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Characterisation of a strongly bioaccumulating hexachloronaphthalene
C h e m o s p h e r e , Vol.15, No.5, pp 619-628, P r i n t e d in G r e a t B r i t a i n
1986
0 0 4 5 - 6 5 3 5 / 8 6 $3.00 + .OO Pergamon Journals Ltd
C H A R A C T E R I S A T I O N OF A S T R O N G L Y B I O A C C U M U L A T I N G HEXACHLORONAPHTHALENE
L i l l e m o r Asplund, Bo J a n s s o n and G ~ r a n S u n d s t r ~ m Special Analytical Laboratory National Environmental Protection Board S-171 25 Solna, S w e d e n Ingvar Brandt D e p a r t m e n t of P h a r m a c o l o g y and T o x i c o l o g y T h e S w e d i s h U n i v e r s i t y of A g r i c u l t u r a l S c i e n c e s B o x 573, S-751 23 Uppsala, S w e d e n U d o A Th B r i n k m a n D e p a r t m e n t of A n a l y t i c a l C h e m i s t r y Free Reformed University 1081 HV Amsterdam, The N e t h e r l a n d s
ABSTRACT Rats w e r e g i v e n a t e c h n i c a l m i x t u r e of p o l y c h l o r i n a t e d n a p h t h a l e n e s (PCN). A d i p o s e tissue and liver samples w e r e a n a l y s e d after I, i0, 30 and 120 days. One of the m i n o r c o m p o n e n t s in the t e c h n i c a l p r o d u c t was the d o m i n a n t PCN c o m p o u n d in the tissues a l r e a d y after i0 days and the only one d e t e c t a b l e a f t e r 120 days. The c o n c e n t r a t i o n of this c o m p o u n d was in all a n a l y s e d a n i m a l s m u c h h i g h e r (up to 140 times) in the liver than in the adipose tissue. In o r d e r to c h a r a c t e r i s e this c o m p o u n d c h r o m a t o g r a p h i c and mass s p e c t r o m e t r i c data w e r e r e c o r d e d for all a v a i l a b l e c h l o r o n a p h t h a l e n e s . T h e s e data i n d i c a t e two p o s s i b l e structures, one of w h i c h was s y n t h e s i s e d in m i n u t e amounts. This compound, 1 , 2 , 3 , 5 , 6 , 7 - h e x a c h l o r o n a p h t h a l e n e , was s h o ~ to have the same c h r o m a t o g r a p h i c and mass s p e c t r o m e t r i c p r o p e r t i e s as the bioa c c u m u l a t i n g compound.
INTRODUCTION
A n a l y s e s of p o l y c h l o r i n a t e d
naphthalenes
(PCN) h a v e r e v e a l e d that
just a few
of the 75 p o s s i b l e c o n g e n e r s are p r e s e n t in samples from the e n v i r o n m e n t
(i).
In m a m m a l i a n t i s s u e there is one h e x a c h l o r i n a t e d n a p h t h a l e n e w h i c h totally d o m i n a t e s the c h r o m a t o g r a p h i c pattern. a g r e a t n u m b e r of c o m p o u n d s
As all t e c h n i c a l PCN p r o d u c t s c o n t a i n
this i n d i c a t e s either a n o t h e r source for these
p o l l u t a n t s or that some of the PCN c o m p o n e n t s are more stable than others. B i o l o g i c a l effects of a few p u r e PCN c o n g e n e r s have b e e n i n v e s t i g a t e d and e n z y m e a c t i v i t i e s w e r e i n d u c e d by hexa-, (2,3),
h e p t a - and o c t a c h l o r o n a p h t h a l e n e s
but d i f f e r e n c e s c o u l d be o b s e r v e d b e t w e e n d i f f e r e n t isomers.
619
620
Polybrominated naphthalenes brominated biphenyls
(PBN) h a v e b e e n found as i m p u r i t i e s
(PBB) used as flame r e t a r d a n t s
a c c i d e n t a l c o n t a m i n a t i o n of animal h a v e b e e n investigated.
food w i t h PBB,
a number of PBN c o n g e n e r s
A m o n g those 2 , 3 , 4 , 5 , 6 , 7 - h e x a b r o m o n a p h t h a l e n e
found to be very p e r s i s t e n t
in e x p e r i m e n t a l
The p r e s e n t i n v e s t i g a t i o n was p e r f o r m e d mulating polychlorinated naphthalenes products
in poly-
(4,5). B e c a u s e of an
animals
has b e e n
(6,7,8).
in o r d e r to find out if the b i o a c c u -
c o u l d be d e r i v e d from the t e c h n i c a l
and to get as m u c h i n f o r m a t i o n as p o s s i b l e of the s t r u c t u r e of these
compounds.
