
unsymmetrical
about
the
log
of
the
LC­
50.
In
probit
analyses
the
percent
kill
is
transformed
to
probit
units.
The
history
of
this
transformation
and
the
statistical
technique
is
outlined
in
Reference
3.~
Bliss
first
proposed
the
name
prabit
for
his
modification
of
Baddum's
normal
equivalent
deviant.
The
probit
of
the
proportion
P
is
defined
as
the
abscissa
which
correspond
to
a
probability
P
in
a
normal
distribution
with
a
mean
of
5
and
a
variance
of
1;
in
mathematical
terms,
the
probit
of
P
is
Y
where
Y­
5e­
1/
2
u2du
The
transformation
may
be
considered
a
stretching
of
the
percent­
kill
scale
to
give
a
probit
scale,
during
which
the
sigmoid
curve
becomes
straightened
The
sigmoid
curve
is
the
result
of
plotting
percent
kill
versus
concentration
thus,
the
probit
straightens,
this
line.
Probits
for
specific
values
of
P
have
been
prepared
by
Bliss
(
1935)
and
reproduced
by
Fisher
and
Yates
(
1948)

in
their
statistical
tables
for
biological,
agricultural,
and
medical
research.

After
experimental
data
on
the
relationship
between
dose
and
mortality
are
obtained,
either
a
graphic
or
a
numerical
technique
can
be
used
to
estimate
the
probit
line.
Both
these
techniques
are
dependent
on
the
probit
transformation
The
graphic
technique
is
much
more
rapid
and
is
sufficient
for
many
Reference
3.
Bliss,
C.
I.,
The
Method
of
Probits
­
A
Correction
Science,
79,
409­
10.

11
purposes,
such
as
preliminary
testing
for
LC­
50
concentration
and
less
 
exacting
needs.
However,
for
more
complex
problems,
for
which
an
accurate
assessment
of
the
precision
of
estimates
is
required,
the
more
detailed
numerical
technique
is
necessary.
In
drawing
a
probit
line
and
judging
i~
sagreement
with
the
data,

only
the
vertical
deviations
of
the
points
must
be
considered.
The
line
must
be
so
placed
that
the
difference
between
the
probit
values
which
are
plotted
and
the
probits
given
by
the
line
at
the
same
dosages
is
as
small
as
possible.

A
linear
least
 
squares
fit
is
made
to
the
probit
data;
extreme
probits
outside
the
range
of
2.5
to
7.5
(
1
to
99
percent)
carry
little
weight
and
should
be
disregarded.
The
fitted
probit
line
is
therefore
a
weighted
regression
line
of
probit
units
versus
the
log
of
the
concentration.
Here
weighting
can
be
thought
of
as
being
related
to
the
reciprocal
of
the
theoretical
variance,
and
the
major
assumption
underlying
the
applied
probit
analysis
is
that
the
treatments
are
independent
of
one
another.

In
the
.
probit
analysis
fiducial
limits
were
set
at
the
95
 
percent
confidence
level.
This
level
was
thought
to
be
descriptive
of
the
biological
analysis,
whereas
a
fiducial
level
lower
than
85­
percent
confidence
was
only
descriptive
of
the
experimental
design.
Therefore,
in
the
analyses
only
the
85­
to
95­
percent
confidence
level
is
indicative
of
the
experiment
and
the
fiducials
shown
are
a
measure
of
the
exactness
of
the
experiment.

Fiducials
were
calculated
when
no
evidence
of
significant
heterogeneity
of
the
points
about
the
regression
line
was
found.
Then,
the
95
 
percent
confidence
level
was
computed
as
a
5­
percent
probability
that
the
deviation
from
the
true
log
of
LC­
50
is
the
estimate
that
will
be
greater
than
and
lower
than
the
true
value.
These
limits
will
correspond
to
a
concentration
and
would
be
equidistant
from
the
estimated
LC­
50.
But,
since
the
limits
derived
are
plotted
on
a
logarithmic
scale,
they
will
not
be
symmetrical
when
placed
on
a
concentration
scale.
The
difference,
however,
is
trivial
for
close
 
fitting
data,
but
with
relatively
larger
standard
errors
or
for
more
extreme
limits,
the
difference
may
be
considerably
greater
(
Reference
4).

Reference
4.
Finney,
0.
J.,
Probit
Analysis,
Cambridge
University
Press,
1962.

12
C.
Diluter
A
modified
Mount
and
Brungs
proportional
diluter
was
used
to
dispense
the
toxicant.
These
diluters
are
composed
of
glass
with
tygon
tubing
for
the
fittings.
The
walls
of
the
diluter
are
made
of
double­
strength
glass
sealed
with
Dow
Corning
silicone
sealant.
The
use
of
glass,
limited
tygon
connections,

and
silicone
sealant
affords
a
unit
of
sturdy
construction
and
a
unit
that
is
easily
disassembled,
relatively
free
from
external
contamination,
and
easily
cleaned
between
and
during
tests.
A
complete
description
of
the
diluter
is
given
by
Mount
and
Brungs
in
Reference
5.

The
modification
to
the
diluter
consisted
of
replacing
the
flow
splitter
with
a
U
 
tube
within
the
box
and
a
Y­
connector
outside
the
box.
The
Yconnector
acted
as
a
flow
splitter
and
the
U
 
tube
provided
a
more
uniform
mix.

The
standard
flow
splitter
was
found
ineffective
because
heavy
bacterial
growth,

stimulated
by
the
toxicant,
clogged
the
system.

Proportional
diluters
have
an
advantage
over
serial
diluters
in
that
one
concentration
is
diluted
to
achieve
all
concentrations;
the
diluter
provides
`
five
different
concentrations
PIUS
a
positive
control.
Also,
this
diluter
system
can
provide
concominant
replicates.
The
diluters
used
at
this
facility
were
gravity
fed
(
H20
and
toxicant)
with
domestic
water
that
had
been
passed
through
a
commercial
dechlorinator.
This
system
was
employed
to
preclude
experimental
losses.
The
toxicant
was
also
fed
into
the
system
on
a
gravity
basis
(
dipping
bird).
The
system
cycles
were
set
at
3.5
minutes
to
maintain
the
dissolved
 
oxygen
and
temperature
levels.

The
calibration
of
the
diluter
system
was
accomplished
by
using
green
food
coloring.
This
dye
was
diluted
(
one
to
one)
with
tap
water.
The
50­
percent
mixture
was
allowed
to
flow
into
the
system
in
the
same
manner
as
the
toxicant.

The
diluter
was
allowed
to
cycle
for
several
minutes
and
then
an
aliquot
was
taken
from
each
of
the
replicate
test
chambers
and
analyzed
for
absorbance
on
Reference
5.
Mount,
D.
I.,
and
Brungs,
W.
A.,
"
A
Simplified
Dosing
Apparatus
for
Fish
Toxicology
Studies,"
Water
Research,
Vol.
1,
1967,
p.
21.

13
a
l3auch
and
Lomb
Model
20
Spectrophotometer
at
510
nm.
A
straight
line
color
plot
was
constructed
to
establish
concentration
in
the
test
chambers
(
Reference
6).

d.
Chemical
Analysis
To
properly
evaluat°
any
toxicant,
monitoring
of
the
system
must
be
continuous.
Routinely
in
this
experimentation,
the
quality
of
the
water
was
tested.
Throughout
the
experiments,
daily
checks
were
made
of
the
pH,

conductivity
temperature,
and
dissolved
oxygen.
The
temperature
and
dissolved
oxygen
were
different
for
each
test,
but
they
were
never
allowed
to
reach
stress
levels
for
any
test
species.
These
tests
were
performed
to
assure
that
the
quality
of
the
test
water
did
not
change.
Additionally,
after
the
intror~

Ction
of
FC­
206,
the
chemical
oxygen
demand
was
measured
routinely
to
insure
that
problems
did
not
arise
within
the
system.

In
addition
to
the
routir~
etests,
water­
quality
tests
were
performed
to
evaluate
the
quality
of
the
water
for
aquatic
testing.
These
tests
were
performed
.
by
the
Environmental
Chemistry
Research
Division,
AFCEC,
located
at
Kirtland
Air
Force
Base,
New
Mexico
and
the
Environmental
Health
Laboratory
located
at
Kelly
Air
Force
Base,
Texas.
The
results
of
these
analyses
are
given
in
Table
1;
they
substantiate
that
the
water
quality
was
in
line
with
the
acceptable
ranges
outlined
for
bioassay
testing.

To
evaluate
the
functioning
of
the
diluters,
a
means
to
measure
the
toxicant
concentration
had
to
be
determined.
It
was
found
that
for
ease
and
economy
of
analysis,
the
Methylene
Blue
Active
Substance
(
MBAS)
Method
was
the
best
approach
(
Reference
2).
Further
studies
showed
that
the
t4BAS
was
a
conservative
component
of
the
FC­
206
and
could
be
used
as
a
reliable
indirect
measure
of
the
toxicant
concentrations.
From
the
analysis,
it
was
found
that
the
toxicant
contained
15,726
mg/!.
MBAS.
Although
t.
he
MBAS
technique
gave
numbers
on
the
low
side
of
those
calculated
from
the
diluters,
it
did
provide
a
means
to
monitor
the
performance
of
the
diluters.
Figures
4
through
8
give
the
results
of
the
MBAS
analyses
for
all
the
FC­
206
toxicant
studies.

Reference
6.
Water
Alnosphere
Analysis,
1974
Annual
Book
of
ASTM
Standards,
Part
23,
American
Society
for
Testing
and
Materials,
1974.

14
Calcium,
mgi!.

Magnesium,
mg/!.

Sodium,
mgi!.

Potassium,
mgi!.

Bicarbonate,
mg/!.

Carbonate,
mg/!.

Sulfate,
mg/
L
Chloride,
mg/!.

Fluoride,
mgi!.

Nitrate
and
Nitrite
as
Nitrogen,
mg/!.

Dissolved
Solids
at
180
°
C,
mg/!.

Calculated
(
Sum),
mg/!.

Hardness
Total,
mgi!.

Noncarbonate,
.
mg/!.

Alkalinity
as
CaCO3,
mgi!.

Carbon
Dioxide,
mg/!.

Sodium
Adsorption
Ratio
Langlier
Ratio
at
25
°
C
Conductivity,
pmhos
at
25
°
C
Color,
Pt­
Co
Units
pH
Manganese,
mg/!.

Iron,
mg/!.

Silica,
mg/!.

Turbidity,
JTU
Units
Table
1.
CHEMICAL
ANALYSIS
OF
DILUTION
t~
JATER
Characteristic
Mean
Characteristic
Mean
(
31
52
8.3
27
2.9
124
0
51
13.8
0.54
1.64
285
278
165
23
143
18
0.9
­
0.2
430
0.875
7.48
0.0025
0.14
30
2.0
Chemical
Oxygen
Demand,
mgi!.

Dissolved
Solids,
mgi!.

Oils
and
Greases
(
Infrared),
mg/!.

Surfactants
(
LAS),
mg/.
e.

Phenols,
mg/!.

