T ab le of Co nte nts
A B S T
RA C T
I I I
A C
KN O
W
LE D G
M
E N T S
I V
L I ST O F F I G U R E S
V
L I ST O F T A B L E S
V I
I N
T R O
DU C T I O N
:L I T E R A T U R E R E V I E
W
A N D B A C K G
RO U N D
1
B
AC
KG
RO U
N D1
P A H
S
1
S
PA
HM
O
DE
L4
M
IC
RO
TO
X -AC U T E
6
M
IC R O T O X
-C H R O N I C
1 1
O B J E C T I V E S
12
M
A T E R I A L S A
ND
M
E
TH O
DS
14
C
H EM
I C A L S
14
P A H S A
M
P L E
P RE
PA R A T I O N
14
M
I C R O T O X
-A C U T E T O X I C I T Y
14
I N D I V I D U A L P A H T
EST I N G
15
P A H
M
I X T U R E T E ST I N G
16
C H R O N I C T O X I C I T Y
16
C H R O N I C T O X I C I T Y T E ST I N G
17
M
E D I A P R E P A R A T I O N
18
P O S IT I V E C O N T R O L P R E P A R A T I O N
18
S A
M
PL E P R E P A R A T I O N
18
T E S T P R O C E D U R E
19
Z P A H
M
O D E L
19
R E
S U L T S A N D D I S C U SS
IO N
2 1
I N D I V I D U A L P A H A C U T E T O X I C I T Y T E S T S
2 1
D U A L
M
I X T U R E S
2 3
O T H E R
M
I X T U R E S
23
S P A H
M
O D E L
2 4
C H R O N I C T O X I C IT Y
2 5
C O N C
L US
IO
N 2 7R
E F E R E N C E S
2 9
A B ST R A C T
T
od
ate ,f
ew r e s e a r ch
er sh
a v e a s s e s s ed
th
e t oxi
ci
ti
e s of P
ol
ycycl i
cA
r om
ati
cH
yd
r ocarb
ons(
P A H
's
)
asm i
xtu r e s, al
th
oug h
th
ati
sh
ow th
ey c om
m
o nl y
oc c u ri
n th
e envi
r o nm
e nt.T h
e to xi
ci
ti
es of
2,3
, and 4
ri
ng
P AH
's w e r e
d
ete rm i
n ed f
o ri
nd i
vid
ual
c om p
ou nd
s.S
yn erg i
sti
c antag
o ni
sts or ad d i
ti
ve ef f
ect s of m i
x tu r es of
two , thr e e , a n
d f
o u rP A H
c om p
ou nd
s wer e a s s e s s ed b y
c omp
ari
ng
th
e to xi
ci
ti
e s of tho s em i
xtu r e s to th
at of i
nd
iv id
u al
co mp
o u nd
s.M
i
c ro t oxm i
xtu r e re s ults we re al
s o c om p
ared
t o th
o s ep
r ed i
ct ed b y
th
eI PA H m
od
el
.T h
e t oxi
ci
ti
e s of
ch
ry
s e n e ,p y
r e n e ,p h
e n a nth
ren e ,f l
u o r ene ,f l
u o r a nth
r e n e, ace n ap h
th
al
e ne,p h
e n a nth
r e n e, a nd
n ap h
th
al
e n e we r e ev al
u at ed
usi
ng
th
eM
i
croto x®
a c ute a n
d
ch
roni
c t oxi
ci
ty a ssay wh i
ch
u s e s ab i
ol
um i
n e s c e ntb
ac te ri
a. Vi b ri
of
i
sh
e ri
, tom
eas u r e to x
i
ci
ty
. Re s ul
t si
nd i
c a te th
at, a si
nd i
vi d
u al
c om p
o u nd
s, th
e a c ute t oxi
ci
ti
e s of P A H
s a r ei
n v e rsel
y r el
ated
to wate r s ol
ub i l i
ty .T h
e r e s e a r ch
al
so sh
o wed
th
a t mi
xtu r es c a nb
e syn e rg i
s ti
c,a ntago n
i
sti
c, o r ad d i
ti
v e.C h
r o ni
c te sts sh
ow ed
th
a tP A H
s wi
th l
owe r wate r s ol
ub i
li
ty r eq
ui
reg
r e ate r conc entra ti
ons th
an o th
e rP A H
s t o sh
ow a to xi
c r e sp
o n s e.L
a stl
y,
f
r om
the r e s e a r ch
, o n e ca n s e e that th
e ad d i
ti
v e c o n c ep
tf
o r PA
Hm
ixtu r e si
sg
e n e r al l
y up
held
.A C K N O
W
L E D G
M
E N T S
I
wo ul d f i
r s tl i k
e to th
ank G
od f
o rg i
vi
ng
me th
e cour ag
e and
d
ete rm i
n ati
o n to a c c om p l i
shm
yg
o al
s. Ih
a v e to th
a nk m
yf
am i l
y a nd f
ri
e nd
sf
o r th
ei
rs u
p p
o rt a nd
e n c o u rag
em
e nt a s well
. El i
z ab
eth G
uth
e ri
e a nd C h
ad
Rop
e rh
elp
ed m
e s om
u ch d
u ri
ng
th
el
a s t t w oy
e ars.T h
ank y
o u som
u ch f
o r e very
th i
ng
.I
wo ul d
al
s ol i k
et o t
h
a nk D
r .P f
a end
e r, w
h
ob
el i
e v ed i
nm
e and
al l
ow
ed m
e to wo rk w i
th h i m
.L I ST O F F I G U R E S
F I
G U
R E 1. PA H S
tr u ctu r e,
W
ate rSo
l
ub i l i
ty, a nf L
of K
owF I G U R E 2
.I
nd i
vi d
ual P A H T
oxi
ci
ty E
xp
re s s ed i
nT
oxi
cU
ni
t sF I
G U
RE 3
. PA H T
o xi
ci
ty v sW
ate rSo
lu
b i l i
tL I
S
TO F T A
B L ES
T A B L E
L
PA
H T o xictyU
ni
ts of
Ind i
vi d
u alC
om
po u nd
sT A
B L E 2
.D
u al
M
i
x tu r e sT A B L
E 3
.P A H T
oxi
ci
tyU
ni
t s of
M
i
xtu r esT
A
BL
E4
. P A HC h