EXPERIMENTAL
F e m a l e S p r a q u e - D a w l e y rats t e c h n i c a l PCN p r o d u c t s bw)
(bw 200-240 g) w e r e given a single oral dose of
(Halowax 1014 and 1051, K o p p e r s C h e m i c a l Co;
in corn oil. The a n i m a l s
120 days after d o s i n g and s p e c i m e n s removed.
20 m g / k g
fed H a l o w a x 1014 w e r e k i l l e d at i, i0, 30 and from liver and i n t r a p e r i t o n e a l
fat w e r e
H a l o w a x 1051 was given to one animal w h i c h was k i l l e d a f t e r 30 days.
The w e i g h t gain of all H a l o w a x - t r e a t e d rats was similar to that of the c o n t r o l rats,
and no signs of t o x i c i t y w e r e observed.
The t i s s u e samples w e r e e x t r a c t e d w i t h a m i x t u r e of n - h e x a n e and a c e t o n e ether
(redistilled),
(Merck)
(redistilled)
f o l l o w e d by a m i x t u r e of n - h e x a n e and d i e t h y l
(9). A f t e r e v a p o r a t i o n of the solvents and d e t e r m i n a t i o n of the
lipid amount,
the residues w e r e r e d i s s o l v e d in 2 , 2 , 5 - t r i m e t h y l p e n t a n e
(redistilled)
and l - b r o m o n a p h t h a l e n e
standard. BDH)
(Merck) was added as an i n t e r n a l
T h e s e solutions w e r e t r e a t e d w i t h c o n c e n t r a t e d s u l f u r i c a c i d
for lipid removal.
environmental
samples
to no i n t e r f e r e n c e
(98%;
The c h a r c o a l c o l u m n c l e a n - u p d e s c r i b e d e a r l i e r for
(i) was not n e c e s s a r y to use in this i n v e s t i g a t i o n due
from c h l o r d a n e s
in the l a b o r a t o r y animals.
Synthesis M i n u t e amounts of a h e x a c h l o r o n a p h t h a l e n e was s y n t h e s i s e d by e x h a u s t i v e c h l o r i n a t i o n of 1 , 5 - d i a m i n o n a p h t h a l e n e w i t h c h l o r i n e gas in dilute carbon tetrachloride isoamylnitrite
s o l u t i o n and s u b s e q u e n t d e a m i n a t i o n by t r e a t m e n t w i t h in t e t r a h y d r o f u r a n
Liquid chromatography
(i0).
(LC)
A n H P L C m e t h o d was used for the f r a c t i o n a t i o n of the i n v e s t i g a t e d PCN product.
A V a r i a n 5000 i n s t r u m e n t e q u i p p e d w i t h a c h a r c o a l column
PX-21A,
460 x 2.3 mm) w i t h t o l u e n e
and 5 mL fractions collected.
(i mL/min)
(Amoco C a r b o n
as the m o b i l e p h a s e was used
621
Gas chromatography/mass spectrometry (GC/MS) The G C / M S a n a l y s e s w e r e p e r f o r m e d on a F i n n i g a n 4021
instrument.
The fused
silica columns w e r e d i r e c t l y c o n n e c t e d into the ion source. H e l i u m was used as c a r r i e r gas.
I n j e c t o r and t r a n s f e r
line t e m p e r a t u r e s w e r e 250°C and the
i n j e c t i o n s w e r e made in a s p l i t l e s s mode. The s t a t i o n a r y phases used w e r e BP-I,
SE-54 and SP-2331. All these gave six peaks c o r r e s p o n d i n g to h e x a c h l o r o -
naphthalenes,
Negative were
but the first p h a s e did not separate the two HpCN isomers.
ions from c h e m i c a l
studied.
ionisation
s o u r c e was 250"C.
internal
(99.95%, AGA,
Stockholm)
S p e c t r a w e r e scanned from -150 to -500 amu. The b i o l o g i c a l
s a m p l e s w e r e a n a l y s e d by m u l t i p l e molecular
in m e t h a n e
The e l e c t r o n e n e r g y was 70 eV and the t e m p e r a t u r e of the ion
ion d e t e c t i o n using two ions in the
ion c l u s t e r for the PCN c o n g e n e r s and the b r o m i d e ions for the standard
(i).
