49.10.

01

Table 959.08A.

AOAC Official Method 959.08

Paralytic Shellfish Poison
Biological Method
First Action 1959
Final Action

Caution: Use rubber gloves when handling materials that may

contain paralytic shellfish poison.

Correction table for weight of mice

Weight of mice, g

Mouse unit

10

0.50

10.5

0.53

11

0.56

11.5

0.59

12

0.62

12.5

0.65

A. Materials

13

0.675

(a) Paralytic shellfish poison standard solution.100 mg/mL

saxitoxin dihydrochloride. Available from Washington Seafood
Laboratory Branch (HFS-426, Office of Seafood, U.S. Food and
Drug Administration, 8301 Muirkirk Rd, Laurel, MD 20708, USA)
as acidified 20% alcoholic solution. Standard is stable indefinitely
in cool place.
( b ) P a r a l y t i c s h e l l f i s h p o i s o n w o r k i n g s t a n d a rd
solution.1 mg/mL. Dilute 1 mL standard solution to 100 mL with
H2O. Solution is stable several weeks at 34C.
(c) Mice.Healthy mice, 1921 g, from stock colony used for
routine assays. If <19 or >21 g, apply correction factor to obtain true
death time (see Table 959.08A). Do not use mice weighing >23 g
and do not re-use mice.

13.5

0.70

14

0.73

B. Standardization of Bioassay

Dilute 10 mL aliquots of 1 mg/mL standard solution with 10, 15,

20, 25, and 30 mL H2O, respectively, until intraperitoneal injection
of 1 mL doses into few test mice causes median death time of
57 min. pH of dilutions should be 2-4 and must not be >4.5. Test
additional dilutions in 1 mL increments of H2O, e.g., if 10 mL
diluted with 25 mL H2O kills mice in 57 min, test solutions diluted
10 + 24 and 10 + 26.
Inject group of 10 mice with each of 2 or preferably 3 dilutions that
fall within median death time of 57 min. Give 1 mL dose to each
mouse by intraperitoneal injection and determine death time as time
elapsed from completion of injection to last gasping breath of mouse.
Repeat assay 1 or 2 days later, using dilutions prepared above
which differed by 1 mL increments of H2O. Then repeat entire test,
starting with testing of dilutions prepared from newly prepared
working standard solution.
Cal cu late me dian death time for each group of 10 mice used
on each di lu tion. If all groups of 10 mice in jected with any 1
di lu tion gave me dian death time <5 or >7 min, dis re gard re sults
from this dilu tion in sub se quent cal cu la tions. On other hand, if
any groups of 10 mice in jected with 1 di lu tion gave me dian
death time fall ing be tween 5 and 7 min, in clude all groups of
10 mice used on that di lu tion, even though some of median death
times may be <5 or >7 min. From me dian death time for each
group of 10 mice in each of selected di lu tions, de ter mine
n u m b e r o f m o u s e u n i t s / m L f r o m S o m m e r s Ta b l e ,
Table 959.08B. Di vide cal cu lated mg poi son/1 mL by mouse
units/1 mL to ob tain con ver sion fac tor (CF value) ex pressing mg
poi son equiv a lent to 1 mouse unit. Cal cu late av er age of
in di vid ual CF val ues, and use this av er age value as ref er ence
point to check rou tine as says. In di vid ual CF val ues may vary
signif i cantly within lab ora tory if tech niques and mice are not
rig idly con trolled. This sit u a tion will re quire con tin ued use of
work ing stan dard or sec ond ary standard, de pend ing on vol ume
of as say work per formed.

14.5

0.76

15

0.785

15.5

0.81

16

0.84

16.5

0.86

17

0.88

17.5

0.905

18

0.93

18.5

0.95

19

0.97

19.5

0.985

20

1.000

20.5

1.015

21

1.03

21.5

1.04

22

1.05

22.5

1.06

23

1.07

C. Use of Standard with Routine Assays of Shellfish

Check CF value periodically as follows: If shellfish products are

assayed less than once a week, determine CF value on each day
assays are performed by injecting 5 mice with appropriate dilution
of working standard. If assays are made on several days during
week, only 1 check need be made each week on dilution of standard
such that median death time falls within 57 min. CF value thus
determined should check with average CF value within 20%. If it
does not check within this range, complete group of 10 mice by
adding 5 mice to the 5 mice already injected, and inject second group
of 10 mice with same dilution of standard. Average CF value
determined for second group with that of first group. Take resulting
value as new CF value. Variation of >20% represents significant
change in response of mice to poison, or in technique of assay.
Changes of this type require change in CF value.
Repeated checks of CF value ordinarily produce consistent
results within 20%. If wider variations are found frequently,
possibility of uncontrolled or unrecognized variables in method
should be investigated before proceeding with routine assays.
D. Preparation of Test Sample