r o ni
c To xi
ci
ty
As s e s s
m
ent ofC
hr onic an d A cute T o xici t y of Pol ycycl ic A r om
aticH
y dr o c a rbo n s(
PA H
s)
a nd P A H
M
i
xtu r e sI N T R O D U C T I O N A N D O B J E C T I V E S
I
ntr od
u cti
o n :L i
teratu re revi
ew a nd b
a ck g
r ou nd
PA
H
sP A H
s arem
ul
ti
-ring
str u ctu r e sh
el d
t og
eth
e rb
y s tab l
e c a rbo n
-car
bo n
b
o nd
sthat can
b
e s ub
sti
tuted
wi
th
eith
e r ni
trog
en , sul f
u r , o r o xy g
e n(F i g
u r e1
)
(
B l
um
e r,19 7
6
)
.T h
ey
o c cu ri
n th
e en vi
ro nm
e n t wh
e n c om p l
ex org
ani
c sub
s t anc e s are exp
o s ed
to
h i g h
t em p
er atu r es orp
r ess u r e(A g
e n cyf
o rT
o xi
cS
ub
st an c e s a nd
Di
s e aseR
eg i
stry,19 9 3
)
.PA H
s are c om
mon c ont am i
na n t s of h
az a rd
ou s wa st e si
t e s and
a rep
r e s entin
ae r
i
al fal l
o ut of
com b
u stio np
r od
u ct s,in
d
u str ial
a nd
s ewag
ele a
k
ag
e, a nd
th
e accid
e ntald is
p
os al
of p
etrole u
m
p
r od
u cts(
C
er nig l i
a,19 92
)
. PA H
s occur n atu ral l y i
n th
een v
ir
onm
entd
ue t o oi l
se ep
s, su rf
a ce r u n-o
f f
, and f
o res t and p
rai
ri
ef i
re s,b
ut th
ei
rc once ntr at
i
o ns c a nb
e ele v a
ted d
u e t oh
um
a n a c ti
vi
ti
e s(
C h
au nd
r y,199 5
)
.P hy
sio c
h
em ic a
l
ch
a ract eris
ti
c s, su ch
a slo
w w a te r s ol
ub il
i
ty
and h ig h
o c t an ol
-water coe
f f i
ci
ents, af f
e c t th
ep
ersi
st en ce of
th
e s e c omp
o u nd
sin
th
e en vir
onm
ent.P A H
s are n o t very
s olu
b l
e , and
th
eir
wa te r s olu
b i l i
ty
d
ecr e ase s wi
th
in c r e
asin
g
m
ol
ec ul
ar wei
gh
t.P A H
sh
ave ah ig h
af f i
nit
y f
or org
ani
cm
a t t e r and
org
ani
c c o nt ai
nin
g
se
d
ime nt, wh
er e th
ey pa rti
ti
o n to th
e orga ni
cf
r a cti
on w h ich
c a n r edu c e theirb i
o a v ai l
ab i l i
ty
.T h
ey
ar eb
u ri
ed
a nd p
e rsi
s t u nti
ld
eg
r ad
ed
,b i
oaccum
ul
a ted
, o rre
m
oved b y d
red g i
ng
(
C
er ni g l i
a,19 9 2
)
.T h
er ei
s th
eref
or e ad i
r ec t rel
a ti
o nsh i p
b
etwee nP A H
to xi
ci
ty
a nd b i
o a v ai l
ab i l i
ty . PA H d
eg
r ad
ati
o ni
s alsoi
nf l
u e n c ed b y
s uch
t
h
ing
s a s tem p
e r atu r e, p H , n utrie nts, o xy ge n, a nd
soi l
typ
e.PA H
s c onsi
s t of 2
-7 b
en ze ne r
i
ng
s ;PA H
s wi
th l
es s th
a nf
ou rb
e nz e n e ri
ng
s may
b
e v ol
ati l i l i
zed
,p
hotooxi d i
zed
, oxi d i
zed
,b i
oa ccum
ul
at ed
, ab
so rb
ed i
n th
e s oi l
,l
e ach
ed
or
b i
od
eg
r ad
ed b y m i
cr o o rg
ani
sm
s(
C
e r ni g l i
a,19 9 2
;P
ark
, e t al
,19 9 0
;B
o s s ert a nd
B
a r th
a,1 9 8 6
)
.P A H
s wi
th f
ou r orm
oreb
enz e n e ri
ng
s arel
e s sb i
od
eg
rad
ab l
e,l
e s sm
ob i l
e , a nd p
e r si
stl
ong
eri
n en vi
ronm
ent al m
a tri
c es.H i g h m
ol
e c ul
ar wei g h
tP A H
sar e
d
es orb
ed
sl
owl y
and
th
e ref
o re a rel
es s a v ai l
ab l
ef
orb i
ol
og i
cal
up
t ak
e .T h
eh
al f
-l i
v es of h i g h m
ol
e c ul
a r wei g h
tP A H
sl i k
eb
en z o[
a]
p y
r e n e a re al
s ol
ong
e r th
anl
owm
ol
e cul
arw
ei g h
tPA H
sl i k
e n ap h
th
al
e ne.T h
e seP A H
s ar e rec al
ci
tr a nt a nd
c a np
e rsi
sti
n th
e en vi
r o nm
e ntf
o rl
ong p
eri
od
s of
ti m
e(
C
er ni g l i
a,19 9 2
)
.R
e c e n tl y
, r e s e a r ch
h
a sb
e e n c o nd
uc t ed
t o te s t th
ed
eg
rad
ab i l i
ty
of
h i g h
erm
ol
ec ul
ar wei g h
tP A H
s.A
stud
yb y
M
a cG i l l i
v ry
and Sh i
ari
si
n19 9 3 f
ou nd y
ea st t oh
a v e th
e ab i l i
ty t od
eg
r ad
ep h
ena n th
r e ne a nd b
e n z o{
a}
a nth
r a c e ne(
M
acG i l l i
v e ry a nd
Sh i
a ri
s,19 9 3
)
. Ith
a s al
sob
e e nf
o u nd
th
a tM
y
cob
a c ti
um
sp
.PY R
-1 h
a s t
h
e ab i l i
ty tom i
n e r al i
zef l
u o ranth
en e, n ap h
th
al
e ne , a nd p y
r enei
np
ure cul
tur e a nd
al
s o e nh
a n c em i