R E S U L T S AND D I S C U S S I O N
T h e i n v e s t i g a t e d product,
H a l o w a x 1014,
contains chlorinated naphthalene
c o n g e n e r s w i t h 4 to 8 c h l o r i n e atoms as the major c o n s t i t u e n t s as shown in F i g u r e IA. The PCN p a t t e r n in the animals one day after dosing was
rather similar to that of the t e c h n i c a l product,
of h e p t a - and o c t a c h l o r o n a p h t h a l e n e s w e r e
(Figure IB)
but the r e l a t i v e levels
lower in the animals.
This may very
w e l l be e x p l a i n e d by a less e f f e c t i v e a b s o r p t i o n of these c o m p o u n d s
(ii).
The h e x a c h l o r o n a p h t h a l e n e w i t h the s h o r t e s t GC r e t e n t i o n time was d o m i n a n t in intraperitoneal
fat I0 days after dosing.
T h i s p a t t e r n was further accen-
t u a t e d a f t e r 30 days and a f t e r 120 days the only peak d e t e c t e d c o r r e s p o n d e d to the h e x a c h l o r o n a p h t h a l e n e was
(Figure IC). The c o n c e n t r a t i o n of this c o m p o u n d
still after this p e r i o d a b o u t 50% of that m e a s u r e d after one day.
In the liver samples the stable h e x a c h l o r o n a p h t h a l e n e was d o m i n a n t a l r e a d y a f t e r one day. The liver c o n c e n t r a t i o n of this c o m p o u n d
(calculated on a
lipid w e i g h t basis) was in all samples h i g h e r than the c o r r e s p o n d i n g concentration compound
in the a d i p o s e tissue. The c o n c e n t r a t i o n ratio of the stable
in liver and a d i p o s e tissue was a b o u t 140 after ten days and 5 after
four months. H a l o w a x 1051 c o n t a i n s m a i n l y octa- and the two h e p t a c h l o r o n a p h t h a l e n e s but a l s o small amounts of h e x a c h l o r o n a p h t h a l e n e
isomers.
In the rat g i v e n this
m i x t u r e one h e x a - and one h e p t a c h l o r o n a p h t h a l e n e w e r e d e t e c t e d after 30 days. T h e r e w e r e no i n d i c a t i o n s of de novo f o r m a t i o n of h e x a c h l o r i n a t e d f r o m the h i g h e r c h l o r i n a t e d congeners.
isomers
622
CIs
CI s
CI7
CI4
ZL
F i g u r e i.
CI8
L
I
I
i
i
10
12
14
16
18
min
Gas c h r o m a t o g r a m s o b t a i n e d w i t h m u l t i p l e ion d e t e c t i o n of m o l e c u l a r ions for p o l y c h l o r i n a t e d naphthalenes. A: H a l o w a x 1014. B: Rat a d i p o s e t i s s u e one day a f t e r a H a l o w a x 1014 dose. C: Rat a d i p o s e t i s s u e 120 days after a H a l o w a x 1014 dose. C h r o m a t o g r a p h i c conditions: 25m x 0 . 2 2 m m id BPI (SGE, Australia), 70°(2min)-20°/min-170°-10°/min-280°C.
623
It has b e e n shown that the m o r e toxic c o n g e n e r s of PCDD/F are b i o a c c u m u l a t e d m o r e than the less toxic c o n g e n e r s found in h i g h e r c o n c e n t r a t i o n s
(12). Furthermore,
the toxic c o m p o u n d s are
in the liver than in a d i p o s e tissue
(13). The
r e s e m b l a n c e b e t w e e n these toxic c o m p o u n d s and the p e r s i s t e n t h e x a c h l o r o naphthalene
is s t r i k i n g and it is e s s e n t i a l to get more k n o w l e d g e about this
compound.
T h e p u r e PCN c o n g e n e r s a v a i l a b l e to us c o n t a i n one to five c h l o r i n e atoms. S e p a r a t i o n of the t e c h n i c a l c o m p l e x m i x t u r e s has earlier b e e n done by H P L C using both straight
(14,15)
and r e v e r s e d p h a s e columns
to i s o l a t e the p e r s i s t e n t H x C N by any of these m e t h o d s and a c h a r c o a l c o l u m n was tested. u s e d in the a n a l y s e s of P C D D / F
(16). The p o s s i b i l i t y seemed to be limited
The a d s o r b e n t used was of the type o f t e n
for c l e a n - u p purposes.