(a) Clams, oysters, and mussels.Thoroughly clean outside of

shellfish with fresh water. Open by cutting adductor muscles. Rinse
inside with fresh water to remove sand or other foreign material.
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Table 959.08B. Sommers Table

Death time:mouse unit relations for paralytic shellfish poison (acid)
Death time

1:00

Mouse units
100

Death time

Mouse units

5:00

1.92

10

66.2

05

1.89

15

38.3

10

1.86

20

26.4

15

1.83

25

20.7

20

1.80

30

16.5

30

1.74

35

13.9

40

1.69

40

11.9

45

1.67

45

10.4

50

1.64

Remove meat from shell by separating adductor muscles and tissue

connecting at hinge. Do not use heat or anesthetize before opening
shell, and do not cut or damage body of mollusk at this stage. Collect
ca 100150 g meats in glazed dish. As soon as possible, transfer
meats to No. 10 sieve without layering, and let drain 5 min. Pick out
pieces of shell and discard drainings. Grind in household-type
grinder with 18 14 in. (36 mm) holes or in blender un til
homogeneous.
(b) Scallops.Separate edible portion (adductor muscle) and
apply test to this portion alone. Drain and grind as in (a).
(c) Canned shellfish.Prepare by blending as in 937.07(b) (see
35.1.01).
E. Extraction

55

8.42

15

1.54

2:00

7.67

30

1.48

05

7.04

45

1.43

10

6.52

7:00

1.39

15

6.06

15

1.35

20

5.66

30

1.31

25

5.32

45

1.28

30

5.00

8:00

1.25

35

4.73

15

1.22

40

4.48

30

1.20

45

4.26

45

1.18

50

4.06

9:00

1.16

55

3.88

30

1.13

Weigh 100 g well-mixed test portion into tared beaker. Add

100 mL 0.1M HCl, stir thoroughly, and check pH. (pH should be
<4.0, preferably ca 3.0. If necessary, adjust pH as indicated below.)
Heat mixture, boil gently 5 min, and let cool to room temperature.
Adjust cooled mixture to pH 2.04.0 (never >4.5) as determined by
BDH Universal Indicator, phenol blue, Congo red paper, or pH
meter. To lower pH, add 5M HCl dropwise with stirring; to raise pH,
add 0.1M NaOH dropwise with constant stirring to prevent local
alkalinization and consequent destruction of poison. Transfer
mixture to graduate and dilute to 200 mL.
Return mixture to beaker, stir to homogeneity, and let settle until
portion of supernate is translucent and can be decanted free of solid
particles large enough to block 26-gage hypodermic needle. If
necessary, centrifuge mixture or supernate 5 min at 3000 rpm or
filter through paper. Only enough liquid to perform bioassay is
necessary.

3:00

3.70

10:00

1.11

F. Mouse Test

05

3.57

30

1.09

10

3.43

11:00

15

3.31

30

1.06

20

3.19

12:00

1.05

25

3.08

13

1.03

30

2.98

14

1.015

35

2.88

15

1.000

40

2.79

16

0.99

45

2.71

17

0.98

50

2.63

18

0.972

Intraperitoneally inoculate each test mouse with 1 mL test

solution from E. Note time of inoculation and observe mice
carefully for time of death as indicated by last gasping breath.
Record death time from stopwatch or clock with sweep second hand.
One mouse may be used for initial determination, but 2 or 3 are
preferred. If death time or median death time of several mice is
<5 min, make dilution to obtain death times of 57 min. If death time
of 1 or 2 mice injected with undiluted sample is >7 min, total of 3
mice must be inoculated to establish toxicity of test solution. If large
dilutions are necessary, adjust pH of dilution by dropwise addition
of dilute HCl (0.1 or 0.01M) to pH 2.04.0 (never >4.5). Inoculate 3
mice with dilution that gives death times of 57 min.

50

9.33

6:00

1.60

1.075

55

2.56

19

0.965

4:00

2.50

20

0.96

G. Calculation of Toxicity

05

2.44

21

0.954

10

2.38

22

0.948

15

2.32

23

0.942

20

2.26

24

0.937

25

2.21

25

0.934

30

2.16

30

0.917

35

2.12

40

0.898

40

2.08

60

0.875

Determine median death times of mice, including survivors, and

from Sommers Table determine corresponding number of mouse
units. If test animals weigh <19 or >21 g, make correction for each
mouse by multiplying mouse units corresponding to death time for
that mouse by weight correction factor for that mouse from
correction table (Table 959.08A); then determine median mouse
unit for group. (Consider death time of survivors as >60 min or
equivalent to <0.875 mouse unit in calculating median.) Convert
mouse units to mg poison/mL by multiplying by CF value.

45

2.04

50

2.00

55

1.96

Minutes:seconds.

mg Poison/100 g meat = (mg/mL) dilution factor 200

Consider any value >80 mg/100 g as hazardous and unsafe for
human consumption.
Reference: JAOAC 42, 263(1959).

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