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The photo-induced toxicity of polycyclic aromatic hydrocarbons to larvae of the fathead minnow (Pimephales promelas )
Chemosphere, Vol.16, No.7, Printed in Great Britain
pp 1395-1404,
1987
0 0 4 5 - 6 5 3 5 / 8 7 $3.00 + .OO P e r q a m o n Journals Ltd.
THE P H O T O - I N D U C E D T O X I C I T Y OF P O L Y C Y C L I C A R O M A T I C H Y D R O C A R B O N S TO LARVAE OF THE F A T H E A D M I N N O W (Pimephales promelas) J a m e s T. Oris* and J o h n P. Giesy, Jr. D e p a r t m e n t of F i s h e r i e s and W i l d l i f e M i c h i g a n State U n i v e r s i t y East Lansing, MI 48824 USA ABSTRACT The toxicity of 12 p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s to larvae of the fathead m i n n o w in the p r e s e n c e of s i m u l a t e d s u n l i g h t was examined. A measure of r e l a t i v e t o x i c i t i e s of the toxic c o m p o u n d s is d e s c r i b e d in w h i c h waveband radiation intensity, molar extinction coefficients, and molar body concentration of PAH are considered. In addition, a structure-lethality r e l a t i o n s h i p has b e e n developed, b a s e d on m o l e c u l a r s t r u c t u r e and p h o t o c h e m i cal properties, that c l a s s i f i e s c o m p o u n d s as b e i n g p h o t o t o x i c or n o n - p h o t o toxic. INTRODUCTION Previous studies (10,ii,12) have d e m o n s t r a t e d that anthracene, a fused, linear 3-ring polycyclic a r o m a t i c h y d r o c a r b o n (PAH), is a c u t e l y toxic to juvenile s u n f i s h u n d e r l a b o r a t o r y and field c o n d i t i o n s of solar ultraviolet radiation (SUVR), and that this t o x i c i t y can be p r e d i c t e d from k n o w l e d g e of SUVR intensity, a n t h r a c e n e c o n c e n t r a t i o n and p h o t o p e r i o d duration. Because PAH b e l o n g to a large class of compounds, it is d e s i r a b l e to determine the extent and l i k e l i h o o d that PAH other than a n t h r a c e n e are c a p a b l e of e l i c i t i n g photo-induced t o x i c i t y to fish. M a n y PAH can be c o n s i d e r e d as p o t e n t i a l l y p h o t o t o x i c (2), and there have b e e n reports d e s c r i b i n g the p h o t o - a c t i v i t y of PAH to m a m m a l s (4) and a q u a t i c o r g a n i s m s (6). There are no known reports, however, concerning the range and extent of the p h o t o - i n d u c e d t o x i c i t i e s of PAH to fish. The p r e s e n t study was c o n d u c t e d to : i) d e t e r m i n e the relative photo-induced toxicities of a v a r i e t y of PAH to fish, and 2) develop a structure-lethality r e l a t i o n s h i p to e s t i m a t e the p h o t o - i n d u c e d t o x i c i t y of a compound.
*To w h o m c o r r e s p o n d e n c e may be addressed, University, Oxford, OH 45056 USA.
1395
at D e p a r t m e n t
of
Zoology,
Miami
1396
M A T E R I A L S AND M E T H O D S C o m p o u n d s and T e s t S o l u t i o n s Anthracene acridine
(ANT),
(ACR),
(PER),
benzo(a)anthracene
benzanthrone
(BAN),
benzo(g,h,i)perylene
benzo(e)pyrene purification without
(BEP),
filtered,
purification.
aerated technique
Appropriate desired
tap w a t e r to
dilutions
concentration
solutions
were
(9).
study
The
organisms
(BGP),
pyrene
phenanthrene
available commercially.
further
coating
and
(BAA),
(PYR),
in
the
shell
tests
(9). the
to
achieve
in
in
all
a
test.
C o n c e n t r a t i o n s of PAH
fluorescence
aqueous detection
was d e s i g n e d such that the final PAH c o n c e n t r a t i o n s Therefore,
PAH c o n c e n t r a t i o n s
weights,
n o r m a l i z e d to the least w a t e r soluble compound,
concentrations
charcoal
w e r e m a d e from these s t o c k s o l u t i o n s
on
PAH
in
of each PAH w e r e o b t a i n e d u s i n g a
a v o i d the use of c a r r i e r s o l v e n t s
w o u l d be equimolar.
nominal
highest
(8) were used
solutions,
selected a
(BAP),
the
All c o m p o u n d s except BAN aqueous
(BBA),
perylene
benzo(a)pyrene
d e t e r m i n e d by r e v e r s e - p h a s e H P L C and
the
(DBA),
(PHE) w e r e o b t a i n e d at
Saturated (14),
benzo(b)anthracene
dibenz(a,h)anthracene
b a s i s of p u b l i s h e d b i o c o n c e n t r a t i o n b o d y b u r d e n of I00 n M / g was
values
selected.
of PAH in w a t e r are p r e s e n t e d in Table
and
BGP.
in
in w a t e r
the were
molecular
On this basis,
Nominal
and
actual
i.
O r g a n i s m s and B i o a s s a y P r o c e d u r e Larvae of the f a t h e a d m i n n o w
(Pimephales promelas)
w e r e o b t a i n e d four days
post-hatching from the M i c h i g a n D e p a r t m e n t of N a t u r a l R e s o u r c e s Surface Water Q u a l i t y Division. L a r v a e w e r e t r a n s f e r r e d by p i p e t t e to a small f l o w - t h r o u g h a q u a r i u m filled w i t h c h a r c o a l were
maintained
filtered,
a e r a t e d tap w a t e r at 24 C.
were fed n e w l y h a t c h e d b r i n e shrimp n a u p l i i ad libitum twice a day. On the s e v e n t h transferred
by
The larvae
in the f l o w - t h r o u g h a q u a r i u m for two days a f t e r t r a n s f e r
pipette
to
s o l u t i o n or d i l u t i o n water.
and
(Artemia salina; M e t a f r a m e Corp.) day post-hatching, larvae were
300 ml Pyrex dishes c o n t a i n i n g
150
ml
of
PAH
T r e a t m e n t s c o n s i s t e d of 20 to 25 larvae per dish
and two d i s h e s per PAH examined, i n c l u d i n g two d i s h e s containing dilution water as S U V R - o n l y controls. Dishes w e r e c o v e r e d w i t h a l u m i n u m foil and the larvae
a l l o w e d a 24 h
p r e - i n c u b a t i o n p e r i o d in the a b s e n c e of
SUVR.
After
the p r e - i n c u b a t i o n period, larvae w e r e fed b r i n e shrimp ad l i b i t u m for 0.5 h, all solutions w e r e t h e n r e p l a c e d and the dishes w i t h larvae w e r e placed in r a n d o m p o s i t i o n s u n d e r a l a b o r a t o r y s y s t e m light b a n k w h i c h s i m u l a t e d natural sunlight
(i0).
Light
was
filtered
w i t h a 5 mil t h i c k n e s s
of
Mylar R
to
eliminate >99% of the r a d i a t i o n of w a v e l e n g t h s s h o r t e r than 315 nm. SUVR intensities were m o n i t o r e d as a p r e v i o u s study (i0) and for all exposures were U V - B (290-336 n m ) = 20 u W / c m 2 and U V - A (336-400 n m ) = 95 u W / c m 2. After the initial p r e - i n c u b a t i o n , s o l u t i o n s w e r e c h a n g e d at 12 h intervals. Larvae were fed b r i n e shrimp ad l i b i t u m once a day for 0.5 h prior to changing solutions. Bioassay dishes w e r e e x a m i n e d for larval m o r t a l i t y at least four times daily. T e s t s w e r e c o n d u c t e d until 100% m o r t a l i t y was a c h i e v e d or for a maximum of 96 h, w h i c h e v e r came first. PAH c o n c e n t r a t i o n s in w a t e r were
1397
determined more
for
at the b e g i n n i n g initial
concentrations measured
in
occurred,
effect
exhibited
of a b i o a s s a y
12
water
concentrations
mortality any
and
h
are
reported
in the
initial
due to PAH toxicity
in the a b s e n c e
in the dark.
at the end of a test
fish l a r v a e
samples
i.
were
Nominal organisms. based
72 +/-
and
as the
and
on
water
of
in
in
i00 n M / g
water
bioconcentration
that no compound(s)
Water
actual (nM/g)
526
525
14.7
5.4
ii1.8
PHE
14.5
i0.0
293.2
BAA
1.9
1.8
6.7
BBA
1.1
1.9
N
DBA
0.25
0.15
0 94
PYR
7.21
25.6
BAP
0.82
5.6
BEP
0.43
2.9
6 0
PER
0.79
1.7
7 5
0.20
and
detected.