Phosphates,
mgi!.

Cadmium,
mgi!.

Chromium
(
Hexavalent),
mg/!.

Chromium
(
Total),
mg/!.

Copper,
mg/!.

Cyanides,
mg/!.

Iron,
mgi!.

Lead,
mg/!.

Silver,
mgi!.

Zinc,
mg/
c.

Mercury,
mg/!.

Total
Organic
Carbon,
mg/!.

Nitrite
Nitrogen,
mg/!.

Ammonia
Nitrogen,
mg/!.

Barium,
mg/!.

Arsenic,
mg/!.

Boron,
mgi!.

Nickel
,
mg/!.

Selenium,
mg/!.
5.0
273.0
0.3
1.1
0.
cJOl
0.3
0.01
0.01
0.05
0.02
0.01
0.1
0.05
0.01
0.05
0.
001
1.0
0.02
0.2
1.0
0.01
0.5
0.05
0.01
3.0
2.5­

2.(
­

1.5
 
1
.
C
­

0.5
0.0
Test
Chamber
1
rirl
Test
Chamber
2
Time
n
Test
Chamber
3
01
E
cO
(
0
0
4.)

(
ci
L
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0
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11
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lHhnhhhnflhHHhlnh
­
Figure
4.
Chemical
Analysis
for
MBAS
of
Test
Water
for
Rainbow
Trout
3.
C
Test
Chamber
4
Test
Chamber
6
01
E
`.
0
c.~
J
C­
i
U­

`
I­

0
~
0
4.)

(
ci
C
`
1,

UC0
C­,

­
J
 
4
2.5
2.0
1.
1.
0.5
o.
n
Test
Chamber
5
i­
i
r~­~~
ri_~
afl
fl
n
n
n
n
11
Time
1
Ji­
II
~:
ffl
HI
Figure
4.
Chemical
Analysis
for
MBAS
of
Test
Water
for
Rainbow
Trout
(
Concluded)
01
E
`.
0
C')
(­~)

U­

`
4­

0
C0
r
4­,

(
ci
4­)

C
ci)

0
C0
c)

­
J
OD
Time
Figure
5.
Chemical
Analysis
for
MI3AS
of
Test
Water
for
Fathead
Minnow
01
E
`.
0
C
C­'

U­

4­

0
C
°
0
4­)

(
ci
L.
.4­)

C
0)

0
C
0
L)

­
J
Time
Figure
5.
Chemical
Analysis
for
MBAS
of
Test
Water
for
Fathead
Minnow
(
Concluded)
1.00
~­
 
 
 
 
0.75
­

0.50
0.25­

1.50
1.25
Test
Chamber
1
Test
Chamber
2
Test
Chamber
3
Test
Chamber
5
Test
Chamber
6
01
E
`.
0
cD
C')

U­

9­

0
C
0
4.)

(
ci
I­
4­)

C
0)

0
C0
C­)
Test
Chamber
4
Ii
­
H­

0.00
I­
I
_
J_
Ti
me
i­
i
H
[
1
fin
Figure
6.
Chemical
Analysis
for
MBAS
of
Test
Water
for
Catfish
3..

01
E
`.
0
C,
C­,

U­

`
4­

0
C0
4­.,

`­
ci
I­
.1­'

C
a)

U
C0
L)
Test
Chamber
1
Test
Chamber
3
Test
Chamber
4
Test
Chamber
5
Test
Chamber
6
2.
2.

1.
1.

­
J
Time
Figure
7.
Chemical
Analysis
for
MBAS
of
Test
Water
for
Bluegill
Test
Chamber
1
Test
Chamber
3
Test
Chamber
4
Test
Chamber
6
Test
Chamber
5
01
`.
0
D
C')

(
U­

(
4­

0
C
0
4.)

(
ci
4­
i
C
0)

UC0
L)
5
 
4­
3­

2
 
1
 
n
Test
Chamber
2
H
Time
Figure
8.
Chemical
Analysis
for
MBAS
of
Test
Water
for
Flagfish
e.
Selection
of
Test
Organisms
Because
it
was
desired
to
test
both
warm­
and
cold­
water
organisms,

the
experimental
animals
were
obtained
from
~
variety
of
sources.
The
parent
stock
of
Jordnella
fioridae
was
obtained
from
the
United
States
Environmental
Protection
Agency,
Duluth,
Minnesota.
This
fish,
which
is
indigenous
to
Florida
was
chosen
for
its
ease
of
breeding
and
handling
under
laboratory
conditions
The
fish
is
unique
in
that
three
generations
can
be
observed
in
one
calendar
year.
This
affords
the
opportunity
to
observe
any
morphological
changes
that
might
occur
in
successive
generations
following
exposure
to
a
toxicant.

Fish
showing
a
southern
midwest
distribution
were
represented
by
Ictaluras
punctatus
(
channel
catfish)
and
Pimaphales
prorne7~
us
(
fathead
minnow).

These
test
animals
originated
at
the
United
States
National
Fish
Hatchery
located
at
Uvalde,
Texas.
Lepornis
macrochirus
(
bluegill
sunfish)
came
from
the
National
Fish
Hatchery
located
at
Dexter,
New
Mexico.
The
only
cold
 
water
vertebrate
species
tested
was
Sairao
gairdneria
(
rainbow
trout).
This
test
group
came
from
the
United
States
National
Fish
Hatchery
located
at
Mescalero,
New
Mexico;
the
eggs
for
these
animals
were
supplied
to
the
hatchery
from
a
commercial
hatchery
in
Northern
California.

The
test
animals
were
examined
to
insure
that
no
disease
was
introduced
into
the
facility.
These
animals
were
given
a
2
 
to
4
 
week
period
to
acclimate
themselves
to
the
new
water
conditions
before
testing
occurred.

The
invertebrate
animals
were
selected
on
the
same
basis
as
the
vertebrates
They
were
as
follows:
Chironomus,
midge
(
larval
stage);
Daphnia
magna,
water
flea
(
first
instar
and
adult);
and
Gastropoda,
snail.
These
invertebrates
were
reared
at
the
CERF
facility.

23
SECTION
IV
EXPERIMENTAL
RESULTS
1.
ECOSYSTEM
Within
1
hour
after
ecosystem
testing
began,
the
population
of
fish
showed
signs
of
behavioral
searching
patterns.
The
animals
in
the
test
channel
migrated
into
the
catch
basin;
i.
e.,
the
population
detected
a
foreign
substance
and
was
reacting
by
looking
for
escape
routes
from
the
affected
area.
As
is
characteristic
of
many
toxic
substances,
this
toxicant
seemed
to
disorient
some
of
the
test
animals.
The
animals
that
chose
the
wrong
route
of
escape
fell
victim
to
the
toxicant
in
less
than
6
hours;
the
remaining
animals
were
able
to
maintain
themselves.
After
12
hours
of
exposure,
the
catch
basin
contained
4660
mg/!.
of
FC­
206;
from
previous
tests
this
amount
was
shown
to
be
a
lethal
dosage
for
this
test
animal.
Because
many
fish
frequented
this
area
after
the
first
day
of
testing,
it
was
felt
that
some
damage
had
already
been
done
to
the
biological
mechanism
for
escape
that
caused
these
animals
to
remain
in
the
toxic
reservoir.
However,
because
they
were
able
to
remove
themselves
from
the
toxicant
for
part
of
the
time,
these
animals
survived.
This
indicates
that
in
nature
if
there
are
means
of
escape
(
avoidance
of
the
toxicant),
total
elimination
of
the
species
will
probably
not
occur.
Throughout
the
21
 
day
test,
56
percent
of
the
total
fish
population
was
destroyed.
Some
dead
animals
were
found
in
the
control
channel
at
station
11;
many
of
these
animals
probably
died
because
they
remained
too
long
in
the
polluted
reservoir.
As
stated
earlier,

this
toxicant
seems
to
act
much
like
a
sedative
and
this
could
account
for
some
of
the
deaths
in
the
reservoir.
Residual
effects
were
shown
in
this
experiment
in
that
after
stopping
the
administration
of
the
toxicant
on
the
eighth
day,

16
percent
of
the
population
still
expired.

Probably
more
apparent
in
this
study
were
the
effects
of
the
toxicant
on
the
invertebrate
population
of
the
polluted
channel.
Within
48
hours
the
polluted
channel
of
the
stream
was
devoid
of
life,
except
for
an
occassional
snail.

The
snails
that
remained
alive
did
so
by
actually
crawling
up
out
of
the
water
from
time
to
time.
Within
96
hours
after
the
test
began,
the
snail
population
had
moved
to
the
control
channel.

24.
Because
of
the
nonavailability
oF
baseline
workups
of
species
diversity
and
population
levels,
it
was
impossible
to
speculate
on
recovery
rates.
It
can
be
noted
that
within
72
hours
the
toxic
test
channel
was
devoid
of
life
while
the
control
channel
was
teaming
with
life.
In
general
,
the
control
side
was
a
true
representation
of
a
typical
seasonal
community
with
no
apparent
ill
effects
due
to
testing.

As
the
toxic
channel
began
to
recover,
an
intense
bacterial
bloom
occurred
this
indicated
that
residual
eutrophic
effects
were
occurring.
Within
96
hours
after
stopping
the
administration
of
the
toxicant,
chemical
analysis
showed
no
MBAS
present.
The
bacterial
bloom
occurred
6
weeks
after
testing
was
stopped.
The
control
side
experienced
an
algae
bloom
devoid
of
bacteria.

The
recovery
of
the
polluted
side
was
hampered
by
the
presence
of
the
bacteria
and
seasonal
cold
weather.
However,
the
polluted
side
compl'etely
recovered
in
the
spring
with
no
ill
effects.
The
bacterial
community
was
replaced
by
a
normal
seasonal
algal
community.

2.
BIOASSAY
TESTS
To
best
evaluate
the
FC
 
206
toxicant,
its
effects
need
to
be
looked
at,

not
by
genus
or
family,
but
by
individual
groups
and
their
habitat
orientation.

There
appeared
to
be
a
direct
correlation
between
the
toxicity
expressed
by
warm­
water,
low­
oxygen
 
level
habitat
organisms
and
cold­
water,
and
high­

oxygendemanding
organisms.
The
rainbow
trout
was
the
most
sensitive
of
the
fish
tested
(
Table
2).
The
sensitivity
of
trout
to
foreign
substances
has
been
shown
in
a
variety
of
other
studies.
The
more
tolerant
organisms,
as
stated
before,
appear
to
be
those
that
do
not
need
high
levels
of
oxygen
to
survive.

Figure
9
shows
graphically
the
tolerance
to
FC­
2O6
expressed
by
all
the
organisms
tested.