H a l o w a x 1014 was chroma-
t o g r a p h e d and the f r a c t i o n s a n a l y s e d by GC. The results are shown in F i g u r e 2. The c o m p o u n d w i t h the l a r g e s t r e t e n t i o n volume was the p e r s i s t e n t HpCN
(7a). The stable H x C N
(6a) a l s o had a large r e t e n t i o n v o l u m e but it was
not p o s s i b l e to isolate this compound. relatively
A possible explanation
small r e t e n t i o n v o l u m e of the o c t a c h l o r o n a p h t h a l e n e
c h l o r o s u b s t i t u t i o n in two p e r i p o s i t i o n s makes the m o l e c u l e retentions
less planar.
for the two H p C N
for the c o u l d be the
(i,8- and 4,5-) w h i c h p o s s i b l y
T h i s could a l s o e x p l a i n the d i f f e r e n t
isomers w i t h c h l o r i n e in one or two peri
positions.
CI 3
cl4 Cl 5 Cl 6
e
f Cl 7
a
b Cl 8
Toluene
I
I
i
,
I
,
100
200
300
400
500
600
F i g u r e 2.
R e t e n t i o n of c o m p o n e n t s
700
in H a l o w a x 1014 on a c h a r c o a l column.
ml
624
T a b l e i. G C / M S data of r e f e r e n c e compounds. in text and F i g u r e i.
Experimental
Structure
rel rt*
C11
2I-
0.85 0.86
C12
1,32,61,52,71,41,21,8-
1 03 1 04 1 04 1 04 1 05 1 06 1 .12
38 96 i00 I00 I00 14 i00
Cl 3
1,361,3, 71,4,61,2,51,2,62,3,71,2,71,2,31,3,8-
121 1.22 1.22 1.25 1.25 1.26 1.26 1.27 1.29
21 18 20 40 59 30 72
I00 i00 i00 i00 i00 i00
i00
0
,3 5,7,2 4,6,4 6,7,3 6,7,2 3,5,3 5,8,2 3,4,2 3,7,4,5,8-
1.37 1.41 1.44 1.44 1.45 1.47 1.47 1.47 1.54
8
1 1 1 1 2 74 2 1 I00
i00 I00 i00 i00 i00 i00 i00 i00 34
1,2,3,5,7-
1.61
2
2
i00
Cl 5
(M-35)-
described
n
C14
(M-69)-
conditions
2
(M-I)-
M-
i00 i00 66 62 93 i00 8
* r e t e n t i o n time r e l a t i v e to p e n t a c h l o r o b e n z e n e
The results
from the GC/MS a n a l y s e s of i n d i v i d u a l c h l o r o n a p h t h a l e n e s
are
c o m p a r e d w i t h those from the c o m p o n e n t s of the H a l o w a x 1014 in T a b l e s 1 and 2 As could be expected,
the lower c h l o r i n a t e d c o m p o u n d s
(CI 1 - C13)
s e n s i t i v i t y w i t h m e t h a n e as the r e a g e n t gas. N o or low m o l e c u l a r
gave low ion intensi-
ties w e r e o b s e r v e d for the lowest c h l o r i n a t i o n degrees and the f o r m a t i o n of chloride
ions due to d i s s o c i a t i v e e l e c t r o n capture is p r o b a b l y the d o m i n a t i n g
process.
The spectra of d i c h l o r o n a p h t h a l e n e s
while
show a d o m i n a t i n g
for the h i g h e r c h l o r i n a t e d h o m o l o g u e s M- is dominant.
(M-H)- ion
625
T a b l e 2. G C / M S data of c o m p o n e n t s in a t e c h n i c a l p o l y c h l o r o n a p h t h a l e n e p r o d u c t (Halowax 1014). E x p e r i m e n t a l conditions d e s c r i b e d in text and F i g u r e I.
rel rt*
(M-102)-
(M-70)-
(M-69)-
(M-68)-
1.37 1.41 1.43 1.45 1.49 1.50 1.53
Cl 4
cl 5
3
1.84 1.87 1.88 1.88 1.92 1.94
c d e f
(M-34)-
2 1
M-
I00 i00 i00 100
3 5 52 i00 i00 i00
1.61 1.64 1.65 1.68 1.69 1.70 1.71
C16 ~
(M-35)-
15
2
1 5 1 2
35
12 45
3 i00 3 i00 i00 i00 18
14
4 68 84 21 I00 4
i00 i00 i00 i00 51 i00
3 8 12 6 14
3
i00 75 I00 57 19 43 i00
C17
2.11
2
19
31
i00
Cl 8
2.41
5
32
30
i00
* r e t e n t i o n time r e l a t i v e to p e n t a c h l o r o b e n z e n e
F u r t h e r e x a m i n a t i o n s of the f r a g m e n t a t i o n p a t t e r n s r e v e a l e d an ion c o r r e s p o n d ing to (M-35)- for the di-, compounds common.
containing
tri- and t e t r a c h l o r i n a t e d naphthalenes.