D.
87 1 486
7
3.7
0.15
52.9
49.5
0.0
in
120.6
ANT
31.6
and
compounds
Organism
(ug/L)
SUVR-only
were spiked
factor, was
(ug/L)
BAN
study
from
for all
actual
BGP
where assess
larvae
of PAH
nominal
ACR
to
the
this
in all
of PAH
were
BGP,
indicates
Water Compound
of PAH
recoveries
PAH
between In tests
No PAH t e s t e d
concentrations
PAH b o d y - b u r d e n s N.D.
mean
12 h old solutions.
Percent
once
period.
(mean +/- SE).
solubility
weights.
and at least
test
to the PAH was p e r f o r m e d
Concentrations
12%
12 h,
the
geometric
of SUVR.
(9).
actual
Nominal
molecular
during
a 96 h d a r k e x p o s u r e
determined
Table
at zero and
solutions
N.D.
N.D.
control
Efficacy The similar
and R e l a t i v e efficacy to that
of
to q u a n t u m
of
mortality
larvae.
The rate
each
of M o r g a n
analagously larval
Potency
Factor phototoxic
compound
and W a r s h a w s k y
yield versus
(6).
in p h o t o c h e m i s t r y the rate
of m o r t a l i t y
versus
of q u a n t a
was
determined
Efficacy
in
( ~ )
and is a d e s c r i p t o r absorbed
a is
of the rate
by a compound
time can be d e s c r i b e d
manner defined
by e q u a t i o n
in 1.
the
1398
n
d(%Mortality)
[(I°lTl ) ( ~kb Ca) ] -~
=
A.~
(i)
dt A
where:
= the average = waveband
n u m b e r of q u a n t a a b s o r b e d p e r t i m e ( U V - B = 3 1 5 - 3 3 6 nm, U V - A = 3 3 6 - 4 0 0
VIS2=420-450
nm, V I S l = 4 0 0 - 4 2 0
nm, and
nm).
Iol
= waveband
Tk ~
= o p t i c a l t r a n s m i t t a n c e of e p i d e r m i s for w a v e b a n d (15). = mean molar e x t i n c t i o n c o e f f i c i e n t of c o m p o u n d in octanol
radiation
waveband
intensity
= depth
Ca
of l a r v a e = 0.2 cm) = molar concentration
n
= n u m b e r of w a v e b a n d s = time
for
(L/mole/cm) (8).
b
t
(uW/cm2)(9).
of
radiation
penetration
in o r g a n i s m
of c o m p o u n d in o r g a n i s m
(b = avg.
diameter
(moles/kg).
(s)
= e f f i c a c y of c o m p o u n d I n t e g r a t i o n of e q u a t i o n
1 yields:
%Mortality =
A. ~
t
+
B
(2)
w h i c h is in the form of a l i n e a r e q u a t i o n where, time,
B is t h e
intercept
and
A.~
is t h e
in p l o t s of % M o r t a l i t y v e r s u s
slope
of t h e
line.
Efficacy,
therefore, can be d e t e r m i n e d a l g e b r a i c a l l y from k n o w l e d g e of the c a l c u l a t e d A and t h e s l o p e of t h e % M o r t a l i t y v e r s u s t i m e c u r v e for e a c h i n d i v i d u a l compound. The Relative P o t e n c y F a c t o r (RPF) is an i n d e x of t h e r e l a t i v e e f f i c a c y of a c o m p o u n d c o m p a r e d to t h e l e a s t e f f i c a c i o u s of t h e c o m p o u n d s tested.
Therefore,
e f f i c a c y is a u n i ~ l e d e s c r i p t o r of the p h o t o t o x i c a c t i v i t y
of a c o m p o u n d
and RPF
gives
compounds used
in this
study.
a relative
i n d e x of a c t i v i t y
for t h e g r o u p
of
RESULTS S i x of t h e 12 c o m p o u n d s t e s t e d e x h i b i t e d a c u t e p h o t o - i n d u c e d toxicity ( T a b l e 2). Median-lethal-time (LT50) v a l u e s r a n g e d f r o m 0.83 h for BAN to 65.1 h for BAA. O n t h e b a s i s of RPF, B A N e x h i b i t e d t h e g r e a t e s t a n d BAP e x h i b i t e d t h e l e a s t l e v e l of c o n c e n t r a t i o n a n d a b s o r p t i o n - s p e c i f i c photoi n d u c e d t o x i c i t y a m o n g t h e c o m p o u n d s t h a t w e r e p h o t o t o x i c ( T a b l e 2). Of the r e m a i n i n g six compounds, four c o m p o u n d s e x h i b i t e d no e f f e c t c o m p a r e d to SUVRonly controls (BEP, DBA, PER, PHE) a n d t h e o t h e r t w o c o m p o u n d s e x h i b i t e d a m a r g i n a l l e v e l ( <20% m o r t a l i t y in 96 h) of p h o t o - i n d u c e d t o x i c i t y (BBA, BGP). M o r t a l i t y in S U V R - o n l y c o n t r o l s was less t h a n 5% in a l l tests. C o n t r a r y to the o r i g i n a l d e s i g n of this experiment, e q u i m o l a r b o d y - b u r d e n s of PAH w e r e not o b t a i n e d (Table i), e v e n t h o u g h PAH c o n c e n t r a t i o n s in w a t e r w e r e r e l a t i v e l y c l o s e to the s e l e c t e d n o m i n a l concentrations. The e q u a t i o n u s e d to c a l c u l a t e
1399
A
and
~
takes
in the
animal,
so e v e n t h o u g h the a c h i e v e m e n t of e q u i m o l a r b o d y - b u r d e n s was desirable,
it was
not entirely
into
account
necessary.
the
concentrations
of c o m p o u n d
Since BBA was not detected
in f i s h t i s s u e
( T a b l e i)
this c o m p o u n d was not u s e d for further analysis.
Table
2.
Tabulated absorbed
values
of m e d i a n
( A ), e f f i c a c y
( ~
for a l l p h o t o t o x i c c o m p o u n d s . o r d e r of r e l a t i v e potency.
Compound
LT50
lethal
times
), a n d R e l a t i v e Compounds
A
(LT50), Potency
are listed
~
average Factor
quanta (RPF)
in d e c r e a s i n g
RPF
(h)
BAN
0.83
0.183
5.46
E-2
337.1
PYR ACR
3.20 4.30
0.372 0.397
1.45 E-2 7.00 E-3
i00.i 48.3
ANT
15.75
0.218
3.12
E-3
21.5
BAA
65.09
0.i00
2.38
E-3
16.4
BAP
40.05
2.913
1.45 E-4
1.0
C o r r e l a t i o n a n a l y s e s w e r e p e r f o r m e d w i t h the m e a s u r e s of m o r t a l i t y and the chemical
characteristics
relationship. coefficients,
The
of
the
factors
compounds
considered
to
determine
included
first and second order m o l e c u l a r
a
structure-activity
octanol-water
c o n n e c t i v i t y indicies,
partition energies
of l o w e s t s i n g l e t e x c i t e d state splitting, e n e r g i e s of l o w e s t t r i p l e t excited state splitting, the difference between singlet and triplet splitting energies, p h o s p h o r e s c e n c e lifetimes, a v e r a g e m o l a r e x t i n c t i o n c o e f f i c i e n t s in octanol for each of the four w a v e b a n d s examined, and the s u m m e d total of all molar extinction coefficients across all wavebands significant univariate correlations were observed between of m o r t a l i t y Because
(LT50,
RPF,
no u s e f u l
A. ~)
( 3 1 5 - 4 5 0 nm). No any of the measures
and any of the a b o v e c h e m i c a l
univariate
predictive
relationships
characteristics. were
observed,
d i s c r i m i n a t e a n a l y s e s (13) w e r e used to c l a s s i f y the c o m p o u n d s as b e i n g either p h o t o t o x i c or non-phototoxic. A l l c o m p o u n d s t e s t e d w e r e d e s i g n a t e d as being t o x i c (TOXIC) or n o n - t o x i c (NOTOX) on t h e b a s i s of b i o a s s a y r e s u l t s , a n d a stepwise discriminant a n a l y s i s w a s p e r f o r m e d to d e t e r m i n e w h i c h v a r i a b l e s c o u l d b e u s e d to b e s t c l a s s i f y t h e c o m p o u n d s i n t o t h e t w o g r o u p s . Stepwise d i s c r i m i n a n t a n a l y s i s d e t e r m i n e d that the b e s t c a n o n i c a l d i s c r i m i n a n t model for c l a s s i f i c a t i o n of t h e c o m p o u n d s c o n s i s t e d of p h o s p h o r e s c e n c e lifetime (PLT) a n d f i r s t o r d e r m o l e c u l a r c o n n e c t i v i t y i n d e x (MCI). Phosphorescence l i f e t i m e e x h i b i t e d the m a i n e f f e c t in the c l a s s i f i c a t i o n w i t h a p a r t i a l r 2 in
1400
the m o d e l
of 0.69
(P > F = 0.003)
m o d e l of 0.39 (P > F = 0.056). calibrate
c o m p a r e d to M C l w i t h a p a r i t a l
r 2 in the
D i s c r i m i n a n t a n a l y s e s w e r e t h e n c o n d u c t e d to
a c l a s s i f i c a t i o n model
to predict photo-induced PAH toxicity.