To
account
for
the
toxicity,
the
mode
of
action
produced
by
the
toxicant
should
be
known.
FC
 
2O6
appears
to
affect
the
transfer
of
oxygen
across
the
gill
membranes.
Before
death
the
vertebrate
organisms
go
through
the
classical
stages
exhibited
when
low
dissolved­
oxygen
levels
are
present;
they
become
alarmed
easily.
Sudden
changes
in
light
intensity
caused
the
fish
to
become
excited
to
the
point
of
bringing
on
premature
death.
The
racing
motion
25
Table
2.
LC­
5O
DATA
FOR
TEST
SPECIES
Species
Age
Test
Period
LC­
5O
(
95%
Confidence
Level)

Fathead
Minnow
Channel
Catfish
Flagfish
Bluegill
Rainbow
Trout
Young
(
1
month)
96
hr
24
hr
Young
(
3
months)
72
hr
Adult
96
hr
36
hr
Young
(
6
weeks)
96
hr
Young
(
2
weeks)
96
hr
Eggs
96
hr
Adult
96
hr
Eggs
144
hr
144
hr
Adult
(
6
months)
72
hr
2527.0
2527.5
2550.2
3378.0
2113.0
1198.0
2113.0
97.0
1635.0
20.0
18.0
1901.6
33.59
77.60
79.82
43.33
37.61
42.43
53.07
37.61
43.77
63.73
51.90
58.97
959.10
1141.0
1666.2
1702.5
1161.9
1700.0
950.0
2
month
start
fry
2
month
start
fry
2
month
start
fry
4
month
start
fry
4
month
start
fry
4
month
start
fry
4
month
start
fry
4
month
start
fry
4
month
start
fry
1
month
start
fry
1
month
start
fry
1
month
start
fry
1
month
1
month
2
months
3
months
3
months
2
months
2
months
29
days
29
days
29
days
76
days
76
days
76
days
76
days
76
days
76
days
28
days
28
days
28
days
96
hr
96
hr
48
hr
48
hr
96
hr
48
hr
96
hr
26
Table
2.
LC­
50
DATA
FOR
TEST
SPECIES
(
Concluded)

Sp2cies
Age
[
Test
Period
(
95%
Confidence
Level)

1
month
96
hr
1
month
96
hr
1
month
soft
96
hr
1
month
soft
96
hr
1
`
month
soft
96
hr
1
month
soft
96
hr
1
month
soft
144
hr
1
month
soft
144
hr
Instars
96
hr
Instars
96
hr
Instars
96
hr
Instars
288
`
hr
Instars
288
hr
Instars
48
hr
Instars
48
hr
Instars
48
96
48
48
48
hr
hr
hr
hr
hr
Instars
96
hr
Instars
48
96
48
hr
hr
hr
6
weeks
90
days
Fry
9~
Tdays
Adult
90
days
Adult
90
days
Adult
90
days
Eggs
96
hr
Adult
96
days
Mayfly
Chironomid
Mayf1
y
Daphnia
Chi
ronomjd
Daphnia
Rainbow
Trout
Flagfish
Combined
Fathead
Flagfish
Combined
Flagfish
Flagfisha
Bluegill
814.64
963.44
1008.1
1023.7
1008.1
889.55
780.27
680.77
69.16
46.00
49.92
198.87
149.61
228.14
153.79
245.96
1922.4
4189.1
4189.1
3973.0
413.0
840.0
4626.0
12,955.0
30.65
303.0
198.0
292.0
298.0
145.0
2921.0
~
With
aeration.

27
7003
4000
­

3000­

2000
­

1000­
~`
3~
4~

13
Figure
9.
Tolerance
of
Test
Species
to
FC­
2O6
Toxicant
2
C,

`.
O
e
C)

o~
.,­

0)
U
C,

C,
Legend
1.
Fathead
(
Adult)
2.
Fathead
(
Fry)
3.
Channel
Catfish
(
Fry)

Do
­
4.
Flagfis.
h
(
Adult)
5.
Flagfish
(
Young)
6.
Flagfish
(
Eggs
­
No
Aeration).
7.
Flagfish
(
Eggs
 
Aeration)
8.
Bluegill
(
Adult)
9.
Rainbow
Trout
(
Fry)
10.
Rainbow
Trout
(
Fry
­
Soft
Water)

5000
 
11.
Mayfly
(
Larva
­
First
Instar)
12.
Water
Flea
(
First
Instar).
13.
Chirnomid
(
Larva
­
First
Instar)

8
6~
nfl.
r,
11
n
28
produced
by
external
disturbances
speeded
up
the
opercular
movements
and
eventually
caused
the
death
of
the
organisms.
Displays
of
escape
behavior
were
common;
the
fish
actually
jumped
from
the
water.
Thus
all
of
the
test
chambers
were
covered
during
this
experiment
to
eliminate
the
biasing
of
the
data
by
animals
jumping
from
the
test
chambers.
There
appeared
to
be
hemorrhaging
of
the
temporal
region
of
the
head.
Before
the
stages
just
described,
the
animals
underwent
other
physiological
changes.
There
was
a
darkening
of
the
skin;
the
animals
became
lethargic;
and
feeding
was
sporadic­­
the
fish
merely
striking
the
food
and
not
actually
eating
it.
In
many
cases
the
cause
of
death
was
probably
starvation.
This
behavior
had
occurred
in
other
studies
in
which
animals
were
subjected
to
MBAS,
LAS,
and
ABS
contamination
(
Reference
7).
It
should
be
added
that
at
no
time
during
the
test
or
when
these
symptoms
were
recorded
was
the
oxygen
level
in
the
test
chambers
to
a
point
at
which
the
animals
should
have
been
stressed.
If
gill
 
associated
anoxia
was
the
mode,
the
trout's
sensitivity
may
be
explained
because
the
organisms
most
tolerant
to
the
FC
 
206
toxicant
were
also
those
most
tolerant
to
the
low
oxygen
levels.

Additional
work
was
performed
to
evaluate
the
eff.
ects
produced
when
trout
fry
were
exposed
to
excessively
high
levels
of
FC­
206
for
from
1
to
6
minutes.

At
a
concentration
of
30,000
mg/.
C,
trout
fry
went
under
water
and
gave
the
appearance
of
sedated
animals
within
30
seconds
after
the
introduction
of
the
toxicant.
Upon
removal
of
the
test
fish,
recovery
occurred
in
3
to
5
minutes.

When
left
in
this
concentration
for
more
than
4
minutes,
the
entire
population
was
lost.
When
exposed
to
60,000
mg/
L.,
the
animals
survived
for
only
2
minutes
before
substantial
losses
were
incurred.
However,
no
permanent
damage
to
the
exposed
animals
was
evident.
These
test
animals
were
observed
for
several
days;

no
losses
or
behavioral
differences
between
the
control
and
the
test
groups
were
noted.
In
the
sublethal
work,
this
was
not
the
case.
After
extended
exposure
to
low
levels
of
toxicant,
the
sick
animals
died
when
placed
in
fresh,
clean
water.
This
shows
that
gradual
irreversible
damage
is
done
by
extended
perio!
is
of
exposure
to
low
levels
of
FC­
206.
This
experiment
showed
that
in
a
natural
situation
the
animals
have
only
a
few
seconds
to
properly
evaluate
the
situation
and
escape
before
the
toxicant
takes
effect.

Reference
7.
Howes,
D.,
Newson,
C.,
and
Tovell,
P.
W.
H.,
"
Effects
of
Water
Hardness
on
the
Toxicity
of
Nonionic
Detergents
to
Fish,"
The
Journal
of
the
International
Association
of
Water
Pollution
Research,
Vol.
9,
January
1975.

29
A
90­
day
study
was
conducted
to
evaluate
the
effects
of
the
FC­
206
toxicant
on
the
spawning
of
selected
test
organisms.
It
was
known
from
previous
work
that
a
concentration
in
excess
of
145
mg/~
e.
would
be
fatal
to
development
and
thus
care
was
taken
to
protect
the
adults
from
these
high
concentrations.

However,
after
1
month
no
eggs
were
produced
at
any
concentration
over
the
control
It
appears
that
the
eggs
initially
produced
in
the
lowest
concentration
were
eggs
that
were
already
prepared
for
spawning.
These
test
fish
were
randomly
selected
from
the
breeder
tanks.
This
test
was
conducted
in
accordance
with
the
methods
outlined
by
Mr.
Don
Allison
of
the
Environmental
Protection
Agency,
National
Water
Quality
Laboratory
(
Reference
8).
The
data
indicate
that
at
concentrations
as
low
as
77
mg/.
tL
egg
production
did
not
occur
after
the
first
30
days
of
the
study.
Since
it
cannot
be
clearly
stated
that
FC­
206
prevents
spawning
(
Figure
10)
further
investigation
is
warranted.

In
an
additional
study,
fathead
minnow
adults
were
encouraged
to
spawn.

Although
this
study
was
conducted
to
obtain
a
90­
day
LC­
50,
spawning
did
occur
in~
the
control
group
and
at
a
concentration
of
52.49
mg/
L
However,
at
the
next
higher
concentration
(
147
mgf~.)
spawning
did
not
occur.
It
appears
that
the
potential
for
the
cessation
of
spawning
exists
when
fish
are
subjected
to
sublethal
dosages
of
FC
 
206
for
extended
periods.
This
theory
is
supported
by
the
testing
of
flagfish
eggs.
Eggs
allowed
to
hatch
without
aeration
or
agitation
showed
much
lower
tolerance
to
FC­
206
than
those
hatched
in
aerated
or
agitated
waters;
agitated
and
aerated
eggs
showed
a
five­
fold
better
hatchability
The
development
of
a
surface
film
appeared
to
interfere
with
oxygen
uptake
and
thus
the
eggs
were
killed.
There
was
also
a
time
delay
in
the
hatching
of
the
eggs
after
the
introduction
of
FC­
206.
This
may
indicate
low
dissolved­
oxygen
availability
to
the
eggs.

It
was
also
found
that
those
invertebrates
normally
found
in
low­

oxygenlevel
water
survived
the
best
during
testing.
The
mayfly
larva
showed
a
very
low
tolerance
to
FC­
206
and
required
high
oxygen
levels
for
survival;
whereas
the
water
flea
was
more
tolerant
to
low
oxygen
levels
and
to
FC
 
206.

Reference
8.
Goode
and
Beane,
Recommended
Bioassay
Procedures
for
Jordanella
fiorid.
ae
Chronic
Testing,
United
States
Environmental
Research
Laboratory,
Duluth,
Minnesota.

30
~
20I
Concentration
of
FC­
206,
mgfC
Figure
10.
Egg
Production
and
Hatchability
of
Pimaphales
promelus
and
Jordanella
floridae
0
50
147.
0
77~
90
31
3000
2500
­

~
2000
°
l500
C.
0'

4­',

~.
100o
 
.4­,,
C:
0),

0
 
Figure
11.
Age
Versus
Lethality
to
FC­
206
for
Vertebrate
Species
The
differences
between
the
LC­
50s
among
different
tests
of
conspecific
is
well
within
the
range
of
acceptability
(
Table
2).
Although
there
is
a
lack
of
sufficient
data
on
soft
water,
it
appears
that
there
is
no
substantial
change
in
the
lethality
of
the
FC
 
206
between
treatments
of
soft
and
hard
water.