For the
six to eight c h l o r i n e atoms the (M-34)- was more
The p e n t a c h l o r o n a p h t h a l e n e s
gave a m i x e d p a t t e r n in this region.
The
loss of 34 mass units in spectra f r o m p o l y c h l o r i n a t e d c o m p o u n d s has earlier been reported
(17) and is p r e s u m e d to c o r r e s p o n d to loss of a c h l o r i n e
r a d i c a l and a d d i t i o n of a h y d r o g e n radical. suggested 1,8-
for this p r o c e s s
(and/or 4,5-) p o s i t i o n s gave m o r e
chlorine
Several m e c h a n i s m s h a v e been
(18). The c o m p o u n d s w i t h c h l o r o s u b s t i t u e n t s
in
intense ions c o r r e s p o n d i n g to a
loss than their isomers.
The relative retentions
in T a b l e 1 i n d i c a t e that the closer the c h l o r i n e
a t o m s are p o s i t i o n e d in the molecule, a homologous
the longer is the r e t e n t i o n time w i t h i n
group. The c o m p o u n d s w i t h 1,8-
s e e m to h a v e extra long retention.
(and 4,5-)
chloro s u b s t i t u t i o n
626
B o t h r e t e n t i o n time and m a s s s p e c t r u m of the stable c o m p o u n d cate that no 1 , 8 - ( a n d / o r 4 , 5 - ) c h l o r o possible hexachloronaphthalenes f u l f i l l e d in two, (IV and III,
substitution
O f the ten
(Figure 3) this s u b s t i t u t i o n p a t t e r n
namely 1,2,3,5,6,7-
is
and 1 , 2 , 3 , 4 , 6 , 7 - h e x a c h l o r o n a p h t h a l e n e
respectively).
The c h l o r i n a t i o n of the 1 , 5 - d i a m i n o n a p h t h a l e n e hexa-,
(6a) thus indi-
is present.
gave three PCN congeners:
( r e t e n t i o n time r e l a t i v e to p e n t a c h l o r o b e n z e n e
1.84;
one
I M- = I00,
I(M_34 )- = 5, one hepta- and o c t a c h l o r o n a p h t h a l e n e . The y i e l d was low and no a t t e m p t s to isolate the p r o d u c t s w e r e made. The GC and MS p r o p e r t i e s of the o b t a i n e d HxCN, w h i c h on the b a s i s of the s y n t h e t i c p a t h w a y s
should have
the s t r u c t u r e IV, agreed v e r y well w i t h the c o r r e s p o n d i n g data f r o m the bioaccumulating
~
c,
c,
c,
c,
Cl
c o m p o n e n t in the PCN products.
c, y
Cl
y
Cl
I
c,
a
c,
a- ~
Cl
II
CI Cl Vl
F i g u r e 3.
" K ='
Cl
c,
c,- y
v
Q Cl VIII
c,
c,
a
c,-~,
Cl
III
CI Cl Vll
The ten p o s s i b l e
c,
CI
IV
V
Cl IX
X
s t r u c t u r e s of h e x a c h l o r o n a p h t h a l e n e .
The data p r e s e n t e d h e r e thus i n d i c a t e that the s t r u c t u r e of the p e r s i s t e n t compound
is 1 , 2 , 3 , 5 , 6 , 7 - H x C N .
This is in a n a l o g y w i t h the s t r u c t u r e s of the
P C D D / F s w h i c h s e e m to be m o s t p e r s i s t e n t 1,2,3,6,7,8-HxCDD a n i m a l s of p r e y
and 1 , 2 , 3 , 6 , 7 , 8 - H x C D F
in the environment.
Both
are d o m i n a n t among the P C D D / F s in
(12).