All
compounds were c o r r e c t l y c l a s s i f i e d when both phosphorescence lifetime and MCI were entered in the discriminant function To
determine
classification study p l u s
the
accuracy
of
the
w a s p e r f o r m e d u s i n g the
(Table 3). classification
criterion,
a
test
ii c o m p o u n d s t e s t e d in the p r e s e n t
an independent set of 17 PAH for which MCI was c a l c u l a t e d and for
w h i c h i n f o r m a t i o n on PLT w a s a v a i l a b l e the t e s t c l a s s i f i c a t i o n , designated non-toxic
had p o s t e r i o r p r o b a b i l i t i e s classification
and
13
classification
Of c o m p o u n d s e x a m i n e d in
12 ( 43% ) w e r e d e s i g n a t e d t o x i c and 16 ( 57% ) w e r e
(Table 4). of
posterior probablities
( T a b l e 4).
Ten of the
12 compounds designated as toxic
of g r e a t e r t h a n 90% for m e m b e r s h i p in the t o x i c the
16
compounds
designated
as
of g r e a t e r t h a n 90% for m e m b e r s h i p
non-toxic
had
in the n o n - t o x i c
(Table 4).
DISCUSSION The
results
anthracene, phototoxic
of
which PAH
these
has
experiments
been
(i0,11,12),
used
demonstrate
in p r e v i o u s
are a c u t e l y
that
studies
phototoxic
to
as
fish.
anthracene ranked fourth out of 12 among compounds tested, level of toxicity among the compounds that were toxic. of r e l a t i v e toxicities, compound
for
the
therefore,
examination
PAH
other
than
a representative Based
on RPF,
exhibiting a median
From the point of view
anthracene appears to be an adequate model of
photo-induced
PAH
toxicity
to
fish.
Anthracene also exhibits a median l e v e l of photo-induced toxicity to Daphnia m a n n a (8) and w i t h few e x c e p t i o n s , the r e l a t i v e t o x i c i t i e s of the v a r i o u s compounds were v e r y s i m i l a r between fish l a r v a e and zooplankton. photodynamic activities
The relative
of PAH t h a t w e r e t e s t e d for t o x i c i t y a g a i n s t b r i n e
shrimp nauplii (6) do not c o r r e l a t e w e l l with the RPF v a l u e s obtained for the same c o m p o u n d s in t h e p r e s e n t study. T h i s d i s a g r e e m e n t in the r e l a t i v e t o x i c i t i e s can be e x p l a i n e d
in p a r t by the fact that,
in t h e p r e v i o u s study
(6), the concentration of PAH in organisms were based on nominal
values.
The
importance of obtaining direct measurements of tissue PAH concentrations is i l l u s t r a t e d by the present study because the selected nominal concentrations of PAH in fish were not all a c c u r a t e l y obtained (Table i). The d e v e l o p m e n t of a c l a s s i f i c a t i o n scheme that can determine whether or not a PAH has the p o t e n t i a l to c a u s e p h o t o - i n d u c e d t o x i c i t y is s i g n i f i c a n t . While the discriminant analysis presented here can only c l a s s i f y a compound as b e i n g t o x i c or n o n - t o x i c and has no p r e d i c t i v e c a p a b i l i t i e s w i t h r e g a r d to r e l a t i v e l e v e l s of t o x i c i t y , it m a y p r o v e to p o s s e s s p o w e r in a s s e s s i n g the p o t e n t i a l for e n v i r o n m e n t a l i m p a c t and in i d e n t i f y i n g g e o g r a p h i c a r e a s of environmental concern. V a l i d a t i o n of the current c l a s s i f i c a t i o n scheme will be d i f f i c u l t . However, comparisons among other studies show that compounds examined in the test c l a s s i f i c a t i o n were designated correctly. compounds
considered
in
M o r g a n and Warshawsky (6)
most All
which were common to the
1401
Table
Phosphorescence
3.
connectivity bioassay
results,
analysis
with
function. within
PLT
Compound
lifetimes
values
(3)
(7)
[MCI],
[PLT],
classification
probability
TOXIC
of
within
and
96 h,
order
of compound
compound
of membership,
= phototoxic
first
classification
by =
based
on
discriminant
linear
NOTOX
molecular
discriminant non-phototoxic
96 h.
MCI
Bioassay
(s)
Classified
Designation:
Posterior
Into:
Probability
Of Membership NOTOX
In:
TOXIC
ACR
0. 150
4.5856
TOXIC
TOXIC
0.0002
9998
ANT
0. 090
4.8094
TOXIC
TOXIC
0.0003
9997
BAN
0. 020
6.2635
TOXIC
TOXIC
0.0108
9892
BAA
0 359
6.2201
TOXIC
TOXIC
0.0185
9815
PYR
0 630
5.5594
TOXIC
TOXIC
0.0295
9705
BAP
0 105
6.9701
TOXIC
TOXIC
0.0621
9379
BGP
0 438
7.7201
NOTOX
NOTOX
0.5451
0 4549
DBA
1 600
7.6308
NOTOX
NOTOX
1.0000
0 0000
BEP
2 120
6.9761
NOTOX
NOTOX
1.0000
0.0000
PHE
2 940
4.8154
NOTOX
NOTOX
1.0000
0.0000
PER
3 500
6.9761
NOTOX
NOTOX
1.0000
0.0000
GENERALIZED D2(IIJ)
=
SQUARED
DISTANCE
(X i - Xj)
C O V -I
FUNCTION (X i - Xj)
NOTOX
TOXIC
NOTOX
0.000000
10.897554
TOXIC
10.897554
0.000000
LINEAR Constant
= -0.5
Xj
DISCRIMINANT
C O Y -I Xj NOTOX
Constant
-40.631639
FUNCTION
Coefficient
Vector TOXIC
-19.831543
MCI
9.257358
6.750125
PLT
8.536444
4.224812
= C O V -I Xj
1402
Table
Phosphorescence
4.
connectivity test
values
classification
hydrocarbons.
TOXIC
to be non-phototoxic,
Compound
PLT
lifetimes (3)
[MCl], for
(7) and
some
= Predicted
selected
(s)
first
tested
Classified Into:
order
of discriminant polycyclic
to be phototoxic,
* = Compounds
MCl
[PLT], results
NOTOX
molecular analysis aromatic
= predicted
in b i o a s s a y s .