Although
the
data
are
inconclusive
to
speculate
on
the
effects
on
the
same
species
at.
different
chronological
periods
of
development,
it
appears
that
there
is
no
substantial
difference
in
the
toxicity
of
FC
 
206
at
different
developmental
stages
in
fish
(
Figure
11).

Normal
respiration
of
Daphnia
is
accomplished
by
the
passage
of
oxygen
across
the
body
membrane.
This
could
account
for
the
high
tolerance
in
that
it
would
be
more
unlikely
that
the
total
body
system
would
be
engulfed
in
a
film
than
it
would
for
a
surface
film
to
develop
on
the
gills
of
a
fish.

Although
tests
were
performed
on
channel
catfish
and
bluegill
fry
for
a
90­

day
period,
an
LC­
50
was
not
determined.
The
concentrations
used
in
these
tests
were
not
sufficient
to
cause
substantial
kills.
There
was
a
difference
in
the
Juvenile
~
Adult~

/
/

I
I
32
rate
of
growth
shown
in
th2
bluegill
tests;
however,
in
the
catfish
experiment,

there
was
no
concrete
evidence
of
any
growth
changes
at
the
levels
tested.
The
levels
established
for
catfish
were
from
18
to
100
mg/­
c­;
the
levels
for
bluegill
were
from
30
to
200
mg/
L
The
nongrowth
differentiation
shown
by
the
catfish
could
reflect
again
that
a
species
tolerant
to
low
oxygen
levels
would
not
be
as
seriously
affected
in
its
feeding
patterns
as
would
an
organism
dependent
on
a
high
oxygen
level.
Therefore,
if
the
animal
is
not
being
affected
in
its
feeding
or
normal
functions
it
would
stand
to
reason
that
its
growth
rate
would
not
change.
In
all
the
experiments
conducted,
except
those
on
the
channel
catfish,
the
organism's
normal
growth
patterns
were
disturbed.
It
is
not
known
at
this
time
if
the
somatogenic
level
of
secretion
was
affected.

However,
it
is
not
felt
that
this
is
the
case,
since
the
channel
catfish
did
not
show
growth
changes.
Examples
of
these
data
are
given
in
Figure
12.
As
can
be
seen
in
the
graphic
representation
for
trout,
these
organisms
appeared
to
show
a
crossover
between
19
and
16
mg/.
e;
this
probably
occurred
because
at
levels
below
20
mg/
Se.,
no
effects
were
being
felt
by
the
test
animals.
The
apparent
crossover
at
37
mg/.
P~
is
brought
about
by
having
too
few
points
to
reflect
a
true
distribution.
Here
the
graph
is
skewed
because
of
one
abnormally
large,
hardy
animal
in
the
test
chamber.

The
data
for
the
flagfish
were
a
good
representative
collection
which
reflected
a
true
growth
pattern
from
juvenile
to
adult.
­
The
atrophy
established
by
the
exposed
animals
was
evident.
These
two
tests
were
chosen
to
reflect
growth
rate
differences
between
a
hardy,
tolerant
species
and
a
very
nontolerant
species.
The
data
presented
in
this
figure
represent
combined
data
from
the
replicates
of
these
tests.

33
Concentration
of
FC­
206,
mg/~
C.

Figure
12.
Growth
Length
and
Weight
Data
for
Sa7.
mo
gairdneria
and
Jordanelia
floridae
0.88
Jordanella
floridae
U
Length
0
Weight
cr,
~

.
C
~
0~
c~
C
~
16
14
­

12
 
10
 
8­

6­

4~.

2­

0
0.8
4.0
0.7
3.5
0.6
3.0
0.
E
­
2.
E
0.4
2.0
0.3
1.5
0.2
1.0
0.1
0.5
Bairno
qairdncria
o
Length
~
Weight
`
1
`
1
`
4
/

`
5
0.0
16
19
29:
37
0.0
0
100
200
250
350
500
34
SECTION
V
CONCLUSIONS
AND
REC0~
MENDATIONS
1.
CONCLUSIONS
1.
FC
 
206
has
devastating
effects
at
concentrations
as
low
as
50
mgJ.
C­
to
some
invertebrate
and
vertebrate
species.
2.
The
family
Salmo
is
the
most
susceptible
of
the
fish
tested
to
the
FC
 
205
toxicant.

3.
Spawning
is
probably
affected
by
FC­
206
even
at
low
levels
(
100
mg/.
C~

or
less).

4.
FC­
205
seriously
affects
the
growth
and
development
of
vertebrate
organisms.

5.
FC­
206
may
retard
the
hatching
of
eggs.

6.
FC
 
205
at
low
levels
probably
prevents
the
formation
and
disposition
of
eggs
by
vertebrate
species.

7.
FC
 
206
does
not
appear
to
cause
genetic
morphological
changes
nor
does
it
appear
to
produce
infertility
in
the
F1
or
F2
generations
of
offsprings
of
fish
subjected
to
low
levels.

8.
Fish
are
capable
of
detecting
low
levels
of
FC­
206
and
will,
if
given
the
opportunity,
escape
from
the
area
of
contamination.

9.
The
use
of
model
stream
systems
to
evaluate
toxicants
is
not
only
feasible
but
gives
data
that
are
more
representative
of
what
is
actually
happening
in
nature.

10.
Algae
populations
are
destroyed
by
FC­
206
at
levels
of
less
than
9420
mg/
L
11.
FC
 
206
appears
to
increase
the
eutrophication
process
of
an
aquatic
system.

2.
RECOMMENDATIONS
1.
From
the
results
of
these
tests,
it
is
recommended
that
the
toxicant
be
used
for
training
purposes
only
when
proper
precautions
to
prevent
water
contamination
have
been
taken.

35
2.
If
feasible,
the
foam
should
not
be
used
during
known
seasons
of
local
spawning.
Training
schedules
should
be
set
up
in
accordance
with
local
fish­
spawning
conditions.
3.
Even
though
FC
 
206
is
biodegradable,
it
should
be
diluted
as
much
as
possible
before
it
is
allowed
to
enter
a
water
supply
or
domestic
treatment
plant.

4.
Additional
follow
 
up
studies
should
be
performed
to
ascertain
the
mode
of
action
produced
by
this
toxicant.

5.
Additional
local
land­
u~
eand
water
 
use
evaluations
should
be
made
prior
to
extensive
training
uses
of
this
product.

6.
Additional
work
should
be
accomplished
on
the
effects
of
this
toxi
 
cant
on
terrestrial
organisms.

36
REFERENCES
1.
Modeling
­
the
Eutrophication
Process,
Ann
Arbor
Science
Publishers,
Inc.,
Ann
Arbor,
Michigan,
1975.

2.
Standard
Methods
for
the
Evaluation
of
Water
and
Wastewatcr,
13th
Edition,

American
Public
Health
Association,
Washington,
D.
C.,
1973.

3.
Bliss,
C.
I..,
The
Method
of
Probits
­
A
Correction
Science,
79,
409­
10.

4.
Finney,
D.
J.,
Probit
Analysis,
Cambridge
University
Press,
1962.

5.
Mount,
0.
I.,
and
Brungs,
W.
A.,
"
A
Simplified
Dosing
Apparatus
for
Fish
Toxicology
Studies,"
Water
Research,
Vol.
1,
1967,
p.
21.

6.
Water
Atmosphere
Analysis,
1974
Annual
Book
of
ASTM
Standards,
Part
23,
American
Society
for
Testing
and
t~
1aterials,
1974.

7.
Howes,
D.,
Newson,
C.,
and
Tovell,
P.
W.
H.,
"
Effects
of
Water
Hardness
on
the
Toxicity
of
Nonionic
Detergents
to
Fish,"
The
Journal
of
the
International
Association
of
Water
Pollution
Research,
Vol.
9,
January
1975.

8.
Goode
and
Beane,
Recorivnended
Bioassay
Procedures
for
Jordanella
floridae
Chronic
Testing,
United
States
Environmental
Research
Laboratory,
Duluth,
Minnesota.

37
(
The
reverse
of
this
page
is
blank.)
APPENDIX
A
ADEOSINE
TRIPHOSPHATE
(
Alp)
ANALYSIS
TO
ASSESS
TOXICITY
OF
AQUATIC
POLLUTANTS
by
Kathryn
G.
Vogel,
Ph.
D.

39
TABLE
OF
CONTENTS
Section
Page
I
INTRODUCTION
41
II
TEST
METHODS
44
1.
Extraction
of
ATP
44
2.
Determination
of
ATP
44
3.
Determination
of
Chlorophyll
a
46
4.
Determination
of
Dry
Weight
47
III
EXPERIMENTAL
RESULTS
AND
ANALYSES
48
1.
Algae
Treated
with
Sodium
Azide
48
2.
Daphnia
Treated
with
FC­
206
48
3.
Fathead
Minnows
Treated
with
FC­
206
50
4.
Pollution­
Monitoring
Methodology
50
5.
Sidestream
Pollution
54
IV
CONCLUSIONS
60
REFERENCES
61
LIST
OF
ILLUSTRATIONS
Figure
Page
A­
l
Growth
and
ATP
Content
of
Unicellular
Green
Algae
(
Chiorella
vuigaris)
in
Pure
Culture
42
A
 
2
Standard
Curve
for
JRB
ATP
Photometer
with
duPont
Luminescence
Reagents
45
A­
3
Azide
Poisoning
of
Chiorella
vulgaris
49
LIST
OF
TABLES
Table
Page
A­
i
AlP
Content
for
Various
Organisms
41
A­
2
Twenty­
Four­
Hour
Effect
of
FC­
206
on
ATP
Content
of
Daphnia
magna
49
A­
3
Seventy­
Two­
Hour
Effect
of
FC­
206
on
ATP
Content
of
Whole
Fathead
Minnows
51
A­
4
Assay
of
Periphyton
Community
from
One
Outdoor
Tank
53
A­
5
Assay
of
Sidestream
Transects
Before
and
After
Pollution
with
FC
 
206
55
A
 
6
Assay
of
Periphyton
Community
Attached
to
Plexiglass
Slides
in
Sidestreams
During
Administration
of
and
Recovery
from
Pollution
with
FC­
2O6
57
40
SECTION
I
INTRODUCTION
1.
BACKGROUND
The
basic
assumption
for
the
development
of
an
adeosine
triphosphate
(
ATP)

analysis
methodology
is
that
every
living
organism
contains
an
approximately
constant
amount
of
ATP
per
unit
of
dry
weight.
A
measure
of
ATP
can
therefore
be
translated
into
units
of
biorr.
ass
(
Reference
1).
An
equally
important
assumption
is
that
only
living
organisms
contain
ATP.
These
assumptions
have
been
tested
and
verified
by
several
workers
(
References
2,
3).
In
our
laboratory
ATP
and
dry
weight
for
several
different
organisms
were
determined
(
Table
A­
i);
these
numbers
were
always
in
the
range
of
0.1
to'l.
O
pg
ATP/
mg
dry
weight.