A m o n g the PBN found in the p o l y b r o m i n a t e d b i p h e n y l s one p e r s i s t e n t c o n g e n e r has b e e n i d e n t i f i e d as 2 , 3 , 4 , 5 , 6 , 7 - h e x a b r o m o n a p h t h a l e n e 1 , 2 , 3 , 5 , 6 , 7 - H x B N has p r e v i o u s l y b e e n s y n t h e s i s e d know,
(8). The
(15) but has,
not b e e n t e s t e d for p e r s i s t e n c e and b i o l o g i c a l activity.
as far as we
627
The similarities between the stable HxCN and the most potent congeners of the PCDD and PCDF families are alarming.
Even if 2,3,7,8-TeCDD was a thousand
times more potent than the HxCN the latter could be a major cause of the total effect due to much higher concentrations to synthesise 1,2,3,5,6,7-hexachloronaphthalene
in the environment.
Attempts
in amounts large enough for
biological testing are therefore underway.
ACKNOWLEDGEMENT
Parts of this study were supported by the Research Committee of the National Environmental
Protection Board.
REFERENCES I.
Jansson B, Asplund L and Olsson M, "Analysis of polychlorinated naphthalenes in environmental samples", Chemosphere 13 (1984) 33.
2.
Campbell M A, Bandiera S, Robertson L, Parkinson A and Safe S, "Hepta-, hexa-, tetra- and dichloronaphthalene congeners as inducers of hepatic microsomal drug-metabolizing enzymes", Toxicology 26 (1983) 193.
3.
Campbell M A, Bandiera S, Robertson L, Parkinson A and Safe S, "Octachloronaphthalene induction of hepatic microsomal aryl hydrocarbon hydroxylase activity in the immature male rat", Toxicology 2-2 (1981) 123.
4.
Hass J R, McConell E E and Harvan D J, "Chemical and toxicologic evaluation of Firemaster BP-6", J. Agric. Food Chem. 26 (1978) 94.
5.
Tondeur Y, Hass J R, Harvan D J, Albro P W and McKinney J D, "Determination of suspected toxic impurities in Firemaster FF-I and Firemaster BP-6 by high resolution gas chromatography-high resolution mass spectrometry", J. Agric. Food Chem. 32 (1984) 406.
6.
Robertson L ~, Thompson K and Parkinson A, "Synthesis, characterization and biological effects of polybrominated naphthalenes", Toxicology 25 (1984) 127.
7.
Birnbaum L S, Darcey D J and McKinney J D, "Hexabromonaphthalene contaminants of polybrominated biphenyls: Chemical composition and disposition in the rat", J. Toxicol. Environm. Health 12 (1983) 555.
8.
Birnbaum L S and McKinney J D, "A persistent hexabromonaphthalene isomer is 2,3,4,5,6,7-hexabromonaphthalene", J. Toxicol. Environm. Health, in press.
9.
Jensen S, Reuterg~rdh L and Jansson B, "Analytical methods for measuring organochlorines and methyl mercury by gas chromatography", FAO/SIDA, Manual of methods in aquatic environment research. Part 9. (Manila, Philippines, May 7 - June 9, 1979). ~orkshop Manual part 3. FAO Fish. Tech. Pap. 212 (1983) 21.
628
I0.
Cadogan J I G and Molina G A, "A simple and convenient deamination of aromatic amines", J. Chem. Soc. Perkin I (1973) 541.
ii.
Birnbaum L S, "The Role of Structure in the disposition of halogenated aromatic xenobiotics", Environmental Health Perspectives 61 (1985) ii.
12.
Nygren M, Rappe C, Lindstr~m G, Hansson M, Bergqvist P-A, Marklund S, Domell~f L, Hardell L and Olsson M, "Identification of 2,3,7,8-substituted polychlorinated dioxins (PCDDs) and dibenzofurans (PCDFs) in environmental and human samples", presented at "Chlorinated dioxins and dibenzofurans in the total environment", ACS National Meeting, Miami, FI, USA, April 1985, abstract No 55.
13.
Gasiewicz T A, Olson J R, Geiger L H and Neal R A, "Absorption, distribution and metabolism of 2,3,7,8-tetrachlorodibenzodioxin (TCDD) in experimental animals", in Human and environmental risks of chlorinated dioxins and related compounds, Ed R E Tucker, Plenum Press, New York 1983, p 495.
14.
Brinkman U A Th, De Kok A, Reymer H G M and De Vries G, "Analysis of polychlorinated naphthalenes by high-performance liquid and thin-layer chromatography", J. Chromatogr. 129 (1976) 193.