n e r al i
za ti
on of
f l
uoranth
ene a nd
p
yr e nei
n sed i m
e nts c o ntai
ni
ng
n ati
v eT he t ox ic,
m
utage nic, a nd cci r cin o
ge n ic n atu re of P A H s is a c o n c ern to bothh
um
an a nd
e n vir o nm
ental he a
l
th
(
A g
e n cy f
o rT
o xi
cS
ub
st anc e a nd D i
s e a seRe
g i
stry
,19 9 3
)
.E
xp
o s u re toPA H
sp
r o v es t ob
e a si g
ni f
ic ant
h
e al
th
ri
sk
t op
e op l
el iv
i
ng i
n th
ei
nd
u stria
l
iz ed
a r ea s of
the
wo rl d
. For ex am p l
e ,P
AH
c o ntami
n ati
on r a ng
e sf
rom 5
ng / g
o
f
soi l in
u nd
ev eloped
a r eas and
1
.79
x10
ng/ g
at a n oi l
r ef i
n e ry(
C
e r nig l
ia,19 9 2
)
.T h
e17 7 5
s tud
yb y P
o t tf
ou nd
ch i m
ney sw eep
s toh
a v e ah ig h i
nci d
enc e of
c a n c er ,p
r e s um
ab l
yd
u e t o th
ei
r e xp
os u r e t o PA
H s(
A g
ency f
o rT
o xi
cS
ub
sta n c e a nd D i
s e a s eR
eg i
s try ,19 9 3
)
.I
n th
e19 3 0
's
B
e n z o{
a}
p y
r e n e wasis
ol
at ed
f
rom
c oal
t ar , acons t
i
tuentf
ou nd in
soot, a nd f
ou nd
t ob
e c ar cin
og
e ni
c .C
on ce n tr ati
ons of P A H
sin
coa
l
t ar c anb
e ord
e rs of m
ag
ni
tud
eg
r e ate r th
a n th
osef
o u nd i
np
etr ol
e um d
e riv e
d
m
at eri
al
s(
M
ach
ad
o e t al
,19 9 3
)
.P
A H
e xp
osu r eh
a s no t onl y b
e enl i
nk
ed
to s e v e ral
typ
e s of
can c e rb
uti
ti
s al
s oi
n volv
ed i
n af f
e c ti
ng
th
eh
em
a t op
oe ti
c and
th
ei m m
u n e sy
st em . PA H
sm
ay af f
ect th
eh
um
a nf
e tu s as wel l
(
Ag
en cy f
orT
oxi
cS
ub
stan c e a nd
Di
sea s eR
eg i
stry
, 19 9 3
)
I
n19 9 3
,A
sp
o stol i
et al
. s tud i
ed
th
e ef f
e c t s o n work
e rs of p
rol
ong
ed
exp
o s u r e t o c ut ti
ng
f l
uid
s ; cut ti
ng f l
ui d
s a r em ix
tur e s contai
nin
g
vari
o u sp
er c ent ag
es of m i
n er al
oi l
s and /
or ,m
or e r a rel
y , synth
etic
oi l
s.T h
es ef l
uid
s ar e u sed m
ain
l y i
n me ch
ani
cal
eng i
ne erin
g
an
d
th
em
et al f
ab
ri
cati
o ni
nd
us try
.I
t w a s c on cl
ud
ed f
r om
th i
s re s ea rch
th
a tP A H
sw
e r em
ai
nl y
re sp
onsi b l
ef
or c ar cin o
g
e ni
c activ
i
ty
(
sk i
n and
re sp i
ratory
t um
or s)
a ssoc
i
a t ed
wi
th
th
e u se of
cut ti
ng fl
uid
ov e r ti m
e .P A H
s,m
or eover , arel i
sted
o n th
eII P
R
eg i
stry ,19 9 3
)
.A
pp
r opri
ate e v al
u ati
o n of p
o te nti
al ad
v e r s e ri
skf
r om
P A H e xp
o s u r ei
sd
ep
e nd
ent o n th
e ap p
r opri
ate to xi
c ol
og i
c al
a ss essm
e n t of PA H
s.T
oxi
ci
ty
asse s sme n ts u si
ng
exp
eri m
e n t al
ani m
al
sh
aveb
ee np
erf
ormed
f
ori
nd i
vi d
ual P A H
s(
K
w an ,D
utk
a, a nd L i
u ,19 9 0
)
. In th
e e n vi
ronme nt,h
owe v e r,PA H
sar e a
l m
o st al waysf
o u nd
as c om p
o u nd m i
xtu res.M
i
x tu r e s existi
n th
e sed im
e ntb
e c a u s es e
d i m
e n t c ont am i
n a ti
o n usu al l y i
n vol
ve s th
e s et tl i
ng
of m
any ch
emi
c al
s(
S
war tz e t al
,19 95
)
.I
th
asb
e e nf
ou nd
th
a tP
AH m i
xtu re s ar e som
eti m
e sm
or e to xi
c th
a ni
nd i
vid
u al
P A H
t oxi
ci
ty
.T h
e com b i
ned
s tr e ss es of m
ul
ti p l
e cont am i
n a nts, o ri
n th i
s casePA H
s,m
ayca u s e e nv
i
r onm
ent al
ef f
ec ts th
a t woul d
no tb
e e xp
e cted
wh
enPA H
s a re tes ted i
nd i
vid
ual l y
.P
A H m i
xtu r es a rem
ore resi
stant t od
eg
r ad
ati
o n a nd
e xh ib i
t a ntag
o ni
sti
c, syn e rg i
sti
c, o ra
dd i
ti
v e ch
a r a cte ri
sti
c s(
C h
aud
ry
,199 5
;K
el l
ey
a nd C
er ni g l i
a,199 5
)
.M
i
x tu r e s th
at e xh ib i
t ant ag
o ni
sti
c ch
ar a c te ri
sti
cs are th
o s e th
a t sh
ow al
e s s t oxi
cre s
p
o n s e th
a n th
e sum
of
th
ei
nd i
vi d
u al
c om p
ou nd
s t e sted
.Sy
n e rg i
s ti
c r esp
o n se s wi l l
o c cu rw h
en th
em i
xt ure to xi
ci
ty i
sg
r ea t er th
an th
at of
th
ei
nd i
vid
ual
c om p
ound
s te s ted
.A dd i