Posterior
Probability
of M e m b e r s h i p NOTOX
in:
TOXIC
Benzo(b)anthracene
0.01
6.2141
TOXIC
0.0000
1.0000
Phenazine
0.08
4.6546
TOXIC
0.0000
1.0000
* Acridine
0.15
4.5856
TOXIC
0.0002
0.9998
* Anthracene
0.09
4.8094
TOXIC
0.0003
0.9997
0.28
5.8541
TOXIC
0.0050
0.9950
0.41
5.8541
TOXIC
0.0100
0.9900
* Benzanthrone
0.02
6.2635
TOXIC
0.0110
0.9890
* Benzo(a)anthracene
0.36
6.2201
TOXIC
0.0185
0.9815
* Pyrene
0.63
5.5594
TOXIC
0.0295
0.9705
* Benzo(a)pyrene
0.ii
6.9701
TOXIC
0.0621
0.9379
Benzo(b)chrysene
0.18
7.6308
TOXIC
0.2800
0.7200
Dibenz(a,c)phenazine
0.29
7.4819
TOXIC
0.2520
0.7480
* Benzo(g,h,i)perylene
0.44
7.7201
NOTOX
0.5451
0.4549
Fluoranthene
0.99
5.5654
NOTOX
0.7940
0.2060
Benzo(k) fluoranthene
0.83
6.9701
NOTOX
0.8900
0.ii00
Benz(b)triphenylene
0.80
7.3867
NOTOX
0.9350
0.0650
Benzo(a)fluorene
2.61
6.0225
NOTOX
1.0000
0.0000
Benzo(b)fluorene
2.24
6.0166
NOTOX
1.0000
0.0000
Chrysene
2.54
6.2261
NOTOX
1.0000
0.0000 0.0000
Benz(c)acridine Benz(a) acridine
Fluorene
5.00
4.5118
NOTOX
1.0000
Dibenz(a,h)acridine
2.31
7.5535
NOTOX
1.0000
0.0000
NOTOX
1.0000
0.0000
Carbazole
8.04
4.4046
Coronene
9.50
8.2142
NOTOX
1.0000
0.0000
Dibenz(a,j)anthracene
2.51
7.3421
NOTOX
1.0000
0.0000
* Dibenz(a,h)anthracene
1.60
7.6308
NOTOX
1.0000
0.0000
* Benzo(e)pyrene
2.12
6.9761
NOTOX
1.0000
0.0000
* Phenanthrene
2.94
4.8154
NOTOX
1.0000
0.0000
* Perylene
3.50
6.9761
NOTOX
1.0000
0.0000
1403
test c l a s s i f i c a t i o n w e r e d e s i g n a t e d c o r r e c t l y as b e i n g t o x i c or non-toxic. but
two
compounds
(fluoranthene
tested
against
D.
and b e n z o ( k ) f l u o r a n t h e n e )
magna
by
Newsted
and
All
Giesy
(8)
m a t c h e d the d e s i g n a t e d c a t e g o r i e s
from
the p r e s e n t study (Table 4). These c o m p o u n d s w e r e of i n t e r m e d i a t e t o x i c i t y to D. m a q n a with LT50 values of 10.8 a n d 13.0 h f o r f l u o r a n t h e n e and benzo(k) f l u o r a n t h e n e ,
respectively.
been
comparisons
misclassified,
Newsted
and Giesy
(8) i n d i c a t e
_manna w i t h e s t i m a t e d fish within general
96 h.
assessment
work
other
these
compounds
phototoxic
appear
compounds
that PAH causing photo-induced
LT50 values
More
Although of
greater
is n e e d e d
to h a v e
studied
toxicity
t h a n 8 to 9 h a r e n o t p h o t o t o x i c in t h i s
a r e a of s t u d y b e f o r e
of PAH p h o t o - i n d u c e d
toxicity
to a q u a t i c
of a c l a s s i f i c a t i o n
criterion
organisms
by
to D. to
a more can be
accomplished. The derivation important
insight
toxicity
of PAH.
into the m o l e c u l a r The major
factor
mechanism
determining
b a s e d on M C I a n d P L T l e n d s
of a c t i o n whether
in the p h o t o - i n d u c e d or n o t a c o m p o u n d
is
classified as b e i n g p h o t o t o x i c is P L T (cf. R e s u l t s ) . P L T is a d i r e c t m e a s u r e m e n t of the r a d i a t i v e energy d i s s i p a t i o n of a m o l e c u l e from the excited triplet
state
excited
states
including light
to t h e may
radiative
and heat.
singlet return
states
the
state
ground
processes
in w h i c h
Non-radiative
processes
photosensitized molecule of e x c i t e d
ground
to
(16). state
energy may
Molecules in
with,
such
number
is d i s s i p a t e d
also
occur,
is p a s s e d to other m o l e c u l e s
in, a n d r e a c t i o n s
a
of
as PAH
in
pathways,
in t h e f o r m of
where
energy
from a
l e a d i n g to the formation
these other molecules.
Radiative
and n o n - r a d i a t i v e p r o c e s s e s o p e r a t e s i m u l t a n e o u s l y and the net d i s s i p a t i o n of e n e r g y f r o m an e x c i t e d s t a t e m o l e c u l e is an i n t e g r a t i o n of t h e s e c o m p e t i n g processes.
The
probability
of
photosensitized
reactions
with
molecules
for
which r a d i a t i v e p r o c e s s e s d o m i n a t e is d i r e c t l y p r o p o r t i o n a l to the l i f e t i m e of the e x c i t e d s t a t e (16). The results of t h e s e e x p e r i m e n t s demonstrate, however, fish
that PAH w i t h
larvae,
proportional
indicating to t h e
short p h o s p h o r e s c e n c e that
length
the
lifetimes
photo-induced
of e x i s t a n c e
are m o r e p h o t o t o x i c to
toxicity
of t h e e x c i t e d
of PAH
is i n v e r s e l y
triplet
state.
The
r a t e of e n e r g y t r a n s f e r in n o n - r a d i a t i v e p r o c e s s e s h a s b e e n s h o w n to be i n v e r s e l y p r o p o r t i o n a l to the r a d i a t i v e l i f e t i m e of e x c i t e d states (i). These facts lead to the s p e c u l a t i o n that the p h o t o - i n d u c e d t o x i c i t y of PAH to fish is d e t e r m i n e d by t h e r a t e of n o n - r a d i a t i v e energy transfer from the excited s t a t e of a p a r t i c u l a r compound. T h e r e f o r e , m e c h a n i s m s w h i c h d e p e n d on the l i f e t i m e of e x c i t e d states, such as d i r e c t i n t e r a c t i o n (5), m o s t l i k e l y do not predominate, transfer,
and
such
reactions as t h e
which
formation
depend of
on the
reactive
rate
and
singlet
efficiency oxygen
(i),
of energy are more
probable.
CONCLUSIONS This
study
has
demonstrated
that
cause p h o t o - i n d u c e d t o x i c i t y to fish,
the
potential
exists
for m a n y
PAH to
and that a n t h r a c e n e has b e e n an a d e q u a t e
1404
model
compound
development
in t h e p r e v i o u s
of
information
studies
a classification
about
the
structure
based
and p h o t o c h e m i s t r y
w h e t h e r or not a PAH has the p o t e n t i a l is a s i g n i f i c a n t a d v a n c e m e n t impact of t h e s e compounds.
of t h i s p h e n o m e n o n
criterion,
in the
(i0, ii, 12).
on
easily
The
obtained
of a compound,
to predict
to c a u s e p h o t o - i n d u c e d t o x i c i t y to fish
assessment
of the p o s s i b l e
environmental
ACKNOWLEDGEMENTS Portions College this the
of
Program,
this
is p u b l i c a t i o n Michigan
research
number
sponsored
by
the
Michigan
Sea
w i t h the g r a n t M A - 8 5 A A - D - S G - 0 4 5 ,
MICHU-SG-86-300.
Agricultural
article number
were
p r o j e c t R/TS-21,
Experiment
Funding
Station,
was
also
from which
Grant
from which
received
this
from
is j o u r n a l
11936.
REFERENCES 1.
Bennett,
R.G.,
and
R.E.
Kellogg.
Photochem.
Photobiol.
7:
571
- 581
(1968). 2.
Dworkin,
3. 4.
Koch, R. T o x i c o l . E n v i r o n . Chem. 6 : 8 7 - 96 (1983). Kochevar, I.E., R.B. A r m s t r o n g , J. E i n b i n d e r , R.R. Harber.
5.
Landrum, meulen
M. J. Gen. P h y s i o l .
Photochem.
41:1099
Photobiol.
P.F., J.P. G i e s y , a n d S. H r u d e y ,
36:
65 - 69
J.T. Otis,
eds., O i l
T e c h n o L o g y , P e r g a m o n Press, 6.
Morgan,
7.
Morgan,
8.
2 5 : 3 1 - 38 (1977). Newsted, J.L., a n d J.P.
9.
(1986). Oris, J.T. PhD Dissertation.
D. W a r s h a w s k y ,
Photochem
Environ.
ii.
Oris, J.T., a n d J.P. G i e s y . E n v i r o n .
12.
Oris,
J.P.
Giesy,
A~uat. Toxicol.
P.M.
Toxicol.
Allred,
D.F.
L.C.
In J.H. V a n d e r -
Chemistry r Biology,
Photobiol.
25:39
- 46
Photochem.
Toxicol.
