(
September
1996)
1
ATTACHMENT
A
MARINE
ACUTE
TOXICITY
TEST
PROCEDURE
AND
PROTOCOL
East
Boston
Terminal
Company
Division
of
Tosco
Corporation
I.
GENERAL
REQUIREMENTS
The
permittee
shall
conduct
acceptable
acute
toxicity
tests
in
accordance
with
the
appropriate
test
protocols
described
below:

!
Mysid
Shrimp
(
Mysidopsis
bahia)
definitive
48
hour
test.

Acute
toxicity
data
shall
be
reported
as
outlined
in
Section
VIII.

II.
METHODS
Methods
to
follow
are
those
recommended
by
EPA
in:

Weber,
C.
I.
et
al.
Methods
for
Measuring
the
Acute
Toxicity
of
Effluents
to
Freshwater
and
Marine
Organisms,
Fourth
Edition.
Environmental
Monitoring
Systems
Laboratory,
U.
S.
Environmental
Protection
Agency,
Cincinnati,
OH.
August
1993,
EPA/
600/
4­
90/
027F.

Any
exceptions
are
stated
herein.

III.
SAMPLE
COLLECTION
A
discharge
sample
shall
be
collected.
Aliquots
shall
be
split
from
the
sample,
containerized
and
preserved
(
as
per
40
CFR
Part
136)
for
the
chemical
and
physical
analyses.
The
remaining
sample
shall
be
dechlorinated
(
if
detected)
in
the
laboratory
using
sodium
thiosulfate
for
subsequent
toxicity
testing.
(
Note
that
EPA
approved
test
methods
require
that
samples
collected
for
metals
analyses
be
preserved
immediately
after
collection.)
Grab
samples
must
be
used
for
pH,
temperature,
and
total
residual
oxidants
(
as
per
40
CFR
Part
122.21).

Standard
Methods
for
the
Examination
of
Water
and
Wastewater
describes
dechlorination
of
samples
(
APHA,
1992).
Dechlorination
can
be
achieved
using
a
ratio
of
6.7
mg/
L
anhydrous
sodium
thiosulfate
to
reduce
1.0
mg/
L
chlorine.
A
thiosulfate
control
(
maximum
amount
of
thiosulfate
in
lab
control
or
receiving
water)
should
also
be
run.

All
samples
held
overnight
shall
be
refrigerated
at
4oC.
(
September
1996)
2
IV.
DILUTION
WATER
A
grab
sample
of
dilution
water
used
for
acute
toxicity
testing
shall
be
collected
at
a
point
away
from
the
discharge
which
is
free
from
toxicity
or
other
sources
of
contamination.
Avoid
collecting
near
areas
of
obvious
road
or
agricultural
runoff,
storm
sewers
or
other
point
source
discharges.
An
additional
control
(
0%
effluent)
of
a
standard
laboratory
water
of
known
quality
shall
also
be
tested.

If
the
receiving
water
diluent
is
found
to
be,
or
suspected
to
be
toxic
or
unreliable,
an
alternate
standard
dilution
water
of
known
quality
with
a
conductivity,
salinity,
total
suspended
solids,
and
pH
similar
to
that
of
the
receiving
water
may
be
substituted
AFTER
RECEIVING
WRITTEN
APPROVAL
FROM
THE
PERMIT
ISSUING
AGENCY(
S).
Written
requests
for
use
of
an
alternative
dilution
water
should
be
mailed
with
supporting
documentation
to
the
following
address:

Director
Office
of
Ecosystem
Protection
U.
S.
Environmental
Protection
Agency­
New
England
1
Congress
Street
Suite
1100
(
CMA)
Boston,
MA
02203
It
may
prove
beneficial
to
have
the
proposed
dilution
water
source
screened
for
suitability
prior
to
toxicity
testing.
EPA
strongly
urges
that
screening
be
done
prior
to
set
up
of
a
full
definitive
toxicity
test
any
time
there
is
question
about
the
dilution
water's
ability
to
support
acceptable
performance
as
outlined
in
the
'
test
acceptability'
section
of
the
protocol.