15.
Brinkman U A Th and De Vries G, "Relationship between structure and retention of polyhalogenated aromatics in two adsorption chromatographic systems", J. Chromatogr. 169 (1979) 167.
16.
Brinkman U A Th and De Kok A, "High-performance liquid chromatographic analysis of the heptachloronaphthalenes present in Halowax 1051", J. Chromatogr. 129 (1976) 451.
17.
Stemmler E A, Hites R A, "Methane enhanced negative ion mass spectra of hexachlorocyclopentadiene derivatives, Anal. Chem. 5/7 (1985) 684.
(Received in Germany 20 February 1986; accepted 5 March 1986)
1986
0 0 4 5 - 6 5 3 5 / 8 6 $3.00 + .OO Pergamon Journals Ltd
C H A R A C T E R I S A T I O N OF A S T R O N G L Y B I O A C C U M U L A T I N G HEXACHLORONAPHTHALENE
L i l l e m o r Asplund, Bo J a n s s o n and G ~ r a n S u n d s t r ~ m Special Analytical Laboratory National Environmental Protection Board S-171 25 Solna, S w e d e n Ingvar Brandt D e p a r t m e n t of P h a r m a c o l o g y and T o x i c o l o g y T h e S w e d i s h U n i v e r s i t y of A g r i c u l t u r a l S c i e n c e s B o x 573, S-751 23 Uppsala, S w e d e n U d o A Th B r i n k m a n D e p a r t m e n t of A n a l y t i c a l C h e m i s t r y Free Reformed University 1081 HV Amsterdam, The N e t h e r l a n d s
ABSTRACT Rats w e r e g i v e n a t e c h n i c a l m i x t u r e of p o l y c h l o r i n a t e d n a p h t h a l e n e s (PCN). A d i p o s e tissue and liver samples w e r e a n a l y s e d after I, i0, 30 and 120 days. One of the m i n o r c o m p o n e n t s in the t e c h n i c a l p r o d u c t was the d o m i n a n t PCN c o m p o u n d in the tissues a l r e a d y after i0 days and the only one d e t e c t a b l e a f t e r 120 days. The c o n c e n t r a t i o n of this c o m p o u n d was in all a n a l y s e d a n i m a l s m u c h h i g h e r (up to 140 times) in the liver than in the adipose tissue. In o r d e r to c h a r a c t e r i s e this c o m p o u n d c h r o m a t o g r a p h i c and mass s p e c t r o m e t r i c data w e r e r e c o r d e d for all a v a i l a b l e c h l o r o n a p h t h a l e n e s . T h e s e data i n d i c a t e two p o s s i b l e structures, one of w h i c h was s y n t h e s i s e d in m i n u t e amounts. This compound, 1 , 2 , 3 , 5 , 6 , 7 - h e x a c h l o r o n a p h t h a l e n e , was s h o ~ to have the same c h r o m a t o g r a p h i c and mass s p e c t r o m e t r i c p r o p e r t i e s as the bioa c c u m u l a t i n g compound.
INTRODUCTION
A n a l y s e s of p o l y c h l o r i n a t e d
naphthalenes
(PCN) h a v e r e v e a l e d that
just a few
of the 75 p o s s i b l e c o n g e n e r s are p r e s e n t in samples from the e n v i r o n m e n t
(i).
In m a m m a l i a n t i s s u e there is one h e x a c h l o r i n a t e d n a p h t h a l e n e w h i c h totally d o m i n a t e s the c h r o m a t o g r a p h i c pattern. a g r e a t n u m b e r of c o m p o u n d s
As all t e c h n i c a l PCN p r o d u c t s c o n t a i n
this i n d i c a t e s either a n o t h e r source for these
p o l l u t a n t s or that some of the PCN c o m p o n e n t s are more stable than others. B i o l o g i c a l effects of a few p u r e PCN c o n g e n e r s have b e e n i n v e s t i g a t e d and e n z y m e a c t i v i t i e s w e r e i n d u c e d by hexa-, (2,3),
h e p t a - and o c t a c h l o r o n a p h t h a l e n e s
but d i f f e r e n c e s c o u l d be o b s e r v e d b e t w e e n d i f f e r e n t isomers.