It
was
also
found
that
increasing
ATP
concentration
is
directly
proportional
to
increasing
cell
number
in
a
pure
algal
culture
(
Figure
A­
i).
Several
experiments
such
as
killing
adult
Daphni~
by
freezing
and
then
thawing
them
for
various
periods
of
time
before
determining
the
AlP
content
showed
that
the
ATP
content
of
an
organism
drops
rapidly
to
a
negligible
amount
after
death.

Table
A­
i.
ATP
CONTENT
FOR
VARIOUS
ORGANISMS
.

Organism
ATP
Cont
pg/
mg
dry
ent,
weight
Filamentous
Green
Algae
(
Ulothrix)
0.1
Snail
Egg
Case
0.1
Daphnia
0.3
­
0.8
Whole
Young
Flagfish
(
Jordanella)
0.6
Whole
Young
Fathead
Minnows
(
Pirneph~
7,
es)
Mixed
Periphyton
Community
0.5
­

0.06
­
0.8
0.2
References
1.
Brezonik,
P.
L.,
Bowne,
F.
X.,
and
Fox,
J.
L.,
"
Application
of
ATP
to
Plankton
Biomass
and
Bioassay
Studies,"
Water
Research,
9,
1975,
pp.
115
 
162.

2.
Hoim­
Hansen,
0.,
"
ATP
Levels
in
Algal
Cells
as
Influenced
by
Environmental
Conditions,"
Plant
Cell
Physiol.,
ii,
1970,
pp.
689
 
700.
3.
Weber,
Cornelius
I.,
"
Recent
Developments
in
the
measurement
of
the
Response
of
Plankton
and
Periphyton
to
Changes
in
Their
Environment,"
Bioassay
Techniques
and
Environmental
C~.
e.
mistry,
Ann
Arbor
Science
Publishers,
1973,
pp.
119
 
138.

41
I­

`
 
I
L)

`
4­
0
S.­
C)
.
c~
E
7
 
6­

5
 
4.

3
 
2
 
Gwth
 
 
ATP
Content
0'~~
C)
 
0.16
 
0.14
 
0.12
o
 
0.10
I
I
 
I
 
/
I
 
0.08
 
0.06
 
0
04
 
0.02
 
0.00
32;
,
/
/

0'
E
I
 
Ui
41
lj
8
12
16
20
­
24
1
28
Time,
day&

Note:
Chiorella
vuigaris
was
grown
at
18
°
Cin
100
m­
e
of
phosphatebuffered
Beijerinck's
medium
(
pH
6.5)
plus
trace
elements
in
cotton­
stoppered
300­
m.
e
Erhlenmeyer
flasks
with
shaking
at
75
oscillations
per
minute.
Cell
number
was
determined
with
a
haemocytometer.
ATP
was
extracted
by
centrifuging
40
m.
C
of
culture
and
immersing
the
cell
pellet
in
5
mL
of
boiling
0.0211
Tris,
pH
7.4.
The
ATP
content
of
the
buffer
was
then
determined.

Figure
A­
l.
­
Growth
and
ATP
Content
of
Unicellular
Green
Algae
(
ChZ.
orella
vulgaris)
in
Pure
Culture
42
Both
dry
weight
and
total
organic
carbon
determinations
combine
measurement
of
living
and
dead
material.
Because
ATP
determinations
measure
only
living
tissue,
the
ATP
method
is
useful
in
distinguishing
living
from
dead
­

organisms.
It
would,
of
course,
be
a
foolish
exercise
to
use
the
method
on
macroscopicaily
visible
organisms,
for
there
are
much
easier
and
cheaper
ways
of
telling
whether
a
fish
is
dead
or
alive.
When
one
is
dealing
with
microorganisms
algae,
or
mixed
communities
of
organisms
that
cannot
be
separated
for
individual
inspection,
however,
a
test
that
can
generate
a
number
that
~
n
 
dicates
living
bioniass
is
no
longer
trivial.
ATP
measurements
can
thus
be
used
both
to
quantify
the
living
organisms
of
a
mixed
periphyton
community
and
to
assess
changes
in
this
community
caused
by
pollution.

2.
OBJECTIVE
The
AlP
project
was
undertaken
for
two
reasons:
(
1)
to
determine
whether
measurement
of
the
AlP
content
of
periphyton
samples
would
give
useful
information
for
assessing
the
toxicity
of
certain
Air
Force
pollutants,
and
(
2)
to
develop
a
methodology
that
would
enable
relatively
untrained
personnel
to
monitor
the
pollution
of
aquatic
ecosystems.

43
SECTION
II
TEST
METHODS
1.
EXTRACTION
OF
ATP
The
living
material
from
which
ATP
was
to
be
extracted
was
immersed
quickly
and
totally
into
boiling
002M
Tris
buffer,
pH
7.75
for
5
minutes.
The
supernatant
was
then
analyzed
for
ATP
content
or
frozen
for
analysis
up
to
3
months
later.
Periphyton
samples
were
concentrated
by
centrifugation.

In
practice
it
is
convenient
to
keep
the
buffer
solution
boiling
in
an
electric
frypan
filled
with
hot
sand.­
The
amount
of
material
added
should
not
be
enough
to
cool
the
buffer.
If
a
large
sample
is
being
extracted,
the
amount
of
buffer
should
be
increased.
Some
water
will
evaporate
from
the
buffer,
but
this
is
not
critical
as
long
as
volume
corrections
are
made
at
the
time
of
the
AlP
assay.
If
the
Iris
is
not
hotter
than
90
°
C,
or
if
the
material
is
not
brought
immediately
to
boiling,
the
amount
of
ATP
extracted
will
be
significantly
reduced.
The
extraction
procedure,
therefore,
is
very
important
to
the
assurance
of
reproducibility
and
accuracy.
­

2.
DETERMINATION
OF
ATP
A
JRB,
Inc.,
ATP
photometer
and
duPont
reagents
were
used.
One
buffer
tablet
and
one
vial
of
enzyme
were
dissolved
in
3
m~
of
distilled
water.
2
0.1
m.
e
of
the
hydrated
enzyme
was
dispensed
into
small
reaction
cuvettes
provided
by
duPont.
The
solution
was
allowed
to
stand
for
30
minutes
before
it
was
assayed.

During
this
period,
the
ATP
photometer
was
turned
on
and
warmed
up.
(
For
this
study
the
instrument
was
usually
operated
at
an
HV
Adjust
of
6.0
V
and
a
Zero
of
4.3
V.)
The
samples
were
then
thawed
and
brought
up
to
a
known
volume.
A
glass
scintillation
vial
with
a
hole
bored
in
its
screwcap
was
used
as
a
cuvette
holder.
The
sample
was
injected
directly
into
the
enzyme
mixture
with
a
l0­
p~
Hamilton
syringe.
The
injection
step
was
critical;
variations
in
the
Footnote
 
2The
3­
m­~
volume
is
recommended
by
duPont.
If
more
water
is
used
the
sensitivity
of
the
assay
is
less,
but
more
assays
are
possible.

44
injection
technique
will
produce
great
variations
in
the
results.
In
this
case,
reproducibility
was
achieved
by
placing
the
needle
tip
against
the
bottorn
of
the
reaction
cuvette
and
dispensing
the
entire
sample
with
one
smooth
and
fairly
rapid
motion
so
that
air
bubbles
­
would
not
be
introduced
with
the
sample.
As
the
injection
was
made,
the
foot
lever
that
activates
a
delayed
count
cycle
of
the
photometer
was
simultaneously
pressed.
During
the
next
15
seconds,
the
pipette
was
set
down,
the
vial
was
smoothly
placed
in
the
counting
chamber,
the
light­
tight
lid
was
placed
over
the
chamber
and
pushed
and
rotated
slightly
until
it
clicked,
and
the
lever
that
exposes
the
photocell
to
the
reaction
was
pulled.
The
instrument
then
measured
emitted
light
for
60
seconds
and
displayed
the
counts
on
a
digital
panel.
At
the
end
of
this
counting
period
the
yellow
"
Reset
SC"
light
came
on.
The
final
display
was
recorded.
The
photocell
lever
was
then
pushed
in
and
the
display
panel
was
reset
to
zero.
The
cuvette
holder
was
then
removed
from
the
holder
and
a
new
cuvette
with
enzyme
was
placed
in
the
holder.

The
ATP
standards
that
were
routinely
used
were
0.002,
0.005,
0.01,
and
0.015
pg
AlP
in
10
p.~
of
distilled
water
(
Figure
A­
2).
If
the
cpm
of
a
sample
2i
2
cH
0ci
 
~
~
t
0.0OO~
0.004
0.008
0.012:
0.016
AlP,
pg
Note:
ATP
standard
(
Sigma
Chemical
Co.)
was
added
in
10
 
p­
C
volume
to
0.1
rme
of
duPont
luminescence
enzyme
reagent.
After
a
15­
second
delay,
emitted
light
was
read
by
the
photometer
for
60
seconds
and
displayed
as
counts
per
minute.
Instrument
settings:
HV
=
6.0
V,
Zero
=
4.3
V.

Figure
A­
2.
Standard
Curve
for
JRB
ATP
Photometer
with
duPont
Luminescence
Reagents
o~~
o
I
I
45
exceeds
300,000,
the
"
Reset
HV"
light
will
come
on
and
all
counting
will
stop.

That
sample
must
then
be
diluted
before
the
assay
can
be
made.

After
completing
the
test,
the
instrument
was
turned
off
and
the
photochamber
was
sealed
to
prevent
damage
to
the
photocell.
Cuvettes
may
be
rinsed
by
hand
or
discarded,
depending
on
the
economics
of
the
situation.
All
glassware
should
be
thoroughly
cleaned
because
ATPase
is
a
ubiquitous
enzyme.

To
determine
the
amount
of
AlP
in
a
sample,
the
cpm
was
located
on
the
standard
graph
produced
during
the
same
run,
and
the
amount
of
AlP
in
the
10­
p­
C.

sample
was
read
from
this.
The
amount
of
ATP
in
the
total
sample
was
calculated
as
follows:
(
AlP/
lU
p~
f.)
x
(
p.
C/
sarnpie)
=
(
ATP/
sample).
For
example,
if
there
is
0.01
pg
of
AlP
in
10
p­
C
of
a.
5­
m~
Csample,
there
is
0.01/
10
x
5000
=

5
pg
ATP
in
the
entire
sample.

A
useful
discussion
and
description
of
the
theoretical
and
practical
aspects
of
this
methodology
may
be
found
in
Reference
4.

3.
DETERMINATION
OF
CHLOROPHYLL
a
Algal
or
periphyton
samples
were
concentrated
by
centrifugation
and
then
added
to
90
percent
V/
V
acetone/
water.
In
routine
assays,
the
periphyton
pellets
were
added
to
5
tnt
of
acetone
in
a
standard
test
tube.
This
acetone/
algal
solution
was
covered
with
parafilm
and
placed
in
the
refrigerator
for
at
least
24
hours.
Extraction
was
assured
if
the
solid
material
at
the
bottom
of
the
tube
was
white
and
the
acetone
was
green.
The
chlorophyll
of
some
algae
cannot
be
extracted
by
this
gentle
procedure
(
Scenedesmus
was
unacceptable
for
laboratory
testing
for
this
reason),
but
most
can
be,
and
all
the
periphyton
samples
were
treated
in
this
manner.