V.
TEST
CONDITIONS
AND
TEST
ACCEPTABILITY
CRITERIA
EPA
New
England
requires
tests
be
performed
using
four
replicates
of
each
control
and
effluent
concentration
because
the
nonparametric
statistical
tests
cannot
be
used
with
data
from
fewer
replicates.
The
following
tables
summarize
the
accepted
Mysid
and
Menidia
toxicity
test
conditions
and
test
acceptability
criteria:
(
September
1996)
3
EPA
NEW
ENGLAND
RECOMMENDED
EFFLUENT
TOXICITY
TEST
CONDITIONS
FOR
THE
MYSID,
MYSIDOPSIS
BAHIA
48
HOUR
TEST1
_________________________________________________________________

1.
Test
type
Static,
non­
renewal
2.
Salinity
25ppt
+
10
percent
for
all
dilutions
by
adding
dry
ocean
salts
3.
Temperature
(
oC)
20oC
+
1oC
or
25oC
+
1oC
4.
Light
quality
Ambient
laboratory
illumination
5.
Photoperiod
16
hour
light,
8
hour
dark
6.
Test
chamber
size
250
ml
7.
Test
solution
volume
200
ml
8.
Age
of
test
organisms
1­
5
days
9.
No.
Mysids
per
test
chamber
10
10.
No.
of
replicate
test
chambers
per
treatment
4
11.
Total
no.
Mysids
per
test
concentration
40
12.
Feeding
regime
Light
feeding
using
concentrated
Artemia
nauplii
while
holding
prior
to
initiating
the
test
13.
Aeration2
None
14.
Dilution
water
Natural
seawater,
or
deionized
water
mixed
with
artificial
sea
salts
15.
Dilution
factor
>
0.5
16.
Number
of
dilutions3
5
plus
a
control.
An
additional
dilution
at
the
permitted
effluent
concentration
(%
effluent)
is
required
if
it
is
not
included
in
the
dilution
series.
(
September
1996)
4
17.
Effect
measured
Mortality
­
no
movement
of
body
appendages
on
gentle
prodding
18.
Test
acceptability
90%
or
greater
survival
of
test
organisms
in
control
solution
19.
Sampling
requirements
For
on­
site
tests,
samples
are
used
within
24
hours
of
the
time
that
they
are
removed
from
the
sampling
device.
For
off­
site
tests,
samples
must
be
first
used
within
36
hours
of
collection.

20.
Sample
volume
required
Minimum
1
liter
for
effluents
and
2
liters
for
receiving
waters
_________________________________________________________________

Footnotes:

1.
Adapted
from
EPA/
600/
4­
90/
027F.

2.
If
dissolved
oxygen
falls
below
4.0
mg/
L,
aerate
at
rate
of
less
than
100
bubbles/
min.
Routine
D.
O.
checks
are
recommended.

3.
When
receiving
water
is
used
for
dilution,
an
additional
control
made
up
of
standard
laboratory
dilution
water
(
0%
effluent)
is
required.
(
September
1996)
5
EPA
NEW
ENGLAND
RECOMMENDED
TOXICITY
TEST
CONDITIONS
FOR
THE
INLAND
SILVERSIDE,
MENIDIA
BERYLLINA
48
HOUR
TEST1
_________________________________________________________________

1.
Test
Type
Static,
non­
renewal
2.
Salinity
25
ppt
+
2
ppt
by
adding
dry
ocean
salts
3.
Temperature
20oC
+
1oC
or
25oC
+
1oC
4.
Light
Quality
Ambient
laboratory
illumination
5.
Photoperiod
16
hr
light,
8
hr
dark
6.
Size
of
test
vessel
250
mL
(
minimum)

7.
Volume
of
test
solution
200
mL/
replicate
(
minimum)

8.
Age
of
fish
9­
14
days;
24
hr
age
range
9.
No.
fish
per
chamber
10
(
not
to
exceed
loading
limits)

10.
No.
of
replicate
test
vessels
per
treatment
4
11.
total
no.
organisms
per
concentration
40
12.
Feeding
regime
Light
feeding
using
concentrated
Artemia
nauplii
while
holding
prior
to
initiating
the
test
13.
Aeration2
None
14.
Dilution
water
Natural
seawater,
or
deionized
water
mixed
with
artificial
sea
salts.

15.
Dilution
factor
>
0.5
16.
Number
of
dilutions3
5
plus
a
control.
An
additional
dilution
at
the
permitted
concentration
(%
effluent)
is
required
if
it
is
not
included
in
the
dilution
series.