(in press)
East Lansing,
6:133-146
MI
(1985).
Chem. 5 : 7 6 1 Grant,
(1977).
Photobiol.
Chem.
M i c h i g a n State U n i v e r s i t y ,
(1985). Oris, J.T., a n d J.P. G i e s y .
and
(1986).
T. A t k i n s o n .
i0.
J.T.,
Walther,
(1982).
a n d P.M. A l l r e d .
(in press)
and
Giesy.
(1958).
and Freshwater:
NY,
D.D., and D. Warshawsky. D.D.,
- 1112
and
- 768 P.F.
(1986).
Landrum.
T.N. V e z i r o g l u , ed., The Biosphere: P r o b l e m s and Solutions, S c i e n c e Publ. B.V., Amsterdam, pp. 639-658 (1984).
In
Elsevier
13. 14.
SAS I n s t i t u t e Inc. 1982. S t a t i s t i c a l A n a l y s i s System, Cary, NC. V e r s t e e g , D.J., a n d J.P. Giesy. Arch. E n v i r o n . C o n t a m . T o x i c o l .
15.
640 (1985). Wan, S. R.R. A n d e r s o n ,
16.
499 (1981). W e l l s , C.H.J. I n t r o d u c t i o n
14:631
-
NY,
146 pp.
a n d J.A. P a r r i s h . to M o l e c u l a r
Photochem.
Photobiol.
Photochemistry.
Halsted
(1972).
(Received in Germany 15 J a n u a r y 1987;
a c c e p t e d 17 March
34:493
1987)
-
Press,
pp 1395-1404,
1987
0 0 4 5 - 6 5 3 5 / 8 7 $3.00 + .OO P e r q a m o n Journals Ltd.
THE P H O T O - I N D U C E D T O X I C I T Y OF P O L Y C Y C L I C A R O M A T I C H Y D R O C A R B O N S TO LARVAE OF THE F A T H E A D M I N N O W (Pimephales promelas) J a m e s T. Oris* and J o h n P. Giesy, Jr. D e p a r t m e n t of F i s h e r i e s and W i l d l i f e M i c h i g a n State U n i v e r s i t y East Lansing, MI 48824 USA ABSTRACT The toxicity of 12 p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s to larvae of the fathead m i n n o w in the p r e s e n c e of s i m u l a t e d s u n l i g h t was examined. A measure of r e l a t i v e t o x i c i t i e s of the toxic c o m p o u n d s is d e s c r i b e d in w h i c h waveband radiation intensity, molar extinction coefficients, and molar body concentration of PAH are considered. In addition, a structure-lethality r e l a t i o n s h i p has b e e n developed, b a s e d on m o l e c u l a r s t r u c t u r e and p h o t o c h e m i cal properties, that c l a s s i f i e s c o m p o u n d s as b e i n g p h o t o t o x i c or n o n - p h o t o toxic. INTRODUCTION Previous studies (10,ii,12) have d e m o n s t r a t e d that anthracene, a fused, linear 3-ring polycyclic a r o m a t i c h y d r o c a r b o n (PAH), is a c u t e l y toxic to juvenile s u n f i s h u n d e r l a b o r a t o r y and field c o n d i t i o n s of solar ultraviolet radiation (SUVR), and that this t o x i c i t y can be p r e d i c t e d from k n o w l e d g e of SUVR intensity, a n t h r a c e n e c o n c e n t r a t i o n and p h o t o p e r i o d duration. Because PAH b e l o n g to a large class of compounds, it is d e s i r a b l e to determine the extent and l i k e l i h o o d that PAH other than a n t h r a c e n e are c a p a b l e of e l i c i t i n g photo-induced t o x i c i t y to fish. M a n y PAH can be c o n s i d e r e d as p o t e n t i a l l y p h o t o t o x i c (2), and there have b e e n reports d e s c r i b i n g the p h o t o - a c t i v i t y of PAH to m a m m a l s (4) and a q u a t i c o r g a n i s m s (6). There are no known reports, however, concerning the range and extent of the p h o t o - i n d u c e d t o x i c i t i e s of PAH to fish. The p r e s e n t study was c o n d u c t e d to : i) d e t e r m i n e the relative photo-induced toxicities of a v a r i e t y of PAH to fish, and 2) develop a structure-lethality r e l a t i o n s h i p to e s t i m a t e the p h o t o - i n d u c e d t o x i c i t y of a compound.
*To w h o m c o r r e s p o n d e n c e may be addressed, University, Oxford, OH 45056 USA.
1395
at D e p a r t m e n t
of
Zoology,
Miami
1396
M A T E R I A L S AND M E T H O D S C o m p o u n d s and T e s t S o l u t i o n s Anthracene acridine
(ANT),
(ACR),
(PER),
benzo(a)anthracene
benzanthrone
(BAN),
benzo(g,h,i)perylene
benzo(e)pyrene purification without
(BEP),
filtered,
purification.
aerated technique
Appropriate desired
tap w a t e r to
dilutions
concentration
solutions
were
(9).
study
The
organisms
(BGP),
pyrene
phenanthrene
available commercially.
further
coating
and
(BAA),
(PYR),
in
the
shell
tests
(9). the
to
achieve
in
in
all
a
test.
C o n c e n t r a t i o n s of PAH
fluorescence
aqueous detection
was d e s i g n e d such that the final PAH c o n c e n t r a t i o n s Therefore,
PAH c o n c e n t r a t i o n s
weights,
n o r m a l i z e d to the least w a t e r soluble compound,
concentrations
charcoal
w e r e m a d e from these s t o c k s o l u t i o n s
on
PAH
in
of each PAH w e r e o b t a i n e d u s i n g a
a v o i d the use of c a r r i e r s o l v e n t s
w o u l d be equimolar.
nominal
highest
(8) were used
solutions,
selected a
(BAP),
the
All c o m p o u n d s except BAN aqueous
(BBA),
perylene
benzo(a)pyrene
d e t e r m i n e d by r e v e r s e - p h a s e H P L C and
the
(DBA),
(PHE) w e r e o b t a i n e d at
Saturated (14),
benzo(b)anthracene
dibenz(a,h)anthracene
b a s i s of p u b l i s h e d b i o c o n c e n t r a t i o n b o d y b u r d e n of I00 n M / g was
values
selected.
of PAH in w a t e r are p r e s e n t e d in Table
and
BGP.
in
in w a t e r
the were
molecular
On this basis,
Nominal
and
actual
i.
O r g a n i s m s and B i o a s s a y P r o c e d u r e Larvae of the f a t h e a d m i n n o w
(Pimephales promelas)
w e r e o b t a i n e d four days
post-hatching from the M i c h i g a n D e p a r t m e n t of N a t u r a l R e s o u r c e s Surface Water Q u a l i t y Division. L a r v a e w e r e t r a n s f e r r e d by p i p e t t e to a small f l o w - t h r o u g h a q u a r i u m filled w i t h c h a r c o a l were
maintained
filtered,
a e r a t e d tap w a t e r at 24 C.
were fed n e w l y h a t c h e d b r i n e shrimp n a u p l i i ad libitum twice a day. On the s e v e n t h transferred
by
The larvae
in the f l o w - t h r o u g h a q u a r i u m for two days a f t e r t r a n s f e r
pipette
to
s o l u t i o n or d i l u t i o n water.
and
(Artemia salina; M e t a f r a m e Corp.) day post-hatching, larvae were
300 ml Pyrex dishes c o n t a i n i n g
150
ml
of
PAH
T r e a t m e n t s c o n s i s t e d of 20 to 25 larvae per dish
and two d i s h e s per PAH examined, i n c l u d i n g two d i s h e s containing dilution water as S U V R - o n l y controls. Dishes w e r e c o v e r e d w i t h a l u m i n u m foil and the larvae
a l l o w e d a 24 h
p r e - i n c u b a t i o n p e r i o d in the a b s e n c e of
SUVR.
After
the p r e - i n c u b a t i o n period, larvae w e r e fed b r i n e shrimp ad l i b i t u m for 0.5 h, all solutions w e r e t h e n r e p l a c e d and the dishes w i t h larvae w e r e placed in r a n d o m p o s i t i o n s u n d e r a l a b o r a t o r y s y s t e m light b a n k w h i c h s i m u l a t e d natural sunlight
(i0).