The
acetone
 
chlorophyll
solution
was
clarified
by
low
 
speed
centrifugation
if
necessary.
The
optical
density
of
the
clear
acetone
solution
was
then
read
Reference
4.
JRB
ATP
 
Photometer
Instruction
Manual,
JRB
Associates,
Inc.,
La
Jolla,
California,
August
1972.

46
at
665
mp
before
and
after
the
solution
was
acidified
with
one
drop
of
concentrated
HC1.
The
amount
of
chlorophyll
a
was
determined
by
the
following
formula
(
Reference
5):

where
Chlorophyll
a(
mg/
m2)
26.73(
665b
­
665a)
x
V
A
665b
=
0D665
before
acidification
665a
=
OD665
after
acidification
V
=
volume
extracted
(
liters)

A
=
area
of
periphyton
substrate
harvested
(
meters2)

For
pure
chlorophyll
a,
the
OD
ratio
665b"
665a
=
1.7.
Pheophytin,
the
degraded
chlorophyll
present
in
dead
material,
shows
no
change
in
optical
density
after
acidification
(
OD
b/
a
ratio
=
1).

Small
porcelain
crucibles
were
cleaned
and
dried
in
the
oven.
Their
tare
`
weight
was
determined
to
the
4th
decimal
place
on
a
Mettler
balance.
The
sample
was
poured
into
the
crucible
and
then
dried
at
85
°
Ffor
48
hours.
The
dry
sample
and
the
crucible
were
weighed
together,
and
the
`
dry
weight
of
the
sample
was
determined
by
subtraction.

Reference
5.
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
13th
Edition,
American
Public
Health
Association,
Washington,
D.
C.,
1973.
in
90
percent
acetone
with
1
 
cm
light
path
4.
DETERMINATION
OF
DRY
WEIGHT
47
SECTION
III
EXPERIMENTAL
RESULTS
AND
ANALYSES
1.
ALGAE
TP.
EAIED
WITH
SODIUM
AZIDE
A
pure
culture
of
the
unicellular
green
algae,
Chiorella
vulgaris,
was
treated
with
0.0511
sodium
azide.
This
compound
is
a
potent
inhibitor
of
oxida
 
tive
phosphorylation
and
is
thus
poisonous
to
respiring
cells.
As
Figure
A­
3
shows,
the
amount
of
ATP
in
the
~
ulture
declined
by
94
percent
over
a
48­
hour
period;
the
number
of
intact
cells
declined
by
only
24
percent.
The
experiment
clearly
shows
that
the
effect
of
a
poison
on
an
algal
culturecan
be
sensitively
measured
by
the
assay
of
AlP
content..
These
data
also
suggest
that
more
than
simple
cell
death
was
measured.
Although
viability
of
the
algal
cells
after
azide
treatment
was
not
directly
assayed,
unicellular
organisms
generally
undergo
rapid
cell
°
lysis
after
death.
It
is
probable
that
the
cells
counted
after
treatment
were
not
dead.
If
this
assumption
is
true,
the
measurements
show
a
decrease
in
the
amount
of
AlP
per
cell
after
the
poison
treatment.
It
would
be
useful
to
be
able
to
~
eterminethe
health
and
vitality
of
a
periphyton
community
or
a
single
larger
animal
simply
by
measuring
its
ATP
content.

2.
DAPHNIA
TREATED
WITH
FC
 
206
The
possibility
that
ATP
content
might
change
in
a
sublethal
fashion
as
a
result
of
treatment
with
an
aquatic
pollutant
was
tested.
Adult
Daphnia
were
treated
with
various
concentrations
of
FC­
206
for
24
hours.
The
amount
of
ATP
in
a
sample
of
the
organisms,
along
with
the
dry
weight
of
organisms
in
that
same
sample,
was
subsequently
determined.
A
number
representing
the
pg
ATP/
mg
dry
weight
was
calculated
for
each
sample.
As
Table
A­
2
shows,
the
amount
of
ATP
was
decreased
by
approximately
50
percent
in
the
samples
treated
for
24
hours
with
30,
120,
600,
and
12,000
mg/­
C
FC­
206.
Ninety­
six­
hour
static
tests
with
FC­
206
demonstrated
an
LC
 
5O
for
adult
Daphnia
of
about
4,000
mg/.
C.
The
AlP
data
indicate
that
a
toxicant
concentration
100­
fold
less
than
the
LC
 
50
caused
a
24­
hour
effect
equal
to
that
of
the
LC­
50.
It
is
tempting
to
conclude
that
this
very
sensitive
method
has
detected
a
subtle
disruption
of
the
48
0.1~~
24
48~
Time
in
NaN
,
hr
­
3
Note:
Sodium
azide
(
0.0511)
was
added
to
a
culture
of
Chiorella.
Aliquots
of
the
culture
were
pulled
at
3,
24,
and
48
hours
for
determination
of
the
number
of
cells
and
AlP
content.

Figure
A.­
3.
Azide
Poisoning
`
of
Chloreila
vulgaris
Table
A­
2.
TWENTY­
FOUR
 
HOUR
EFFECT
OF
FC
 
206
ON
ATP
CONTENT
OF
DAPHiVI.
4
MAGNA
Concentration
of
FC­
206,
mg/.
C
,
ATP
pg/
mg
Cont
dry
ent,
weight
­­­
0.86
0.75
30
0.38
0.42
120
0.74
0.50
600
0.41
0.51
12,000
0.38
0.24
Note:
There
were
no
deaths
during
the
course
of
this
experiment
Daphnia
were
removed
from
the
FC­
206,
divided
into
two
roughly
equal
groups
(
about
100
organisms
in
each),
and
immersed
in
boiling
0.0211
Tris
buffer
for
5
minutes.
The
dead
organisms
were
dried
for
measurement
of
dry
weight
while
the
AlP
content
of
the
buffer
was
determined.
O.
3~
 
 
S.­

L)

L4~
0
c'J
C­,
0.2
­
1.6
1
.4
1.2
 
~
__

0.8
0.
1
 
to
x
E
(
I,

Ia
L)

ft
~­

 
u.
u.
0
5­
~
~`
ci)
o.'­
t
­~

E
ATP
Content
 
 
Growth
0
12
0.2
36
49
organism's
metabolism.
Until
a
great
deal
of
additional
work
has
been
done
on
the
range
of
control
variability
and
the
effects
of
environmental
changes
not
due
to
the
toxicant,
however,
such
a
conclusion
must
be
considered
premature

3.
FATHEAD
MINNOWS
TREATED
WITH
FC­
206
A
similar
experiment
was
performed
with
whole
fathead
minnows.
Young
fish
were
kept
for
72
hours
in
10­
gal
tanks
containing
FC­
206
in
concentrations
of
1000
and
1500
mg/­
C
(
96­
hr
LC
 
50
=
2500
mg/­
C);
a
control
tank
was
also
maintained.

At
the
end
of
the
3­
day
period,
it
was
clear
that
the
fish
kept
at
concentrations
of
1000
mg/'
C
had
been
affected
by
the
toxicant;
several
in
this
group
had
died,
and
others
were
gasping
and
showing
unusual
swimming
behavior.

Individual
living
fish
from
each
group
were
immersed
in
10­
mt
of
boiling
buffer
to
extract
ATP
and
then
weighed.
The
pg
AlP/
mg
dry
weight
for
fish
in
each
group
was
calculated.
The
average
value
of
this
ratio
for
fish
in
both
treatment
groups
was
0.54
(
Table
A­
3),
32
percent
less
than
the
average
control
value
of
0.80.
Again,
it
is
tempting
to
speculate
that
the
AlP
methodology
had
detected
a
subtle
effect.
However,
the
very
slight
decrease
measured
and
the
many
overlapping
individual
values
in
each
group
indicate
that
the
measured
change
is
not
significant.

Initially
it
was
hoped
that
measurements
of
ATP
content
would
be
useful
in
assessing
the
health
of
an
organism
being
affected
by
a
toxicant.
The
work
has
not
verified
this
hope.
In
fact,
ATP
is
not
stored
by
animal
tissues.
In
a
healthy
cell,
the
amount
of
AlP
produced
is
determined
by
the
amount
of
adeosine
diphosphate
(
ADP)
generated.
When
more
ADP
is
present,
more
of
it
is
rephosphorylated
to
restore
the
ATP
used.
This
very
fine
regulation
occurs
in
seconds
in
the
mitochondria;
thus,
it
is
not
surprising
that
the
overall
ATP
content
is
so
constant.
Severe
perturbations
from
normal
patterns
of
energy
use
need
not
necessarily
be
reflected
in
a
detectable
change
in
ATP
content.

4.
POLLUTION­
MONITORING
METHODOLOGY
The
main
goal
in
the
development
of
this
technique
was
that
of
defining
a
50
­
Table
A
 
3.
SEVENTY
 
Tt­!
O­~
OUREFFECT
OF
FC
 
206
ON
ATP
CONTENT
OF
WHOLE
FATHEAD
MINNOWS
Concentration
of
FC­
206,
mg/­
C
ATP
Content,
pg/
mg
dry
weight
Mean
±
S.
D.

 
 
1,000
1,500
0.59,
1.35,
1.16,
0.68
0.50,
0.74,
0.64,
0.69
0.54,
0.59,
0.59,
0.51,
0.46
0.55,
0.67,
0.46,
0.46
0.55,
0.63,
0.60,
0.51
0.41,
0.65
0.79
±
0.30
0.54
±
0.07
0.56
±
0.09
Note:
At
1,500
mg/­
C
fish
were
obviously
affected
and
some
died.
Only
living
fish
were
used
for
the
AT?
assay.
ATP
was
extracted
by
immersing
each
live
fish
in
boiuinn
0.0211
Iris
(
pH
7.8)
for
10
minutes.
The
fish
were
then
removed
from
the
buffer,
dried
at
70
°
Cfor
6
days
and
weighed.
ATP
content
of
the
buffer
was
assayed.
The
approximate
dry
weight
of
the
fish
was
100
mg.

methodology
for
monitoring
the
effects
of
toxicants
on
a
natural
fresh­
water
community.
It
was
hoped
that
a
capability
could
be
developed
for
assessing
damage
to
the
populations
in
an
artificial
stream
stressed
with
toxicants.

This
same
methodology
might
be
useful
in
the
systematic
monitoring
of
natural
waterways.

A
surface
periphyton
community
was
chosen
to
test
this
methodology.

Specifically
that
means
those
protozoans,
algae,
and
small
invertebrates
which
attach
themselves
to,
or
are
closely
associated
with,
growth
on
a
glass
or
plexiglass
slide
suspended­
just
below
the
surface
of
the
body
of
water
being
tested.