ti
vet ox
i
ci
ty
r e sp
o ns e s are th
ose th
ath
a v em i
xt ur e t oxi
ci
ty eq
u al
to th
e s um of
th
ei
nd i
vi d
u al
co
m p
ou nd
to xi
ci
ty.A
1990
s tud
y wi
th b
en zo(
a)p y
ren e(
B A P
)
showed
th
a t sy
ne rg i
s ti
cto x
i
ci
ty resp
o n ses oc cu r r ed
wh
en BA P
wa s te st ed i
n ab i
nary m i
xtu r e of p
ol y
ch
lor ina ted
ar o
m
a ti
ch y d
r o c arb
ons(
D
utk
a and K
w a n,19 82
a nd D
o n n el l y
a t al
,199 0
)
,X
PA
H
M
od
el
T h
eS PA H
M
od
el
wa sd
ev el
op
ed i
n19 95 by D
r.R
ob
ertS
wartz to e s ti m
at e th
et ox
i
ci
ty
of P A H
c o
m p
ou nd d
ata.B
ef
o r e th is, c o ntam
in ated P A H site s we r e e v alu ated b
a s ed
on ind
ivid
u alP A H
to xi
ci
ty.T h
eS P A H m
od
el
assum
e s th
atf
ield
col
le c t ed m i
xtur e s ex e rt a t oxi
c str esst
h
atis
d
ue t o th
e ad d i
tiv e t o
xi
ci
ty
of
th
ei
nd i
vid
ual
com p
ou nd
sp
r e sent.I
tis th
eref
or ep
ossi b le t op
r ed i
ct PA H
to xi
city b
y calculat ing
the t ox ic u ni
ts of thei
nd i
vi du al c om po u nd
s,i
n th is case , u si
ng
am p h ip
od da ta
.T
oxi
c unit
s(
TU
)
were cal
c ula t
ed b
e cause th
ey
canb
ede
riv ed f
rom al l
ty p
e s of
to x
i
ci
tym
e a s u r em
e nts a nd
correc ted f
o r the sol
ub i
li
ti
e s of
the c om p
ou nd
.T h
eT U is
b
ased
o n t
h
e s ol
ub i l i
ty l
im it s o
f
th
eP A H
sin
th
e ad d it
i
vit
y m
od
el
.I
t canb
ed
efi
n ed
a s th
e u ni
te
q
u al
t o the c o n c e
nt ratio n
of
a che
m i
c al
tha tk il
ls5 0
%
of
tes t sp
e ci m
ensi
n a t oxic
it
y t e s t(Sp
ro ug
e,19 70
)
.T U
wer e cal
c ula t
ed b y m
ult
ip l y i
ng
the
P A H
wa te r s ol
ub i l i
ty b y
th
eL C
5 0o
f
th
ePA H f
or e ach in
d i
vid
ual
c om p
ou nd
.T
od
a t e ,13 PA H
sh
aveb
ee n t e s ted
wit
h
p
rom i
si
ng
r e s ult
s.T h
e total
n um b
e r of
t o xic
u ni
ts of
thePA H
s(
I T U
)
wa s c alc u
l
a t ed
,assu
m i
ng
ad d it
i
vi
ty
,b
y ad d i
ng
th
ei
nd i
vi d
ual T U
s of
e a ch
c om p
o u nd
(
S
wartz et al
,19 95
)
.T h
eI P A H m
od
el
was veri f
i ed b y
com p
a ri
ng m
ortal i
ty p
r ed i
ct ed by them
od
el
w
i
th
th
em
ort al it
y
ob
s erv ed in s ed i m
e n t t oxic
i
ty
te stsi
nfi
eld
-c o
l l
e c t ed
s am p l
e s.I
t wa sf
ou nd
th
at th
em
od
el
acc ur a tel y p
r ed ic t ed
the t oxic
it
y
of PA H
-c ontam i
n a ted
s ed i m
ent s wh
enP A H
s we re th
ep
ri
nci p
al
c ont am i
n ant.H
ow e v e r, w
h
en aPA H
wa s not th
ep
ri
nci p
alc o n t a
m i
n ant, sig
ni fi
c a n td i ff
eren ce sb
e twe en th
ep
r ed i
cit e
d
and
th
e ob
ser v ed
to xic
it
y
wer eo
b
se rved
(
S
war tz e t al
, 19 95
)
.T h
erei
s an86
.6
%
c orr e sp
ond
ence and
no si g
nif
ica ntd if f
er enceb
e twe e np
r ed i
cted
to x
i
cit
y f
r om
th
em
od
el
and
a c tual
ob
ser ved
to xi
ci
ty
atP A H
co ntami
nated
sit
es whe
reM^ s e
d i m
e nt qu al ity gui d
el
in e s s u ch
a s :E P A S
ed i m
e ntQ
u al ityC
rite ria(
E
P AA S
Q
C
)
f
o r th
ep
r ot e c tio
n of b
enth ic org
anis
m
s.A pp
arentE f f
e ctT h
re shol d
(
AE T
)
,St
at e of
W
a sh i
ng
t onC l
ean up S
cr eeni
ng L
evel
s( W
A C S L
)
,S
t a t e of
W
ash i
ng
t onS
ed i m
e ntQ
ual i
ty S
ta nd
a rd
s(
W
A S
Q
S
)
,E ff
e ct s r a ng
e-
L
ow(
E R L
)
,
E f f
ec t s rang
e-h i g -h
(
E F H
)
, and Sc
re eni
ng L
evel
C
o n c e ntr ati
o n s(
S
LC
) (
S
wa rtz et al,19 9
5)
.T h i
s ap p
ro a ch i
s sti l l b
ei
ng
stud i
ed
as a way
t op
r ed ic t
P A H m i
xtu re t oxi
cit
y i
ns e
d i m
e nt, a nd
th
e r e a r e som
e u n c e rtai
nty
a nd l i m i
tati
o n s toit
.F
o r e x am p l
e, t
h i
s mod
el
d
o e s n ot tak
ei
nt o a c c ou nt the
un c ert ai
ntie s
b
e twe enm
ari
ne and
e s t u ari
ne am p h ip
od
sin
P A
H
se n sit
i
vit
y.T h
e S P AH
M
od
el
al
sodo e
s n ot a c o u ntf
o r th
ep
ot enti
al
ef f
e cts of