Light
was
filtered
w i t h a 5 mil t h i c k n e s s
of
Mylar R
to
eliminate >99% of the r a d i a t i o n of w a v e l e n g t h s s h o r t e r than 315 nm. SUVR intensities were m o n i t o r e d as a p r e v i o u s study (i0) and for all exposures were U V - B (290-336 n m ) = 20 u W / c m 2 and U V - A (336-400 n m ) = 95 u W / c m 2. After the initial p r e - i n c u b a t i o n , s o l u t i o n s w e r e c h a n g e d at 12 h intervals. Larvae were fed b r i n e shrimp ad l i b i t u m once a day for 0.5 h prior to changing solutions. Bioassay dishes w e r e e x a m i n e d for larval m o r t a l i t y at least four times daily. T e s t s w e r e c o n d u c t e d until 100% m o r t a l i t y was a c h i e v e d or for a maximum of 96 h, w h i c h e v e r came first. PAH c o n c e n t r a t i o n s in w a t e r were
1397
determined more
for
at the b e g i n n i n g initial
concentrations measured
in
occurred,
effect
exhibited
of a b i o a s s a y
12
water
concentrations
mortality any
and
h
are
reported
in the
initial
due to PAH toxicity
in the a b s e n c e
in the dark.
at the end of a test
fish l a r v a e
samples
i.
were
Nominal organisms. based
72 +/-
and
as the
and
on
water
of
in
in
i00 n M / g
water
bioconcentration
that no compound(s)
Water
actual (nM/g)
526
525
14.7
5.4
ii1.8
PHE
14.5
i0.0
293.2
BAA
1.9
1.8
6.7
BBA
1.1
1.9
N
DBA
0.25
0.15
0 94
PYR
7.21
25.6
BAP
0.82
5.6
BEP
0.43
2.9
6 0
PER
0.79
1.7
7 5
0.20
and
detected.
D.
87 1 486
7
3.7
0.15
52.9
49.5
0.0
in
120.6
ANT
31.6
and
compounds
Organism
(ug/L)
SUVR-only
were spiked
factor, was
(ug/L)
BAN
study
from
for all
actual
BGP
where assess
larvae
of PAH
nominal
ACR
to
the
this
in all
of PAH
were
BGP,
indicates
Water Compound
of PAH
recoveries
PAH
between In tests
No PAH t e s t e d
concentrations
PAH b o d y - b u r d e n s N.D.
mean
12 h old solutions.
Percent
once
period.
(mean +/- SE).
solubility
weights.
and at least
test
to the PAH was p e r f o r m e d
Concentrations
12%
12 h,
the
geometric
of SUVR.
(9).
actual
Nominal
molecular
during
a 96 h d a r k e x p o s u r e
determined
Table
at zero and
solutions
N.D.
N.D.
control
Efficacy The similar
and R e l a t i v e efficacy to that
of
to q u a n t u m
of
mortality
larvae.
The rate
each
of M o r g a n
analagously larval
Potency
Factor phototoxic
compound
and W a r s h a w s k y
yield versus
(6).
in p h o t o c h e m i s t r y the rate
of m o r t a l i t y
versus
of q u a n t a
was
determined
Efficacy
in
( ~ )
and is a d e s c r i p t o r absorbed
a is
of the rate
by a compound
time can be d e s c r i b e d
manner defined
by e q u a t i o n
in 1.
the
1398
n
d(%Mortality)
[(I°lTl ) ( ~kb Ca) ] -~
=
A.~
(i)
dt A
where:
= the average = waveband
n u m b e r of q u a n t a a b s o r b e d p e r t i m e ( U V - B = 3 1 5 - 3 3 6 nm, U V - A = 3 3 6 - 4 0 0
VIS2=420-450
nm, V I S l = 4 0 0 - 4 2 0
nm, and
nm).
Iol
= waveband
Tk ~
= o p t i c a l t r a n s m i t t a n c e of e p i d e r m i s for w a v e b a n d (15). = mean molar e x t i n c t i o n c o e f f i c i e n t of c o m p o u n d in octanol
radiation
waveband
intensity
= depth
Ca
of l a r v a e = 0.2 cm) = molar concentration
n
= n u m b e r of w a v e b a n d s = time
for
(L/mole/cm) (8).
b
t
(uW/cm2)(9).
of
radiation
penetration
in o r g a n i s m
of c o m p o u n d in o r g a n i s m
(b = avg.
diameter
(moles/kg).
(s)
= e f f i c a c y of c o m p o u n d I n t e g r a t i o n of e q u a t i o n
1 yields:
%Mortality =
A. ~
t
+
B
(2)
w h i c h is in the form of a l i n e a r e q u a t i o n where, time,
B is t h e
intercept
and
A.~
is t h e
in p l o t s of % M o r t a l i t y v e r s u s
slope
of t h e
line.
Efficacy,
therefore, can be d e t e r m i n e d a l g e b r a i c a l l y from k n o w l e d g e of the c a l c u l a t e d A and t h e s l o p e of t h e % M o r t a l i t y v e r s u s t i m e c u r v e for e a c h i n d i v i d u a l compound. The Relative P o t e n c y F a c t o r (RPF) is an i n d e x of t h e r e l a t i v e e f f i c a c y of a c o m p o u n d c o m p a r e d to t h e l e a s t e f f i c a c i o u s of t h e c o m p o u n d s tested.
Therefore,
e f f i c a c y is a u n i ~ l e d e s c r i p t o r of the p h o t o t o x i c a c t i v i t y
of a c o m p o u n d
and RPF
gives
compounds used
in this
study.
a relative
i n d e x of a c t i v i t y
for t h e g r o u p
of
RESULTS S i x of t h e 12 c o m p o u n d s t e s t e d e x h i b i t e d a c u t e p h o t o - i n d u c e d toxicity ( T a b l e 2). Median-lethal-time (LT50) v a l u e s r a n g e d f r o m 0.83 h for BAN to 65.1 h for BAA. O n t h e b a s i s of RPF, B A N e x h i b i t e d t h e g r e a t e s t a n d BAP e x h i b i t e d t h e l e a s t l e v e l of c o n c e n t r a t i o n a n d a b s o r p t i o n - s p e c i f i c photoi n d u c e d t o x i c i t y a m o n g t h e c o m p o u n d s t h a t w e r e p h o t o t o x i c ( T a b l e 2). Of the r e m a i n i n g six compounds, four c o m p o u n d s e x h i b i t e d no e f f e c t c o m p a r e d to SUVRonly controls (BEP, DBA, PER, PHE) a n d t h e o t h e r t w o c o m p o u n d s e x h i b i t e d a m a r g i n a l l e v e l ( <20% m o r t a l i t y in 96 h) of p h o t o - i n d u c e d t o x i c i t y (BBA, BGP). M o r t a l i t y in S U V R - o n l y c o n t r o l s was less t h a n 5% in a l l tests. C o n t r a r y to the o r i g i n a l d e s i g n of this experiment, e q u i m o l a r b o d y - b u r d e n s of PAH w e r e not o b t a i n e d (Table i), e v e n t h o u g h PAH c o n c e n t r a t i o n s in w a t e r w e r e r e l a t i v e l y c l o s e to the s e l e c t e d n o m i n a l concentrations. The e q u a t i o n u s e d to c a l c u l a t e
1399
A
and
~
takes
in the
animal,
so e v e n t h o u g h the a c h i e v e m e n t of e q u i m o l a r b o d y - b u r d e n s was desirable,
it was
not entirely
into
account
necessary.
the
concentrations
of c o m p o u n d
Since BBA was not detected
in f i s h t i s s u e
( T a b l e i)
this c o m p o u n d was not u s e d for further analysis.
Table
2.
Tabulated absorbed
values
of m e d i a n
( A ), e f f i c a c y
( ~
for a l l p h o t o t o x i c c o m p o u n d s . o r d e r of r e l a t i v e potency.