Samplers
used
consisted
of
a
plastic
support
that
holds
eight
standard
microscope
slides
approximately
1
inch
apart.
This
unit
was
attached
to
a
wire
frame
having
two
foam
floatation
pieces
on
a
stabilizing
loop;
this
allowed
the
slides
to
remain
suspended
just
below
the
surface
of
the
water.
The
whole
unit
can
move
around
with
currents,
however,
so
the
orientation
to
sunlight
is
variable
on
a
day
 
to­
day
basis.
The
slides
were
harvested
at
specific
time
intervals
or
when
the
amount
of
growth
on
each
slide
seemed
sufficient
for
an
assay.

Slides
were
removed
from
the
sample
holders
outdoors
and
dropped
into
individual
petri
dishes
containing
enough
water
to
cover
them.
They
were
then
brought
into
the
laboratory,
where
the
growth
on
both
sides
was
immediately
scraped
off
51
by
hand.
The
edge
of
a
clean
slide
was
used
as
the
scraping
instrument.
It
was
assumed
that
these
samples
were
equivalent
unless
visual
inspection
clearly
showed
this
to
be
untrue
(
e.
g.,
a
large
snail
egg
case
on
one
sample).

Material
from
one
slide
was
used
for
determination
of
chlorophyll
a,
dry
weight,

or
ATP.
The
periphyton
material
was
concentrated
by
a
short
spin
in
a
table
 
top
centrifuge.
The
supernatant
was
discarded,
and
the
loose
pellet
could
then
be
dropped
into
5
nit
of
90­
percent
acetone
for
chlorophyll
extracting,
5
rn.~.
of
boiling
0.0211
Tris
for
ATP
extraction,
or
a
tared
crucible
for
dry
weight
determination
A
small
aliquot
of
the
material
was
also
examined
microscopically
to
obtain
a
qualitative
picture
of
the
population
being
measured.

Measurements
of
this
kind
were
performed
in
a
largc
outdoor
tank
over
a
period
of
several
months.
All
of
the
data
collected
on
this
tank
is
shown
in
Table
A­
4.
The
extremely
preliminary
nature
of
this
data
must
be
stressed,
for
it
was
gathered
while
the
methodology
was
being
developed.
The
purpose
was
to
determine
whether°
this
method
could
be
used
to
docurnentthe
presence,
health,

and
composition
of
a
periphyton
community.
Several
things
were
learned.

The
first
series
of
measurements
was
performed
on
slides
harvested
from
the
tank
on
successive
days.
The
data
show
that
the
amount
of
material,
ATP,

and
chlorophyll
a
accumulated
on
the
slides
had
probably
reached
its
maximum
before
day
seven
and
did
not
change
substantially
during
the
next
seven
days.

In
addition,
the
measurements
have
a
respectable
degree
of
reproducibility
(
pg
ATP/
mg
dry
weight
was
between
0.1
and
0.3).

A
number
was
developed
that
is
the
ratio
of
pg
ATP/
mg
chlorophyll
a
measured
per
square
meter
of
slide
surface
harvested.
This
ratio
is
similar
to,
but
represents
a
major
improvement
upon,
the
Autotrophic
Index
suggested
by
Weber
(
Reference
3).
The
Autotrophic
Index
is
the
ratio
of
dry
weight
of
organic
matter
to
chlorophyll
a.
Because
it
is
a
measure
of
the
amount
of
material
that
contains
chlorophyll,
it
can
assess
only
the
proportion
of
the
sample
that
is
autotrophic.
The
number
we
have
developed
uses
ATP
as
the
numerator,
thus
avoiding
the
problems
inherent
in
dry
weight
measurements.
By
comparing
ATP
content
to
chlorophyll
a
content,
we
are
comparing
two
parameters
which
are
present
only
in
living
material;
accumulation
of
dead
organisms
and
inorganic
material
will
not
bias
the
result.
Since
AT?
is
contained
by
autotrophic
and
heterotrophic
52­
Table
A­
4.
ASSAY
OF
PERIPHYTON
COMMUNITY
FROM
ONE
OUTDOOR
TANK
Harvest
Growth,
Date
days
Dry
weight,'
mg
AT?,
pg
ATP/
rng,
pg
ATP/
rn2,
pg
Chlorophyll/
rn2,
mg
ATP/
rng
chlorop
pg
of
­

hyll,

8/
18
7
18.8
2.00
0.110
533
7.30
73
8/
19
8
16.2
4.10
0.253
1100
8.50
130
8/
20
9
15.5
3.90
0.250
1033
6.50
159
8/
21
10
19.1
4.80
0.251
1280
12.50
102
8/
22
11
11.7
2.60
0.222
702
7.70
91
8/
25
14
20.2
3.40
0.170
907
6.40
142
10/
8
7
12.6
5.20
0.410
1400
10.50
133
10/
8
30
5.5
8.80
0.160
2400
19.40
124
10/
14
7
2.5
7.90
0.320
2121
14.20
149
10/
14
36
76.0
5.10
0.067
1300
11.70
111
10/
31
17
34.0
2.00
0.059
535
15.50
34
11/
7
24
50.0
4.40
0.088
1197
24.00
50
11/
14
14
22.0
3.40
0.160
913
18.00
51
11/
18
11
14.2
2.00
0.143
548
14.00
39
11/
25
7
3.0
1.60
0.530
214
5.10
`
42
1/
13
36
7.6
0.28
0.037
37
0.89
42
1/
14
34
1.8
0.22
0.120
30
0.29
100
1/
21
57
3.1
0.28
0.090
38
1.26
30
2/
15
34
2.7
0.15
0.056
20
0.38
50
4/
23
50
38.0
3.60
0.095
973
24.00
40
4/
30
27
47.0
2.30
0.049
624
9.30
67
53
organisms
in
approximately
similar
amounts
per
unit
of
dry
weight,
while
chlorophyll
a
is
found
only
in
autotrophic
organisms,
the
ratio
will
be
an
expression
of
the
proportion
of
autotrophic
living
material
in
the
sample.
This
ratio
has
been
tested
with
data
from
different
periphyton
communities
and
appears
to
be
potentially
useful
as
a
predictor
of
the
composition
of
the
community
For
example,
measurements
presented
in
Table
A­
4
were
from
a
tank
that
contained
fish
that
were
being
fed
daily.
The
high
nutrient
condition
of
the
water
provided
food
for
a
variety
of
heterotropic
organisms,
which
were
obvious
whenever
samples
were
examined
microscopically.
The
brown,
scurnmy
growth
appearing
on
the
slides
consisted
primarily
of
diatoms;
however,
associated
with
them
were
numerous
amoebae,
ciliates,
nematodes,
and
rotifers.
The
ATP/
chlorophyll
ratio
for
these
communities
ranged
from
100
to
150.
In
November,
when
the
cold
weather
became
severe,
this
ratio
dropped
to
about
50.
Observation
of
the
periphyton
during
this
period
showed
that
along
with
the
very
much
reduced
rate
of
algal
accumulation,
a
change
in
the
composition
of
the
population
had
also
occurred.
The
small
invertebrates
were
no
longer
present;
the
protozoan
popu
 
la.
tion
was
greatly
reduced
and
was
now
composed
primarily
of
flagellates.
The
lowered
ATP/
chlorophyll
ratio
indicated
that
an
increased
percent
of
the
popu
 
lation
was
now
autotrophic.
In
midwinter,
the
amount
of
growth,
as
measured
by
accumulation
of
mass
and
AlP
on
the
slides,
was
extremely
small;
but
the
population
that
did
accumulate
had
an
ATP/
chlorophyll
ratio
and
an
observed
composition
similar
to
those
of
the
late
fall.
In
April,
the
return
of
warm
weather
brought
a
bloom
of
algal
growth,
which
was
measured
as
an
increase
in
periphyton
ATP
and,
particularly,
chlorophyll.
Other
tanks
which
were
not
receiving
fish
food
had
highly
autotrophic
populations.
The
AlP/
chlorophyll
ratio
of
samples
from
these
tanks
ranged
from
25
to
50,
even
during
the
summer
months.

Samples
of
pure
green
algae
(
both
mats
of
filarnentous
Iilothrix
and
the
unicel
 
lular
ChZ.
orella)
had
a
ratio
of
12
­
to
20.

5.
SIDESTREAM
POLLUTION
The
AT?
analysis
technique
was
developed
so
that
a
methodology
for
assessing
the
toxicity
of
certain
compounds
on
an
aquatic
ecosystem
could
be
obtained.

The
feasibility
of
the
methodology
was
given
a
preliminary
trial
during
the
pollution
of
one
sidestream
system
i~
an
artificial
stream.
One
arm
of
a
54
sidestream
was
polluted
with
FC­
206.
Four
days
prior
to
the
initial
addition
of
FC­
2O6,
the
entire
contents
of
an
8­
inch
cross
section
of
the
sidestream
at
approximately
station
3
(
see
Figure
3
in
body
of
report)
was
siphoned
into
a
bucket.
The
sides
and
rocks
were
rubbed
free
of
attached
material.

Approximately
8
£
.
of
material
was
then
poured
through
two
layers
of
nylon
screening
to
separate
coarse
from
fine
material.
Dry
weight,
chlorophyll
a,
and
AT?
content
of
the
coarse
material
were
analyzed.
The
AT?
content
of
the
fine
material
was
also
measured.
These
data
(
top
line
of
Table
A­
5)
show
that
twice
as
much
AlP
~°
ias
present
in
the
coarse
material
as
in
the
fine;
that
the
pg
AlP/
mg
dry
weight
was
in
the
anticipated
range;
and
that
the
Alp/
chlorophyll
ratio
was
very
high,
indicating
the
presence
of
many
nonphotosynthetic
organisms.
After
FC­
206
had
been
administered
for
8
days,
similar
transects
were
siphoned
off
from
the
middle
of
the
polluted
and
the
unpolluted
arms
of
the
sidestream.

Similar
analyses
of
these
samples
showed
that
the
mass
of
material
on
the
polluted
side
had
not
been
decreased
significantly,
but
that
the
AlP
content
had
been
reduced
by
87
percent,
indicating
that
most
of
the
previously
living
material
had
been
killed.
The
chlorophyll
content
also
dropped
76
percent.
The
Table
A­
5.
ASSAY
OF
SIDESTREAM
TRANSECTS
BEFORE
AND
AFTER
POLLUTION
WITH
FC
 
206
Dry
Chioro
h
11
ATP,
pg
ATP/
mg
of
ATP/
mg
of
weight,
~
`
dry
weight,
chlorophyll,

______________
g
pg
Coarse
Filtrated
pg
pg
Before
addi­
1.410
667
78.0
47
0.090
188
tion
of
FC
 
206
Polluted
arm
1.388
160
2.5
14
0.011
106
after
addition
of
FC­
206
for
8
days
Unpolluted
arm
2.860
2346
7.0
22
0.012
12
after
addition
of
FC­
2O6
to
opposite
side
for
8
days
­
_____________
______
__________
____________
_____________

Note:
The
entire
contents
of
the
sidestream
for
an
approximate
length
of
8
inches
were
siphoned
into
a
bucket
and
filtered
through
a
nylon
screen;
aliquots
of
the
coarse
material
were
assayed
for
dry
weight,
chlorophyll
a,
and
AT?
content;
aliquots
of
the
filtrate
were
also
assayed
for
AT?
content.

