Diazinon
DCI:

Fish
Chronic
Toxicity
Testing
Results
EPA
EFED
Meeting
May
30,
2006
Makhteshim­
Agan
of
North
America
L.
Pruett,
R.
Everich,
C.
Habig
Currently
available
studies
include:

°
Fish
early
life
stage
(
fathead
minnow)

°
Fish
full
lifecycle
(
fathead
minnow)

°
Fish
partial
lifecycle
(
brook
trout)

Previous
data
was
reviewed
 
New
study
focused
on
sensitive
endpoints
which
were
not
adequately
addressed
in
available
studies
Summary
of
Previous
Results
 
Minnow
ELS
(
NOEC
90
ppb;
<
90
ppb)

°
Most
sensitive
endpoint:
Growth
°
No
significant
effects
on
survival,
hatching
up
to
1.6
mg/
L
 
Minnow
FLC
(
NOEC
~
3.0
ppb)

°
Sensitive
endpoints
 
Hatching
of
F1
fish
from
exposed
F0
parents
 
Growth
of
F1
from
exposed
F0
parents
 
Trout
PLC
(
NOEC
<
0.55
ppb;
current
EFED
NOEC)

°
Sensitive
endpoint:
Growth
F1
from
exposed
F0
parent
Results
of
Diazinon
Partial
Life­
cycle
test
and
ELS
Range
Finding
Test
The
objective
of
the
range­
finding
testing
was
to
assess
the
effects
of
Diazinon
on
the
most
sensitive
biological
endpoints
as
determined
in
previous
fish
chronic
toxicity
studies.

The
endpoints
of
concern
were
as
follows:

°
Adult
survival
(
F0
fish)

°
Reproductive
success
of
F0
fish
as
defined
by:

 
number
of
spawns
 
total
number
of
eggs
 
eggs
per
spawn
 
egg
hatchability
°
Fry
survival
(
F1
Fish
Test,
28­
days
post­
hatch)

°
Fry
growth
(
standard
length
and
blotted
wet
weight)
Methods
°
Proportional
diluter
system
(
Mount
and
Brungs)

 
Syringe
dispenser
for
the
intermittent
introduction
of
control
and
Diazinon
test
solutions
into
each
test
chamber.

°
The
accuracy
of
the
diluter
(
delivery
within
10%
of
target)
was
verified.

°
Proper
operation
of
the
proportional
diluter
and
all
mechanical
systems
was
verified
twice
each
day
during
the
range­
finding
test.

 
No
malfunctions.

°
Diluter
stock
solutions
 
Prepared
weekly
resulted
in
a
test
solution
concentration
of
8.0
µ
g
a.
i./
L.

 
Subsequent
dilutions
of
this
test
solution
were
prepared
by
the
diluter
system
at
nominal
concentrations
of
4.0,
2.0,
1.0,
and
0.50
µ
g
a.
i./
L.
Diazinon
Partial
Life­
cycle
test
and
ELS
Exposure
Chamber
Design
for
the
Partial
life­
Cycle
Exposure
of
Fathead
Minnow
(
Pimephales
promelas)
to
Diazinon
TEST
SOLUTION
ENTRY
Analytical
Verification
°
Sample
analysis
performed
with
MDS
Sciex
API
4000
LC­
MS/
MS
system
°
The
concentrations
of
diazinon
analyzed
in
test
solutions
and
from
the
diluter
stock
solution:

 
day
­
1,
0,
1,
14,
54,
83,
and
117
of
the
range­
finding
test.