n atu r a
l
v a ri
ab l
e s s u ch
a s sed im
e ntg
r ai
n si
z e, total
o rg
a ni
c c a rb
o n, a nd
r ed
o xp
ote nti
al
(
S
wartz et al
,19 95
)
.M
i
cr oto x"
-Ac u t e
Si
nc ei
t'
s
d
evel
op
me nti
n19 7 9 b
yB
e ck m
a nI
n str um
e nt s, th
eM
i
c ro t ox* a s s ay
h
a sb
een us ed
t od
e t er mi
ne and m
oni
t or s oi l
a nd
s ed i m
ent, wa s t ew at er , su rf
a c e wa te r ,a n
d
al
s og
ro u nd
wate r t oxi
cit
y(
B
ul ic
h
,19 8
4 ;S
om
a su nd
o r am
et ai
,19 90
)
.S
om
e s t a te ,an
d
f
ed
er al
reg
ul
at ory
ag
encie s
us e th i
s sy
stem
i
n s c r e e ni
ng
t e s t s tom
o nit o
ren v
ir o
nm
ent al p
ol l
ut ant s(
So
m
a su nd
o r am
e t al
,19 9 0
)
.M
ic
ro t ox *is
us ed b y
th
eC i
ty
o
f C h
at ta n o og
a t o r a nk
al l
thel
o c al i
nd
u stri
al d i
s ch
a rg
e si
n o rd
e r of
r el
ati
v e t oxi
cit
yan
d i
n ord
er of
rela t
i
ve to xi
ci m p
a c t on th
eC i
ty
'
s wa s t ewa t er tr e a t
m
entp l
ant(
K
u r z,19 84
)
.M
i
c ro t ox *h
a sb
e en r e c om m
end
ed b y T h
eA m
e ri
c a nS
o cie t
y f
o rT
e s ti
ng
^
M
ic r ot ox *f
or te sti
ng tr e ated
ef f l
u e nt s,f
o rp
red i
c ti
ng
land
tr e at ab i l i
ty
o f org
a n icw
a ste san
d f
orb i
oa s se s sme nt of
w a s t ed i
sp
o s al
si
t e s(
M
u nk i
t tri
ck
e t al
,19 9 1
)
.T
ai
w anh
a sb
eg
u n t o u s e th
eM
i
c r o t ox * sy
s tem
to s cree ni
tsi
nd
us tri
al
was t ewat ers(
H
a o et al
,19 9 6
)
.M
i
cr oto x^
i
s a t oxi
ci
ty
test th
ath
a sb
e c om
e o ne of
th
e s tand
ard
t oxi
ci
ty
a s s ay
s(
T a rkp
e a et al, 19 8 6
)
.M
i
cro t ox^
m
ea su res th
ed
e c rea s ed
b i
ol
um i
n e s c e nce of
th
eb
a cte ri
aVi b
r iof
i
s ch
eri i
n th
ep
re s e n c e of
a t oxi
c ag
ent.T h
el
u ci f
e r ase e n zy
mef
r om th i
sb
ac te ri
au ses re
d
u ced f l
avi
n a nd
o xyg
en top
r od
u c el i g h
t(
C h
ang
et al
, 19 8 1
)
.A p h
ot om
et erm
e asu r e s th
ed
e c rea s e of l i g h
t a nd g i
ves a r e ad i
ng
of
th
ep
er c e ntl ig h
tl
o ss,(
G
am m
a)
.A
nE f f
e ctC
o n centr ati
o n o rE C
50i
s th
e np
r od
uced b
yg
r ap h i
ng
th
el
og
of
th
eG
am m
ar es
p
on se ver su s th
el
og
of
th
e s am p l
e con centr ati
on .T h
eE C
5 0
i
s th
e ch
em i
c al
o re nv
i
r onm
e n t al
s am p l
e c onc entr a ti
o n ca usi
ng
a5 0
%
d
e cr e as ei
n th
eM
i
c r o tox *R
e ag
e n tl i g h
t outp
u t u nd
erd
efi
ned
co nd i
ti
o ns of
ti m
e a nd
te st temp
er atu r e(
M
i
c rob i
cs,19 8 9
)
.T h
ed
e cr ea sed i
n t en si
ty
of b i
ol
um i
ne s ce ncei
sp
r op
o r ti
o n al
to th
e to xi
ci
ty of
sp
eci f i
cc
h
em i
c al
s or e n vi
r o nme nt al
s amp l
es.T h
e sm
al l
e r th
eE C
5 0,
t
h
em
o re t oxi
c th
ec
h
em i
c al
o r e n vi
ronm
e nt al
s am p l
e .R
ese ar ch h
a s sh
own th
a t th
e r e sul
ts of
M
i
cro t ox *are co
m p
arab l
e t o o th
era s sa
y
s.C
a n na-
M
i
c
h
a el i d
oui
n19 9 3
, c om p
ared
M
i
cro t ox *t o a
g
a s ch
rom
a t og
rap h
y"
fi
ng
erp
ri
n t sc re eni
ng
t e s t" .H
e c om p
a red
the to xi
c ef f
ects of 7 m i
xtu r es of
vol
ati l
eo r
g
ani
cp
ol l
ut ants(
V O C
)
. Ind
o ing
so,h
ef
ou nd
th
at theM
i
c r ot ox*
-m
•
2 2 0 ti
m
e sm
o r e s e n sitiv e th
a n theG C /
FI
D fo r a r om
aticm
ix tu r e s. T h is te st wa s als of
ou nd m
or e s e n si
tiv e
fo
rd
etec ti
ng
r eg
ul
a rp
etrol
eum
and d ie s e
l
t oxic
i
ty.R
e s e ar ch h
a s al
s ob
e end
o ne c om p
ari
ng
M
ic r
o to x* tof
ish,D
ap h
ni
a , and
othe rm ic r
ob i
al
t oxi
ci
tym
eth
od
s.M
ic r oto x "v s. F ish. T he
2
4
-to -
9 6
-h
r f is
h
LethalC
o n c e ntr ation 50
(
LC
5 0
)
acuteb io a
s say i
s one of
th
em
o st wi d
el
y used
a cu te toxi
ci
t y t e s ts.T h
e reh
a sb
e en ext ensiv
ere se a r c
h
sh
owi
ng
th
atM
ic r
o tox *is
a s sensit
iv
e a s th i
s tes t, e sp