Compound
LT50
lethal
times
), a n d R e l a t i v e Compounds
A
(LT50), Potency
are listed
~
average Factor
quanta (RPF)
in d e c r e a s i n g
RPF
(h)
BAN
0.83
0.183
5.46
E-2
337.1
PYR ACR
3.20 4.30
0.372 0.397
1.45 E-2 7.00 E-3
i00.i 48.3
ANT
15.75
0.218
3.12
E-3
21.5
BAA
65.09
0.i00
2.38
E-3
16.4
BAP
40.05
2.913
1.45 E-4
1.0
C o r r e l a t i o n a n a l y s e s w e r e p e r f o r m e d w i t h the m e a s u r e s of m o r t a l i t y and the chemical
characteristics
relationship. coefficients,
The
of
the
factors
compounds
considered
to
determine
included
first and second order m o l e c u l a r
a
structure-activity
octanol-water
c o n n e c t i v i t y indicies,
partition energies
of l o w e s t s i n g l e t e x c i t e d state splitting, e n e r g i e s of l o w e s t t r i p l e t excited state splitting, the difference between singlet and triplet splitting energies, p h o s p h o r e s c e n c e lifetimes, a v e r a g e m o l a r e x t i n c t i o n c o e f f i c i e n t s in octanol for each of the four w a v e b a n d s examined, and the s u m m e d total of all molar extinction coefficients across all wavebands significant univariate correlations were observed between of m o r t a l i t y Because
(LT50,
RPF,
no u s e f u l
A. ~)
( 3 1 5 - 4 5 0 nm). No any of the measures
and any of the a b o v e c h e m i c a l
univariate
predictive
relationships
characteristics. were
observed,
d i s c r i m i n a t e a n a l y s e s (13) w e r e used to c l a s s i f y the c o m p o u n d s as b e i n g either p h o t o t o x i c or non-phototoxic. A l l c o m p o u n d s t e s t e d w e r e d e s i g n a t e d as being t o x i c (TOXIC) or n o n - t o x i c (NOTOX) on t h e b a s i s of b i o a s s a y r e s u l t s , a n d a stepwise discriminant a n a l y s i s w a s p e r f o r m e d to d e t e r m i n e w h i c h v a r i a b l e s c o u l d b e u s e d to b e s t c l a s s i f y t h e c o m p o u n d s i n t o t h e t w o g r o u p s . Stepwise d i s c r i m i n a n t a n a l y s i s d e t e r m i n e d that the b e s t c a n o n i c a l d i s c r i m i n a n t model for c l a s s i f i c a t i o n of t h e c o m p o u n d s c o n s i s t e d of p h o s p h o r e s c e n c e lifetime (PLT) a n d f i r s t o r d e r m o l e c u l a r c o n n e c t i v i t y i n d e x (MCI). Phosphorescence l i f e t i m e e x h i b i t e d the m a i n e f f e c t in the c l a s s i f i c a t i o n w i t h a p a r t i a l r 2 in
1400
the m o d e l
of 0.69
(P > F = 0.003)
m o d e l of 0.39 (P > F = 0.056). calibrate
c o m p a r e d to M C l w i t h a p a r i t a l
r 2 in the
D i s c r i m i n a n t a n a l y s e s w e r e t h e n c o n d u c t e d to
a c l a s s i f i c a t i o n model
to predict photo-induced PAH toxicity.
All
compounds were c o r r e c t l y c l a s s i f i e d when both phosphorescence lifetime and MCI were entered in the discriminant function To
determine
classification study p l u s
the
accuracy
of
the
w a s p e r f o r m e d u s i n g the
(Table 3). classification
criterion,
a
test
ii c o m p o u n d s t e s t e d in the p r e s e n t
an independent set of 17 PAH for which MCI was c a l c u l a t e d and for
w h i c h i n f o r m a t i o n on PLT w a s a v a i l a b l e the t e s t c l a s s i f i c a t i o n , designated non-toxic
had p o s t e r i o r p r o b a b i l i t i e s classification
and
13
classification
Of c o m p o u n d s e x a m i n e d in
12 ( 43% ) w e r e d e s i g n a t e d t o x i c and 16 ( 57% ) w e r e
(Table 4). of
posterior probablities
( T a b l e 4).
Ten of the
12 compounds designated as toxic
of g r e a t e r t h a n 90% for m e m b e r s h i p in the t o x i c the
16
compounds
designated
as
of g r e a t e r t h a n 90% for m e m b e r s h i p
non-toxic
had
in the n o n - t o x i c
(Table 4).
DISCUSSION The
results
anthracene, phototoxic
of
which PAH
these
has
experiments
been
(i0,11,12),
used
demonstrate
in p r e v i o u s
are a c u t e l y
that
studies
phototoxic
to
as
fish.
anthracene ranked fourth out of 12 among compounds tested, level of toxicity among the compounds that were toxic. of r e l a t i v e toxicities, compound
for
the
therefore,
examination
PAH
other
than
a representative Based
on RPF,
exhibiting a median
From the point of view
anthracene appears to be an adequate model of
photo-induced
PAH
toxicity
to
fish.
Anthracene also exhibits a median l e v e l of photo-induced toxicity to Daphnia m a n n a (8) and w i t h few e x c e p t i o n s , the r e l a t i v e t o x i c i t i e s of the v a r i o u s compounds were v e r y s i m i l a r between fish l a r v a e and zooplankton. photodynamic activities
The relative
of PAH t h a t w e r e t e s t e d for t o x i c i t y a g a i n s t b r i n e
shrimp nauplii (6) do not c o r r e l a t e w e l l with the RPF v a l u e s obtained for the same c o m p o u n d s in t h e p r e s e n t study. T h i s d i s a g r e e m e n t in the r e l a t i v e t o x i c i t i e s can be e x p l a i n e d
in p a r t by the fact that,
in t h e p r e v i o u s study
(6), the concentration of PAH in organisms were based on nominal
values.
The
importance of obtaining direct measurements of tissue PAH concentrations is i l l u s t r a t e d by the present study because the selected nominal concentrations of PAH in fish were not all a c c u r a t e l y obtained (Table i). The d e v e l o p m e n t of a c l a s s i f i c a t i o n scheme that can determine whether or not a PAH has the p o t e n t i a l to c a u s e p h o t o - i n d u c e d t o x i c i t y is s i g n i f i c a n t . While the discriminant analysis presented here can only c l a s s i f y a compound as b e i n g t o x i c or n o n - t o x i c and has no p r e d i c t i v e c a p a b i l i t i e s w i t h r e g a r d to r e l a t i v e l e v e l s of t o x i c i t y , it m a y p r o v e to p o s s e s s p o w e r in a s s e s s i n g the p o t e n t i a l for e n v i r o n m e n t a l i m p a c t and in i d e n t i f y i n g g e o g r a p h i c a r e a s of environmental concern. V a l i d a t i o n of the current c l a s s i f i c a t i o n scheme will be d i f f i c u l t . However, comparisons among other studies show that compounds examined in the test c l a s s i f i c a t i o n were designated correctly. compounds
considered
in
M o r g a n and Warshawsky (6)
most All
which were common to the
1401
Table
Phosphorescence
3.
connectivity bioassay
results,
analysis
with
function. within
PLT
Compound
lifetimes
values
(3)
(7)
[MCI],
[PLT],
classification
probability
TOXIC
of
within
and
96 h,
order
of compound
compound
of membership,
= phototoxic
first
classification
by =
based
on
discriminant
linear
NOTOX
molecular
discriminant non-phototoxic
96 h.
MCI
Bioassay
(s)
Classified
Designation:
Posterior
Into:
Probability
Of Membership NOTOX
In:
TOXIC
ACR
0. 150
4.5856
TOXIC
TOXIC
0.0002
9998
ANT
0. 090
4.8094
TOXIC
TOXIC
0.0003
9997
BAN
0. 020
6.2635
TOXIC
TOXIC
0.0108
9892
BAA
0 359
6.2201
TOXIC
TOXIC
0.0185
9815
PYR
0 630
5.5594
TOXIC
TOXIC
0.0295
9705
BAP
0 105
6.9701
TOXIC
TOXIC
0.0621
9379
BGP
0 438
7.7201
NOTOX
NOTOX
0.5451
0 4549
DBA
1 600
7.6308
NOTOX
NOTOX
1.0000
0 0000
BEP
2 120
6.9761
NOTOX
NOTOX
1.0000
0.0000
PHE
2 940
4.8154
NOTOX
NOTOX
1.0000
0.0000
PER
3 500
6.9761
NOTOX
NOTOX
1.0000
0.0000
GENERALIZED D2(IIJ)
=
SQUARED
DISTANCE
(X i - Xj)
C O V -I
FUNCTION (X i - Xj)
NOTOX
TOXIC
NOTOX
0.000000
10.897554
TOXIC
10.897554
0.000000
LINEAR Constant
= -0.5
Xj
DISCRIMINANT
C O Y -I Xj NOTOX
Constant
-40.631639
FUNCTION
Coefficient
Vector TOXIC
-19.831543
MCI
9.257358
6.750125
PLT
8.536444
4.224812
= C O V -I Xj
1402
Table
Phosphorescence
4.
connectivity test
values
classification
hydrocarbons.