55
ratio
of
pg
ATP/
mg
dry
weight
was
low,
probably
because
of
the
dead
material
now
represented
in
the
denominator.
The
ratio
of
ATP/
chlorophyll
was
over
100,

indicating
the
presence
of
many
nonautotrophic
organisms.
Observation
of
this
sidestream
indicated
that
virtually
all
the
normal
stream
organisms
had
been
killed
or
had
migrated
to
a
less
polluted
location
in
the
system.
The
general
appearance
was
that
of
greyish
 
brown
scum
devoid
of
vertebrate
or
invertebrate
animals
and
containing
very
little
floating
algal
material.

The
unpolluted
arm
had
not
been
assayed
prior
to
pollution;
thus,
a
strict
before
and
after
comparison
was
not
possible.
Our
visual
impression
was
that
the
two
arms
of
the
sidestream
had
similar
amounts
and
types
of
growth
before
pollution.
After
FC­
206
was
administered,
the
mass
of
material
in
the
unpolluted
arm
was
increased,
and
the
chlorophyll
content
was
greatly
increased
(
nearly
4
times).
The
amount
of
AlP
in
the
unpolluted
side
was
low,
as
was
the
ratio
of
AlP/
chlorophyll.
This
low
ratio
is
typical
for
cultures
of
pure
algae.
The
high
chlorophyll
and
low
AlP/
chlorophyll
ratio
confirm
the
visual
observation
that
a
massive
bloom
of
algae
had
occurred
in
that
part
of
the
sidestream
receiving
very
low
levels
of
the
toxicant.
At
this
point,
it
is
not
possible
to
state
that
the
low
toxicant
level
was
a
cause
of
the
bloom,

but
it
would
be
a
feature
to
watch
carefully
in
subsequent
testing.
FC
 
206
was
added
to
laboratory
cultures
of
Chiorella
vulgar­
is
in
concentrations
ranging
from
200
to
10,000
mg/
L.
It
did
not
kill
this
algal
species,
even
at
the
highest
concentration.
At
the
lower
concentrations,
culture
growth
and
morphology
were
indistinguishable
from
those
of
the
controls.
At
concentrations
above
1500
mg/
Z
the
cultures
soon
became
quite
cloudy
and
the
growth
rate
was
reduced.
Closer
observation
revealed
a
heavy
growth
of
rodshaped
motile
bacteria
The
pH
of
the
medium
dropped
to
5.2.
This
pH
change
alone
could
have
been
responsible
for
the
reduced
growth
rate.

Four
days
before
the
FC­
2O6
was
administered,
plexiglass
slides
(
total
surface
=
125
cm2)
were
suspended
in
the
sidestream
at
stations
1,
3,
5,
10,

12,
and
14
(
see
Figure
3
in
body
of
report).
They
were
removed
after
8
days
of
exposure
to
the
pollutant.
The
periphyton
attached
to
the
slides
was
scraped
off
and
analyzed
for
AlP
and
dry
weight.
These
data
are
presented
in
the
top
half
of
Table
A
 
6.
Since
this
was
the
first
time
that
these
slides
and
this
technique
had
been
used,
it
can
be
concluded
only
that
the
amount
of
AT?
at
stations
56
­
Table
1­
6.

01
ASSAY
OF
PERIPHYTON
COMMUNITY
ATTACHED
TO
PLEXIGLASS
SLIDES
IN
SIDESTREAMS
DURING
ADMINISTRATION
OF
AND
RECOVERY
FROM
POLLUTION
WITH
FC­
206
Stationa
ATP/
slide,
Dry
weight/
slide,

~
­­"­.~`~­­"
ATP/
mg,

pg
Chlorophyll
a/
r~
2,

pg
ATP/
rng
of
chlorophyll
a,

pg
Four
Days
Prior
Plus
Eight
Days
During
Addition
of
FC­
206
(
10/
24
­
11/
5)

Polluted
1
35
Unpolluted
10
12
14
0.075
0.150
0.200
0.200
1.130
17.0
13.0
5.4
`

`
9.1
5.0
45.0
5.3
0.0043
0.0120
0.3000
0.0200
0.1100
­
1.8
Fifteen­
Day
Recovery
(
11/
5
­
11/
20)

Polluted
1
35
Unpolluted
10
12
14
3.800
2.500
4.000
1.000
1.400
2.600
31.0
17.0
27.0
14.0
60.0
`

29.0
0.1200
0.1500
0.1500
0.0700
0.0240
0.0890
`
4.0
1.8
6.5
,
4.4
12.8
10.6
76
112
49
18
9
20
aSee
Figure
3
in
body
of
report
for
location
of
stations.
1
through
10
was
low
and
dry
weights
were
extremely
variable.
Inconsistencies
in
growth
on
different
slides
suspended
at
the
same
station
were
visual
ly
obvious
therefore,
the
variations
measured
are
not
surprising.
That
so
very
little
AT?
was
measured
in
the
17
mg
of
material
taken
from
station
1
indicates
that
most
of
the
killed
organisms
remained
attached,
at
least
at
this
point.

New
slides
were
positioned
at
each
station
after
additions
of
FC
 
205
had
been
halted
and
the
sidestream
had
been
flushed
with
water
from
the
main
stream.

The
slides
were
left
in
place
for
15
days
and
then
harvested
°
for
analysis
of
AlP,
mass,
and
chlorophyll
content.
These
data
show
that
the
amount
of
AT?

in
the
polluted
arm
was
substantial
 
 
greater,
in
fact,
than
that
in
the
unpolluted
arm.
The
dry
weights,
though
variable,
were
all
in
the
same
range.
The
greatest
differences.
occurred
in
measurements
of
chlorophyll
a
where
it
was
clear
that
stations
12
and
14
had
a
very
high
chlorophyll
content.
The
ATP/

chlorophyll
ratio
indicated
that
stations
1
through
5
had
supported
the
attachment
of
a
heterotrophic
community;
stations
10
through
14
had
supported
virtually
pure
algal
communities.

These
data
were
at
first
very
puzzling.
Visual
inspection
suggested
that
everything
in
the
polluted
arm
was
dead;
while
at
the
same
time
the
unpolluted
arm
contained
an
unchanged
or
perhaps
enhanced
periphyton
community.
The
measurements
however,
suggested
that
the
polluted
arm
was
supporting
a
nearly
normal
mixed
periphyton
community.
The
numbers
alone
would
lead
one
to
conclude
that
FC­
206
had
had
no
detrimental
effect
on
that
ecosystem.
Microscopic
examination
of
material
from
both
arms
solved
the
puzzle.
The
scumniy,
brown
material
in
the
polluted
arm
proved
to
be
covered
with
a
grey
layer
of
bacteria
These
bacteria
were
attached
to
everything
and
were
multiplying
rapidly
In
this
scum
of
bacteria
and
dead
algal
material,
there
were
few
living
algae
and
even
fewer
living
protozoans.
Invertebrates
were
absent.
Thus,

the
high
AlP
content
and
high
ATP/
chlorophyll
ratio
had
resulted
from
the
rapid
growth
of
bacteria.
These
bacteria
are
probably
the
same
as
those
found
in
the
laboratory
tests
of
Chiorella
(
species
identification
was
not
done)
and
had
probably
been
introduced
into
the
sidestream
with
the
FC­
206.

During
the
initial
recovery
period,
the
abundant
nutrient
available
from
the
organisms
killed
by
the
FC
 
206
created
conditions
favoring
growth
of
the
bacteria
It
is
unfortunate
that
it
was
not
possible
to
further
analyze
this
58
sidearm
to
document
the
time
and
progression
of
the
organisms
responsibie
for
the
eventual
recovery
from
the
FC­
2O6
pollutant.

Although
these
results
are
preliminary­,
measurement
of
AT?,
dry
weight,

and
chlorophyll
a
clearly
offers
an
attractive
tool
for
assessing
the
effects
of
a
toxicant
on
an
aquatic
ecosystem.
It
should
be
stressed
again,
however,

that
such
measurements
must
be
closely
correlated
with
the
intelligent
observation
of
the
population
being
measured.
The
numbers
alone
would
indicate
that
FC­
206
had
caused
only
temporary
damage
to
the
periphyton
community.

In
fact,
a
kill
followed
by
a
bacterial
takeover
occurred,
and
complete
recovery
probably
took
months.

59
SECTION
IV
CONCLUS
IONS
There
exists
a
sensitive,
reliable
method
of
quantitating
AT?.
This
compound
is
present
only
in
living
organisms
and
can
be
used
to
assess
the
living
biomass
of
a
particular
sample.
The
method
is
potentially
valuable
in
the
field,
where
the
biggest
problems
will
be
reproducible
sampling
and
transportatIon
of
samples
to
the
laboratory
without
loss
of
AT?.

The
ratio
pg
ATP/
mg
dry
weight
seems
to
decrease
in
organisms
subjected
to
FC
 
206
in
sublethal
concentrations.
A
great
deal
more
work
on
the
range
of
variability
in
control
situations
must
be
done
before
this
method
will
be
useful
in
assessing
the
health
of
treated
organisms.

The
ratio
pgATP/
mg
chlorophyll
a
can
provide
information
about
the
composition
of
the
population
assayed.
It
is
superior
to
the
Autotrophic
Index
because
only
living
material
is
included
in
the
numerator.
The
ratio
will
be
100
to
150
for
mixed
periphyton
communities
and
about
20
for
populations
of
pure
algae.
This
number,
taken
alone,
is
not
meaningful
as
a
measure
of
pollution.

Measurement
of
AT?
content,
chlorophyll
a,
and
dry
weight
of
periphyton
samples,
when
combined
with
intelligent
observation,
can
give
useful
quantitative
information
when
the
effects
of
a
pollutant
on
an
aquatic
ecosystem
are
to
be
assessed.

60
REF
ER
EflC
E
S
1.
Brezonik,
P.
L.,
Bowne,
F.
X.,
and
Fox,
J.
L.,
"
Application
of
AT?
to
Plankton
Biomass
and
Bioassay
Studies,"
Water
Research,
9,
1975,
pp.
115
 
162.
­

2.
Holm
 
Hansen,
0.,
"
AT?
Levels
in
Algal
Cells
as
Influenced
by
Environmental
Conditions,"
Plant
Cell
Physiol.
,
11,
1970,
pp.
689­
700.

3.
Weber,
Cornelius
I.,
"
Recent
Developments
in
the
Measurement
of
the
Response
of
Plankton
and
Periphyton
to
Changes
in
Their
Environment,"
Bioassay
Techniques
and
Env­
iron'nental
Chemzstry,
Ann
Arbor
Science
Publishers
1973,
pp.
119­
138.

4.
JRB
ATP­
Photorneter
Instrumentation
Manual,
JRB
Associates,
Inc.,
La
Jolla,
California,
August
1972.

5.
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
13th
Edition
American
Public
Health
Association,
Washington,
D.
C.,
1­
973.

61
(
The
­
reverse
of
this
page
is
blank.)