e ci
al l
y wh
en usi
ng
th
ef la t
h
e ad m i
nnow(
Ind
ora to et al
,C
u rti
s et al
,19 82
,E
.V
.S
.C
onsul
t ants,
19 8 9
,B
ul i
ch
e t a
l
,19 80
,M
unk it t
ric
k
e t al
,19 9 1
)
.I
n19 9 3
,D
eZ
wa rt and
S l
o of
d
ete rm in
ed
the t oxic
i
ty
of
15
ch
em i
c als
b y
co
m p
a ri
ng
theM
ic r
o to x *a c ute a ss a
y
to that of g
ree n al g
ae,D
ap h
nia
, a nd fi
sh
.T h
eir
r e su
l
t s sho wed
th
eM
ic
r ot ox a s say
t ob
e c om p
arab le
to the o ther t e s ts, e sp
eci
al l y
th
eacute t ests c on
d
uc ted
wi
th fi
sh
.D i f f
e r e nc e sb
e twe en th
efi
sh
a nd
M
i
cr o t ox®
s ens
i
tiv
i
tyw
ere wi
th in one o rd
er of
mag
ni
tud
e .T h
eC i
ty
of C h
at t a n oog
ah
a s n ot onl y f
ou nd
th
e t e s t t obe
m
o re se nsi
tiv
e,b
uta
ls o
l
e s s exp
ensiv e and
ti m
e c onsum i
ng
.C
om p
ar ed
t o th
eS
tate of T
e n n ess ee, w
h i
ch
a v e r a
g
ed 10
o rle s s
b io
a ssays ay
e ar usi
ng
the LC
5 0fi
sh
t oxi
cit
y
te st,C h
a t ta n oog
ac on
d
uc ted 160
t e s t s usi
ng
M
ic r
otox*in
l
e ss tha n
6 m
onth
s ti m
e(
K
u r z,19 8 4
)
.M
i
c r otox'^ vs.D
ap h
nia
.T h
e c om p
ara tiv
e s e n sit
iv
it
y
of
M
ic
ro t ox® an
d
D
ap h
ni
ais
d
ep
e nd
e nt o n th
e c om p
ou nd
t e s t ed
.M
ic r
o t ox®
i
s c om p
a r ab l
e toD
ap h
nia
e x a
m
p le ,D
ap
hnia ism
o r e s e nsitiv e t o am m
onia, cya nide , he x a ch lo r o e thi a n e ,p
e n tad i
one, and
s od i
um i
au ry l
.M
i
cro t o x *, o n t
h
e o th
erha n
d is
m
o re se nsi
tiv
e toc
h l
orof
o rm
, styr en e a nd h i g h l y
s ub
sti
tut ed
org
a nic s
(
M
u nk i
t tri
ck
et al
,19 9
1)
.M
i
c r o to x "vs. o the r
M
i
c rob ia
l
to xic
i
ty m
e th
od
s. In 19 8 1, D utk
a a nd K
wanc o
m p
a red
M
i
c r ot ox* t o thr ee othe rm
icr ob ial
to xic
ity te st s:Sp
irill
um
v ol
uta n s,P
seud
om
a sf
lu
o re s c e n s, and A
erom
ona shy d
rop h i l
a .T he
M
ic r o
t ox *te st
p
r o ved
t ob
et
h
em
o s t s ensi
tiv
e t o th
e ch
em i
cal
s tes ted
(
wi
th
th
e e x c ep
ti
o n of H g
'
^' *"
,
N i
"^" ^
,
P b
"^ "*"
,
an
d N
-N i
tr o s od i
e th y l
am i
ne)
.M
ic r
o t ox ®wa s
p
rov e n tob
e am
o r e r el i
ab l
e a nd
s ensi
tiv
ete s t
w h
e n com p
a red
t o th
e r es az u ri
n r ed
uctio
n(
R R
)
m
eth
od
and
th
ed i
ssolv
ed
oxyg
end
ep l
e ti
on(
D O D
)
t e s t(
G
r een e e t al
,19 85
)
.M
i
cro t ox *a
l
s o tak
e sl
e s s ti m
e th
an o th
e rb i
oa ss ay
s and h
a s a s t a ti
s tic
al
ad
vant ag
e sin
c e r esul
ts ar eb
a sed
o n usi
ng
over10
^b
acte ria
.W
h
enM
cF
et er s t e s t ed
th
e two org
ani
sm
T
ch
anp
ro c ed
u r e(
al g
ae and
lu
m i
nes c e ntb
ac te ri
a)
wi
th
M
i
c r oto x *h
ef
o u nd
M
ic r o
t ox'
t o
b
e am
ore s ensi
tiv
e te s t .R
el
ativ e sensi
tiv
i
ty t oM
i
cr ot ox *h
a sb
e en c omp
ared
t o a va ri
e ty of b i
o a s s ay
m
e th
od
sf
or th
e toxi
ci
ty
of
ch
em i
cal
s, s ed i m
ent s, and
com p l
ex ef f l
uent s.W
h
enc o
m p
ared
t of i
sh
and D
ap h
ni
a stud i
e s, s ever al
r e s e arch
e r s r each
ed m
a ny
of
th
e s am
em
aj
o r c oncl
u si
ons.M
u nk i
t tri
ck
s s um m
a ri
z e s th
e s e con clu s
io n s
asf
ol l
ow s(
M
u nk i
t tri
ck
e t al
,19 9 1
)
:•
O
rg
a nic
s -M
i
cro to x *is
as s e nsi
tiv e
a s oth
er a s say
sf
o r th
em
eas u rem
ent of p
u r eo r
g
a nic
s.M
ic r
o to x *4
»
^>>Vi|
m
ulti
ch lo ri
n ated b
e n z e n e s, ph
e n ol
s, a nd
eth
anol
s.T h
e sys tem i
sl
es s s en si
ti
v e t oc
y
ani d
e, ch l
orof
o rm
, o rp h
e n ol
.• In o r
g
a ni
cs -M
i
cr oto x *i
s n ot a s s e n si
ti
v e a s oth
er te st s tom
o s ti
n o rg
a ni
c s . Iti
sh
owever ,m
ore se nsi
ti
ve t om
ercu ry
, ars eni
c , a nd
c ob
al
t.•
M
u ni
cip
al
W
as te s -M
i
c ro to x*
i
s af
av o r ab l
eb i
oa s s ayf
o r mo ni
to ri
ng m
u ni
ci p
al
was tes.