°
Control
and
QC
fortified
samples
were
also
analyzed
at
each
sample
period.
Analytical
Verification
 
Results
7.58
2.90
1.43
0.886
0.378
<
MQL
0
5.97
2.86
1.52
0.766
0.318
<
MQL
­
1
Level
5
(
8.0)

Level
4
(
4.0)

Level
3
(
2.0)

Level
2
(
1.0)

Level
1
(
0.50)

Control
Study
Day
8.33
3.29
2.12
0.840
0.440
<
MQL
54
6.48
3.44
1.62
0.880
0.416
<
MQL
14
8.70
3.61
1.80
0.998
0.440
<
MQL
1
%
Nominal
Mean
(
SD)

117
83
0
0
<
MQL
<
MQL
85
0.427
(
0.0281)

0.460
0.426
93
0.932
(
0.080)

1.05
0.938
99
88
93
7.89
(
0.890)

3.50
(
0.440)

1.85
(
0.295)
8.80
4.24
2.20
7.44
3.52
1.94
MQL
=
0.118
µ
g
a.
i./
L
The
day
 
1
values
were
not
included
in
the
calculation
of
the
mean
measured
concentrations
or
standard
deviations
F0
Exposure
 
Study
Design
and
Parameters
°
Initiation
on
October
13,
2005Duration:
116
days
°
25
sub­
adult
fathead
minnows
that
were
59
days
old
at
test
initiation.

Fish
spawned
and
reared
at
ABC
Laboratories.

°
F0
fish
paired
December
6,
2005
after
54
days
of
exposure.

°
Daily
Observations:

 
Mortality
 
abnormal
morphology
or
behaviors
 
Spawns
on
tiles
 
Number
of
eggs
per
spawn
°
All
other
fish
sacrificed
and
growth
measurements
were
recorded
(
standard
length
and
blotted
wet
weight).
F0
Exposure
 
Results
7.89
3.50
1.85
0.932
0.427
Control
7.89
3.50
1.85
0.932
0.427
Control
Mean
Measured
Concentration
(
µ
g
a.
i./
L)
1.868
(
0.457)

43
(
2.5)

4
Male
1.965
(
0.743)

44
(
4.5)

8
Male
1.635
(
0.479)

43
(
3.6)

5
Male
1.730
(
0.381)

43
(
2.6)

10
Male
1.919
(
0.661)

44
(
3.4)

5
Male
Mean
Blotted
Wet
Weight
in
grams
(
standard
deviation)

Mean
Standard
Length
in
mm
(
standard
deviation)

N
Gender
NA
NA
0
Female
1.01
(
0.139)

36
(
2.1)

2
Female
1.701
(
0.616)

42
(
3.5)

7
Male
0.747
(
NA)

33
(
NA)

1
Female
1.095
(
0.226)

36
(
3.3)

5
Female
1.132
(
NA)

38
(
NA)

1
Female
1.002
(
0.049)

36
(
0.58)

3
Female
F0
Exposure
 
Results
189
(
45.2)

2,905
(
1,135)

15
(
2.8)

0.427
254
(
44.3)

4,296
(
2,605)

16
(
8.0)

Control
Mean
Number
of
Eggs
per
Spawn
(
Standard
Deviation)

Mean
Number
of
Eggs
(
Standard
Deviation)

Mean
Number
of
Spawns
(
Standard
Deviation)

Mean
Measured
Concentration
(
µ
g
a.
i./
L)
226
(
22.8)

3,401
(
696)

15
(
3.9)

0.932
205
(
42.3)

3,809
(
2,224)

18
(
9.4)

7.89
192
(
54.6)

3,238
(
2,207)

16
(
6.9)

3.50
148
(
42.2)
*

3,120
(
1,250)

21
(
5.4)

1.85
*
Statistically
significant
difference
(
Dunnett's
Test;
 
=
0.05)
was
observed
between
the
test
substance
treatment
and
the
control.
Hatchability
Trials
 
Study
Design
and
Parameters
°
The
initial
hatchability
trial
performed
from
December
22,
2005
through
January
4,
2006
°
Initiated
with
40
impartially
selected
embryos
°
Second
Hatchability
trial
was
the
initiation
of
the
ELS
testing.
Performed
from
January
4
or
5
through
February
6
or
7,
2006
for
all
treatments
except
for
Level
2B
which
started
on
January
10,
2006
and
terminated
on
February
11,
2006
°
Initiated
with
40
impartially
selected
embryos
that
were
thinned
to
25
randomly
selected
well­
eyed
embryos
on
test
day
3
°
Daily
Observations:

 
Number
of
dead
embryos
 
Number
of
live
fry
 
Number
of
dead
fry
 
Calculated
Percent
Hatch
Hatchability
Trials
 
Results:

7.89
3.50
1.85
0.932
0.427
Control
Mean
Measured
Concentration
(
µ
g
a.
i./
L)
100
95
100
95
100
100
100
100
100
97
100
100
Mean
Percent
Hatch
­

Second
Trial
(
ELS)

Mean
Percent
Hatch
­

Initial
Trial
Early
Life­
Stage
(
ELS)
Study
Design
and
Parameters:

The
ELS
testing
was
initiated
on
January
4
terminated
on
February
11,

2006
°
Initiated
with
40
impartially
selected
embryos
that
were
thinned
to
25
randomly
selected
well­
eyed
embryos
on
test
day
3
°
Duration:
28­
days
post­
hatch
°
Observations:

 
Number
of
dead
embryos
 
Number
of
live
fry
 
Number
of
dead
fry
 
Calculated
Percent
Hatch
 
Calculated
Percent
Post­
hatch
Survival
 
Standard
Length
Measurements
 
Blotted
Wet
Weight
Measurements
Early
Life­
Stage
 
Results
0.121
(
0.034)

18
(
1.8)

98
0.427
0.131
(
0.031)

19
(
1.4)

100
Control
Mean
Blotted
Wet
Weight
in
grams
(
standard
deviation)

Mean
Standard
Length
in
mm
(
standard
deviation)

Mean
Percent
Post­
Hatch
Survival
Mean
Measured
Concentration
(
µ
g
a.
i./
L)
0.140
(
0.046)

19
(
2.1)

82
*

7.89
0.126
(
0.041)

18
(
2.5)

94
3.50
0.113
(
0.031)

18
(
1.6)

98
1.85
0.135
(
0.036)

18
(
1.6)

96
0.932
*
Statistically
significant
difference
(
Dunnett's
Test;
 
=
0.05)
was
observed
between
the
test
substance
treatment
and
the
control
Conclusions
°
At
the
concentrations
tested,
0,
0.427,
0.932,
1.85,
3.50,
and
7.89
µ
g
a.
i./
L,
the
only
biologically
significant
effect
of
the
diazinon
exposure
was
to
the
post­
hatch
survival
during
the
ELS
portion
of
the
study.

°
The
definitive
testing
would
follow
the
same
general
procedures
outlined
above,
but
would
double
the
replication
utilized
in
this
range­
finding
testing,
thus
increasing
the
statistical
power
of
the
testing.

°
Suggested
nominal
concentrations
could
include
0,
1.0,
2.0,
4.0,
8.0,

and
16
µ
g
a.
i./
L.

°
At
these
concentrations,
the
effects
on
the
post­
hatch
survival
should
be
increased
at
the
8.0
and
16
µ
g
a.
i./
L
treatment
levels
thus
providing
more
than
one
treatment
level
with
partial
effects.

°
Even
with
the
increase
in
the
concentrations,
the
likely
NOEC
level
should
fall
between
2.0
or
4.0
µ
g
a.
i./
L.
Concerning
additional
fish
lifecycle
testing
 
Given
the
results
of
currently
available
chronic
fish
studies,
it
is
very
likely
the
NOEC
from
a
new
fish
lifecycle
study
will
be
2.0
to
4.0
ppb
­
higher
than
the
0.55
ppb
chronic
toxicity
endpoint
EFED
is
currently
using
 
How
will
EFED
will
use
data
from
a
new
study
for
risk
assessment
if
the
NOEC
is
greater
than
the
currently
available
chronic
toxicity
endpoint?