TOXIC
to be non-phototoxic,
Compound
PLT
lifetimes (3)
[MCl], for
(7) and
some
= Predicted
selected
(s)
first
tested
Classified Into:
order
of discriminant polycyclic
to be phototoxic,
* = Compounds
MCl
[PLT], results
NOTOX
molecular analysis aromatic
= predicted
in b i o a s s a y s .
Posterior
Probability
of M e m b e r s h i p NOTOX
in:
TOXIC
Benzo(b)anthracene
0.01
6.2141
TOXIC
0.0000
1.0000
Phenazine
0.08
4.6546
TOXIC
0.0000
1.0000
* Acridine
0.15
4.5856
TOXIC
0.0002
0.9998
* Anthracene
0.09
4.8094
TOXIC
0.0003
0.9997
0.28
5.8541
TOXIC
0.0050
0.9950
0.41
5.8541
TOXIC
0.0100
0.9900
* Benzanthrone
0.02
6.2635
TOXIC
0.0110
0.9890
* Benzo(a)anthracene
0.36
6.2201
TOXIC
0.0185
0.9815
* Pyrene
0.63
5.5594
TOXIC
0.0295
0.9705
* Benzo(a)pyrene
0.ii
6.9701
TOXIC
0.0621
0.9379
Benzo(b)chrysene
0.18
7.6308
TOXIC
0.2800
0.7200
Dibenz(a,c)phenazine
0.29
7.4819
TOXIC
0.2520
0.7480
* Benzo(g,h,i)perylene
0.44
7.7201
NOTOX
0.5451
0.4549
Fluoranthene
0.99
5.5654
NOTOX
0.7940
0.2060
Benzo(k) fluoranthene
0.83
6.9701
NOTOX
0.8900
0.ii00
Benz(b)triphenylene
0.80
7.3867
NOTOX
0.9350
0.0650
Benzo(a)fluorene
2.61
6.0225
NOTOX
1.0000
0.0000
Benzo(b)fluorene
2.24
6.0166
NOTOX
1.0000
0.0000
Chrysene
2.54
6.2261
NOTOX
1.0000
0.0000 0.0000
Benz(c)acridine Benz(a) acridine
Fluorene
5.00
4.5118
NOTOX
1.0000
Dibenz(a,h)acridine
2.31
7.5535
NOTOX
1.0000
0.0000
NOTOX
1.0000
0.0000
Carbazole
8.04
4.4046
Coronene
9.50
8.2142
NOTOX
1.0000
0.0000
Dibenz(a,j)anthracene
2.51
7.3421
NOTOX
1.0000
0.0000
* Dibenz(a,h)anthracene
1.60
7.6308
NOTOX
1.0000
0.0000
* Benzo(e)pyrene
2.12
6.9761
NOTOX
1.0000
0.0000
* Phenanthrene
2.94
4.8154
NOTOX
1.0000
0.0000
* Perylene
3.50
6.9761
NOTOX
1.0000
0.0000
1403
test c l a s s i f i c a t i o n w e r e d e s i g n a t e d c o r r e c t l y as b e i n g t o x i c or non-toxic. but
two
compounds
(fluoranthene
tested
against
D.
and b e n z o ( k ) f l u o r a n t h e n e )
magna
by
Newsted
and
All
Giesy
(8)
m a t c h e d the d e s i g n a t e d c a t e g o r i e s
from
the p r e s e n t study (Table 4). These c o m p o u n d s w e r e of i n t e r m e d i a t e t o x i c i t y to D. m a q n a with LT50 values of 10.8 a n d 13.0 h f o r f l u o r a n t h e n e and benzo(k) f l u o r a n t h e n e ,
respectively.
been
comparisons
misclassified,
Newsted
and Giesy
(8) i n d i c a t e
_manna w i t h e s t i m a t e d fish within general
96 h.
assessment
work
other
these
compounds
phototoxic
appear
compounds
that PAH causing photo-induced
LT50 values
More
Although of
greater
is n e e d e d
to h a v e
studied
toxicity
t h a n 8 to 9 h a r e n o t p h o t o t o x i c in t h i s
a r e a of s t u d y b e f o r e
of PAH p h o t o - i n d u c e d
toxicity
to a q u a t i c
of a c l a s s i f i c a t i o n
criterion
organisms
by
to D. to
a more can be
accomplished. The derivation important
insight
toxicity
of PAH.
into the m o l e c u l a r The major
factor
mechanism
determining
b a s e d on M C I a n d P L T l e n d s
of a c t i o n whether
in the p h o t o - i n d u c e d or n o t a c o m p o u n d
is
classified as b e i n g p h o t o t o x i c is P L T (cf. R e s u l t s ) . P L T is a d i r e c t m e a s u r e m e n t of the r a d i a t i v e energy d i s s i p a t i o n of a m o l e c u l e from the excited triplet
state
excited
states
including light
to t h e may
radiative
and heat.
singlet return
states
the
state
ground
processes
in w h i c h
Non-radiative
processes
photosensitized molecule of e x c i t e d
ground
to
(16). state
energy may
Molecules in
with,
such
number
is d i s s i p a t e d
also
occur,
is p a s s e d to other m o l e c u l e s
in, a n d r e a c t i o n s
a
of
as PAH
in
pathways,
in t h e f o r m of
where
energy
from a
l e a d i n g to the formation
these other molecules.
Radiative
and n o n - r a d i a t i v e p r o c e s s e s o p e r a t e s i m u l t a n e o u s l y and the net d i s s i p a t i o n of e n e r g y f r o m an e x c i t e d s t a t e m o l e c u l e is an i n t e g r a t i o n of t h e s e c o m p e t i n g processes.
The
probability
of
photosensitized
reactions
with
molecules
for
which r a d i a t i v e p r o c e s s e s d o m i n a t e is d i r e c t l y p r o p o r t i o n a l to the l i f e t i m e of the e x c i t e d s t a t e (16). The results of t h e s e e x p e r i m e n t s demonstrate, however, fish
that PAH w i t h
larvae,
proportional
indicating to t h e
short p h o s p h o r e s c e n c e that
length
the
lifetimes
photo-induced
of e x i s t a n c e
are m o r e p h o t o t o x i c to
toxicity
of t h e e x c i t e d
of PAH
is i n v e r s e l y
triplet
state.
The
r a t e of e n e r g y t r a n s f e r in n o n - r a d i a t i v e p r o c e s s e s h a s b e e n s h o w n to be i n v e r s e l y p r o p o r t i o n a l to the r a d i a t i v e l i f e t i m e of e x c i t e d states (i). These facts lead to the s p e c u l a t i o n that the p h o t o - i n d u c e d t o x i c i t y of PAH to fish is d e t e r m i n e d by t h e r a t e of n o n - r a d i a t i v e energy transfer from the excited s t a t e of a p a r t i c u l a r compound. T h e r e f o r e , m e c h a n i s m s w h i c h d e p e n d on the l i f e t i m e of e x c i t e d states, such as d i r e c t i n t e r a c t i o n (5), m o s t l i k e l y do not predominate, transfer,
and
such
reactions as t h e
which
formation
depend of
on the
reactive
rate
and
singlet
efficiency oxygen
(i),
of energy are more
probable.
CONCLUSIONS This
study
has
demonstrated
that
cause p h o t o - i n d u c e d t o x i c i t y to fish,
the
potential
exists
for m a n y
PAH to
and that a n t h r a c e n e has b e e n an a d e q u a t e
1404
model
compound
development
in t h e p r e v i o u s
of
information
studies
a classification
about
the
structure
based
and p h o t o c h e m i s t r y
w h e t h e r or not a PAH has the p o t e n t i a l is a s i g n i f i c a n t a d v a n c e m e n t impact of t h e s e compounds.
of t h i s p h e n o m e n o n
criterion,
in the
(i0, ii, 12).
on
easily
The
obtained
of a compound,
to predict
to c a u s e p h o t o - i n d u c e d t o x i c i t y to fish
assessment
of the p o s s i b l e
environmental
ACKNOWLEDGEMENTS Portions College this the
of
Program,
this
is p u b l i c a t i o n Michigan
research
number
sponsored
by
the
Michigan
Sea
w i t h the g r a n t M A - 8 5 A A - D - S G - 0 4 5 ,
MICHU-SG-86-300.
Agricultural
article number
were
p r o j e c t R/TS-21,
Experiment
Funding
Station,
was
also
from which
Grant
from which
received
this
from
is j o u r n a l
11936.
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