• In
d
u stri
al
W
a stes -M
i
c ro to x® c a n al
s ob
e used
t o s c ree nf
o r th
e r el
ati
v e to xi
ci
ty
of
c o
m p l
ex ef f l
u e nts.•
S
ed i m
e ntC
ont am i
na ti
o n -T h
e s e ns
i
ti
vi
ty
of
M
i
cro to x *va r
i
e s wi
th
c ont am i
n a nte x tra c t
i
o n te ch
ni q
u e(
M
u nk i
t tri
ck
et al
,1 9 9 1
)
.M
i
cro t ox ®no t o n
l y h
a sb
e e n u s ed
to te sti
nd i
vi d
ual
to xi
ca nt s,b
u th
as al
s ob
e e nu s e
d
to te st s om
em i
xtu res.M
a ny h
avef
ou nd m i
x tu r e s t ob
e the s um
of
th
ei
r t oxi
cp
art s or a sb
ei
ng
add i
ti
ve(
S
el l
er s and R
am)
.H
ow ever,
i
th
a sb
e end i
s c o v ered
ov ert
i m
e th
a tm i
xtu r e s can r eact syn e rg i
sti
cal l y
a nd
antag
o ni
sti
cal l
y a s wel l
.In
C
an na-M
i
c
h
a el i d
ou 's stu
d y
of
v ol
a ti l
e org
ani
cp
ol l
uta nts,h
ef
ound
m i
xtu re s t o sh
ow ad d i
ti
ve eff
e c ts(
C
an n a-M
i
ch
a el i d
ou ,
19 9 3
)
.B
a s ed
o n exp
o su ret
i m
e, a nd
th
ep
r op
or ti
o n of
ch
em i
cal
com p
on e nt,h
e avy
m
etal
m i
xtu r e s w er ec a t e
g
o ri
zed
a s sy
n e rg i
s ti
c , antag
oni
s ti
c, or ad d i
ti
ve.T h
e r esul
t s sh
owed
th
a t af
t er a4 5
-m i
nut e exp
o s u re,C d
-Z
ni
nt eracte
d
sy
ne rg i
s ti
cal l y
,C d
-N i i
nt eracte
d
ant ag
oni
sti
cal l y
,a n
d C
o -N i
a nd A
s-Pb i
nt er a c t e
d
ad d i
ti
vel
y(
S
el l
er s a nd R
am
)
.W
al k
e r s tud i
ed
th
et o x
i
ci
ty
of
zi
n c a nd p
e nta ch l
o r op h
ol
(
P C P
)
i
n19 87
a nd d
ete rm i
n ed
th
at th
ei
n t e r a cti
o n sb
e twe e n th
e tw o w e r e add i
ti
v e.if^
m
D
utka a nd K wa n(
1 9 8 1)
als o te stedm
ixtu r e s w ithM
i
c r oto x®
. T he
y, l i
k
eC
an na-M
i
ch
a el i d
ouf
ou nd
th
a tm i
xt ure sp
rod
uc ed
re sp
o nses th
a tw
e r e sy
ne rg i
s ti
c ,a nt a
g
oni
s ti
c, or add i
ti
v e ,b
a s ed
o n th
e co n c e ntr ati
o n s of
th
ei
nd i
vi d
u al
ch
em i
c al
a nd
t
h
ei
ncub
ati
o n o r co nta ct ti m
e.F
or e xam p l
e,d i f f
e r e nt c on c e ntr ati
o ns of p h
e n ol
w er ec o
m b i
n ed
wi
th
the s am
e c o n c e ntr ati
o n of s od
ium l
a u ry l s ulp h
ate.C
o ntr ol
s sh
ow ed
th
atp h
e n ol h
ad
ad
ec re a s ed
t oxi
c ef f
e ct wi
th i
ncre ased i
ncub
ati
on ti m
e; th
e t oxi
c ef f
e ct of
so
d i
um
l
au ry l
sul p h
a t e w a s s t ab l
e ov e r a15
-m i
n ut e
p
e ri
od
.W
h
e n th
e two wer ec o
m b i
ned
,h
owe v e r, th
e r e wa s ani
ncre a sei
n t oxi
c ef f
ect. Th
e2 0 p p m p h
e nol
co
m b i
na ti
o n sh
ow ed
a n ad d i
ti
v e r e sp
o n s e, wh i l
e2
.5 p p
mp h
e nol
com b i
nati
o n wi
th 0
.
5
p p m
of
s od i
um l
a u ryl
sul f
at e sh
owed
a synerg i
s ti
c r esp
o nse.A
n a nt ag
o ni
sti
c r e sp
o nsew
a s s e en wh
enh i g h
c o n c e ntr ati
ons of H g
"^"*"
(
0
.0 7 5 p p
m)
we re c omb i
n ed
wi
th Pb
^ "
^
(
5
p p m
)
.M
i
c r o t ox*
i
s s ensi
ti
ve, r ap i d
, a nd i
n exp
e nsi
v e rel
ati
ve to oth
e r assays(
Rib
oan
d K
ai
se r ,19 87
)
.T h
e t a sk
of b
r e ed i
ng
t e st org
a ni
smsi
s el i m i
na t ed i
n th i
s sy
s t em
.A
ssay
s th
at t ak
e two or th
reed
ay
s u si
ng
s t and
ard
t oxi
ci
ty
t est c anb
e cond
uc t ed i
nf i
veto t
h i
r ty m i
nu te s wi
th
c om p
a rab l
e r e sul
ts(
D
eZ
w art,1 9 8 3
)
.M
i
cr otox * -C h
roni
cT
h
eC h
r o ni
c t oxi
ci
ty t e s ti
s a n ew ad d i
ti
o n to th
eM
i
c r oto x *a s sa
y
s
y
stem
.T h
er ef
o r e, th
e r eh
a s n otb
eenm
u ch p
ub l i
sh
ed
c o nce r ni
ng
th i
s t e s t.C h
r o ni
cto x
i
ci
ty
t e sts a r e u s ed
t op
r ed i
c t ad
vers eb i
ol
og i
c al
ef f
e c t s th
a t r e sult w
h
e n an o rg
a ni
sm
i
s e xpo s ed
o v e ri
ts e ntir el if
e cycl
e(
M
i
crob i
c s, 19 96)
.L i k
e th
e a c ute t est, t
h
em
M
i
cro to x *c
h
r o ni
c to xi
ci
ty t esth
asb
e en sh
own t ob
e a s s e n si
ti
v e o rm
o r e s en si
ti
v e th
a no t
h
er ch
r oni