17.
Effect
measured
Mortality­
no
movement
on
gentle
prodding.
(
September
1996)
6
18.
Test
acceptability
90%
or
greater
survival
of
test
organisms
in
control
solution.

19.
Sampling
requirements
For
on­
site
tests,
samples
must
be
used
within
24
hours
of
the
time
they
are
removed
from
the
sampling
device.
Off­
site
test
samples
must
be
used
within
36
hours
of
collection.

20.
Sample
volume
required
Minimum
1
liter
for
effluents
and
2
liters
for
receiving
waters.
_________________________________________________________________

Footnotes:

1.
Adapted
from
EPA/
600/
4­
90/
027F.

2.
If
dissolved
oxygen
falls
below
4.0
mg/
L,
aerate
at
rate
of
less
than
100
bubbles/
min.
Routine
D.
O.
checks
recommended.

3.
When
receiving
water
is
used
for
dilution,
an
additional
control
made
up
of
standard
laboratory
dilution
water
(
0%
effluent)
is
required.

VI.
CHEMICAL
ANALYSIS
At
the
beginning
of
the
static
acute
test,
pH,
salinity,
and
temperature
must
be
measured
at
the
beginning
and
end
of
each
24
hour
period
in
each
dilution
and
in
the
controls.
The
following
chemical
analyses
shall
be
performed
for
each
sampling
event.

Minimum
Quanti­
fication
Parameter
Effluent
Diluent
Level
(
mg/
L)

pH
x
x
­­­
Salinity
x
x
PPT(
0/
00)
Total
Residual
Oxidants*
1
x
x
0.05
Total
Solids
and
Suspended
Solids
x
x
­­­

Ammonia
x
x
0.1
Total
Organic
Carbon
x
x
0.5
Total
Metals
Cd
x
0.001
Cr
x
0.005
Pb
x
0.005
(
September
1996)
7
Cu
x
0.0025
Zn
x
0.0025
Ni
x
0.004
Al
x
0.02
Superscript:

*
1
Total
Residual
Oxidants
Either
of
the
following
methods
from
the
18th
Edition
of
the
APHA
Standard
Methods
for
the
Examination
of
Water
and
Wastewater
must
be
used
for
these
analyses:

­
Method
4500­
Cl
E
Low
Level
Amperometric
Titration
(
the
preferred
method);
­
Method
4500­
CL
G
DPD
Photometric
Method.

or
use
USEPA
Manual
of
Methods
Analysis
of
Water
or
Wastes,
Method
330.5.

VII.
TOXICITY
TEST
DATA
ANALYSIS
LC50
Median
Lethal
Concentration
An
estimate
of
the
concentration
of
effluent
or
toxicant
that
is
lethal
to
50%
of
the
test
organisms
during
the
time
prescribed
by
the
test
method.

Methods
of
Estimation:

!
Probit
Method
!
Spearman­
Karber
!
Trimmed
Spearman­
Karber
!
Graphical
See
flow
chart
in
Figure
6
on
page
77
of
EPA
600/
4­
90/
027F
for
appropriate
method
to
use
on
a
given
data
set.

No
Observed
Acute
Effect
Level
(
NOAEL)

See
flow
chart
in
Figure
13
on
page
94
of
EPA
600/
4­
90/
027F.

VIII.
TOXICITY
TEST
REPORTING
The
following
must
be
reported:

!
Description
of
sample
collection
procedures,
site
description;

!
Names
of
individuals
collecting
and
transporting
samples,
times
and
dates
of
sample
collection
and
analysis
on
chainof
custody;
and
(
September
1996)
8
!
General
description
of
tests:
age
of
test
organisms,
origin,
dates
and
results
of
standard
toxicant
tests;
light
and
temperature
regime;
other
information
on
test
conditions
if
different
than
procedures
recommended.
Reference
toxicity
test
data
must
be
included.

!
Raw
data
and
bench
sheets.

!
All
chemical/
physical
data
generated.
(
Include
minimum
detection
levels
and
minimum
quantification
levels.)

!
Provide
a
description
of
dechlorination
procedures
(
as
applicable).

!
Any
other
observations
or
test
conditions
affecting
test
outcome.

!
Statistical
tests
used
to
calculate
endpoints.