c t e s t s(
M
i
crob i
cs,199 6
)
.T h i
s t e s t al
so tak
esl
es s ti
me th
an oth
ers,
2 2
h
ou r s.T h i
s te s t u s e s th
eM
od
el 5 0 0 A
nal y
zer t od
ete c tl i g h
t loss.T
o a nal y
ze th
er e s ults o
f
th
eM
i
c r oto x ®chr o n
i
c to xicity te st, th
e s of
twa r e system T
o xC
al
c™
,
i
s u s ed
.It
i
ncl
ud
e s su ch
s tati
sti
cal
op
ti
o n s a s: ap
ar am
etri
ch
yp
o thesi
s te st, a nonp
a ram
e tri
ch y p
oth
e si
s te st, a nd p
oi
nt e sti m
ate s ca nb
e u s ed
tod
et erm i
n e th
eN
oO b
ser v ed E f f
e c tC
o nce n tra ti
o n(
N O E C
)
a nd
th
eL
owe stO b
s e r ved E f f
ectC
o n c e ntra ti
o n(
L O E C
)
.T h
eN
O
EC i
s th
eh i g h
e st c o nce ntr a ti
on te st ed
th
at c au se s n o s tati
sti
c al l
ym
eas u rab l
e ef f
ec to n t
h
e te st sy
stem
. Th
e LO E C
, on t
h
e oth
e rh
a nd
,i
sd
i el
owe st conc entra ti
o n tes t ed
t
h
a t c aus es a stati
s ti
c al l y m
e asu rab l
e ef f
e c t o n th
e te st sy
stem
(
M
i
c rob i
c s,19 9 6
)
.O b
j
ecti
v esT h i
s r e s ear ch
wa sd
e si g
ned
t o c om p
a re th
e t oxi
ci
ty
of
PA H
s a si
nd i
vid
u al
c om
p
ou nd
s a nd
a sm i
xtu r e s, a s th
ey ar ef
o u nd i
n th
e en vi
r o nm
e nt.P A H m i
xtu r e to xi
ci
ty
h
as notb
e e n stud i
ed
a sf
ul l y
a s th
at of i
nd i
vi d
ual
com p
ou nd
s.T h
eM
i
c ro t ox*
to x
i
ci
ty
tes t,b
o th
a cute and
ch
roni
c , wer e us ed
t od
e te rm i
n e t oxi
ci
ty
of PA H
s and
th
ei
nt er a cti
o ns of
th
em i
xtures.T h
e sp
e ci f i
c ob
j
ecti
ves of
th i
s r e s ea rch p
r oj
e c t w ere t o :1
.A
s se s s th
e a c u te and
ch
r o ni
c toxi
ci
ty
of P A H
si
nd i
vi d
ual l y
a nd
a sm i
xtu r e s usi
ng
t
h
eM
i
c r ot ox *a s sa
y
sy
s t em
.^,
g
^
2. T o dete r
m
in e i f the to x icity r e spo n s e o f P A Hm
ix tu r e s is pr ed ictab le ba s ed o n datag
e n era t ed
wi
th i
nd i
vid
ual
c om p
ou nd
s u si
ng
th
e ad d i
ti
v em
od
el
(
S
wartz e t al
,19 9 5
)
.3
.T
o e v al
u ated i f f
e r e n c e sb
etwe e n acute a nd
ch
ro ni
c t oxi
ci
ty a ssay r e s ul
t s.m
M
A T E R I A L S A N D
M
E T H O D S
C h
em i
c al
sE i g h
tP A H
s wer e eval
u at ed by
th
eM
i
cro t ox '*b i
o a s s ay
: nap h
th
al
ene, ch
ry
se ne,p y
r e n e, a nth
r a c e n e,f l
u o r a nthr e n e,f
lu o r e n e, ace n ap h
th
e n e(
A l d
ri
ch C h
em i
c al C
o . ,M
il
w auk
ee ,W
I
)
and p h
ena nth
re ne(
S ig m
aC h
em i
c al C
o.,S
t.L
oui
s,M
O
)
,(
F i g
u re1
)
.P
A
H
s am p l
ep
r ep
a r ati
onS
ol
uti
o n s of i
nd i
vid
ual P A H
s wer ep
r ep
ared b y d i
s s ol
vi
ng
e ach P A H i
n t wos e
p
a ra te sol
v e nts,m
eth
anol
and
a2
%
N
aC l
s ol
uti
o n a t th
ei
r r e sp
ecti
v e wa t er s ol
ub i l i
ty
c onc ent rat
i
ons.F
or2 % N
aC l
s ol
uti
ons, satu ra ted
s ol
uti
ons of
ea ch i
nd i
vi d
u al P A H w
e r ep
r ep
a r ed b
yf i
r st weig h i
ng th
e ch
em i
c al
t o atl
e as t1
.5
ti m
es th
e sta nd
ard
sol
ub i l i
tyi
n10
m L
of d i
s til l
ed
,d
ei
o ni
z ed
w ate r. Th
e s atu r at ed N
aC
l sol
uti
o n s we r e mi
x ed
well
a nd
a
l l
owed
to s e ttl
e .S
up
ern a t ant wa s r em
oved
t om
ak
e th
e s am p l
em i
xt ure.S
ol
u ti
ons of
P A H
and m
eth
anol
we rep
r ep
ar ed b y d i
s sol
vi
ng
th
ew
ei g h
t of
th
e ch
emi
c al
ne c es s ary top
rovi d
ew
a te r s ol
ub i l i
ty
of
th
ePA H i
n10 m l
of m
eth
a nol
.T
o xi
ci
ty c om p
ari
son s werem
ad
eb
etw eenN
aC l
s ol
uti
o n s and m
e th
a n ol
s ol
uti
on sf
or e a ch i
nd i
vid
u al P A H
.M
i
croto x'
-
A
c ute to x
i
ci
t yT h
eM
i
crotox a cut e t oxi
ci
ty
te s t eval
uate s t oxi
ci
ty b y
c om p
ari
ng
th
el
um i
n e sce nce of
Vi b
ri
ofi
s ch
e ri
al
o n e to th
el
um i
n e s c e n c ei
n th
ep
r e sen c e of
ap
a rti
cul
a r ch
em i
c al
or e n vi
r o nm
ental
s am p l
e.T h
i
s as say
canb
e c om p l
eted i
n5
to3
0
m
inute s.T h
eM
i
cr oto x®
sys t e