619
620
Polybrominated naphthalenes brominated biphenyls
(PBN) h a v e b e e n found as i m p u r i t i e s
(PBB) used as flame r e t a r d a n t s
a c c i d e n t a l c o n t a m i n a t i o n of animal h a v e b e e n investigated.
food w i t h PBB,
a number of PBN c o n g e n e r s
A m o n g those 2 , 3 , 4 , 5 , 6 , 7 - h e x a b r o m o n a p h t h a l e n e
found to be very p e r s i s t e n t
in e x p e r i m e n t a l
The p r e s e n t i n v e s t i g a t i o n was p e r f o r m e d mulating polychlorinated naphthalenes products
in poly-
(4,5). B e c a u s e of an
animals
has b e e n
(6,7,8).
in o r d e r to find out if the b i o a c c u -
c o u l d be d e r i v e d from the t e c h n i c a l
and to get as m u c h i n f o r m a t i o n as p o s s i b l e of the s t r u c t u r e of these
compounds.
EXPERIMENTAL
F e m a l e S p r a q u e - D a w l e y rats t e c h n i c a l PCN p r o d u c t s bw)
(bw 200-240 g) w e r e given a single oral dose of
(Halowax 1014 and 1051, K o p p e r s C h e m i c a l Co;
in corn oil. The a n i m a l s
120 days after d o s i n g and s p e c i m e n s removed.
20 m g / k g
fed H a l o w a x 1014 w e r e k i l l e d at i, i0, 30 and from liver and i n t r a p e r i t o n e a l
fat w e r e
H a l o w a x 1051 was given to one animal w h i c h was k i l l e d a f t e r 30 days.
The w e i g h t gain of all H a l o w a x - t r e a t e d rats was similar to that of the c o n t r o l rats,
and no signs of t o x i c i t y w e r e observed.
The t i s s u e samples w e r e e x t r a c t e d w i t h a m i x t u r e of n - h e x a n e and a c e t o n e ether
(redistilled),
(Merck)
(redistilled)
f o l l o w e d by a m i x t u r e of n - h e x a n e and d i e t h y l
(9). A f t e r e v a p o r a t i o n of the solvents and d e t e r m i n a t i o n of the
lipid amount,
the residues w e r e r e d i s s o l v e d in 2 , 2 , 5 - t r i m e t h y l p e n t a n e
(redistilled)
and l - b r o m o n a p h t h a l e n e
standard. BDH)
(Merck) was added as an i n t e r n a l
T h e s e solutions w e r e t r e a t e d w i t h c o n c e n t r a t e d s u l f u r i c a c i d
for lipid removal.
environmental
samples
to no i n t e r f e r e n c e
(98%;
The c h a r c o a l c o l u m n c l e a n - u p d e s c r i b e d e a r l i e r for
(i) was not n e c e s s a r y to use in this i n v e s t i g a t i o n due
from c h l o r d a n e s
in the l a b o r a t o r y animals.
Synthesis M i n u t e amounts of a h e x a c h l o r o n a p h t h a l e n e was s y n t h e s i s e d by e x h a u s t i v e c h l o r i n a t i o n of 1 , 5 - d i a m i n o n a p h t h a l e n e w i t h c h l o r i n e gas in dilute carbon tetrachloride isoamylnitrite
s o l u t i o n and s u b s e q u e n t d e a m i n a t i o n by t r e a t m e n t w i t h in t e t r a h y d r o f u r a n
Liquid chromatography
(i0).
(LC)
A n H P L C m e t h o d was used for the f r a c t i o n a t i o n of the i n v e s t i g a t e d PCN product.
A V a r i a n 5000 i n s t r u m e n t e q u i p p e d w i t h a c h a r c o a l column
PX-21A,
460 x 2.3 mm) w i t h t o l u e n e
and 5 mL fractions collected.
(i mL/min)
(Amoco C a r b o n
as the m o b i l e p h a s e was used
621
Gas chromatography/mass spectrometry (GC/MS) The G C / M S a n a l y s e s w e r e p e r f o r m e d on a F i n n i g a n 4021
instrument.
The fused
silica columns w e r e d i r e c t l y c o n n e c t e d into the ion source. H e l i u m was used as c a r r i e r gas.
I n j e c t o r and t r a n s f e r
line t e m p e r a t u r e s w e r e 250°C and the
i n j e c t i o n s w e r e made in a s p l i t l e s s mode. The s t a t i o n a r y phases used w e r e BP-I,
SE-54 and SP-2331. All these gave six peaks c o r r e s p o n d i n g to h e x a c h l o r o -
naphthalenes,
Negative were
but the first p h a s e did not separate the two HpCN isomers.
ions from c h e m i c a l
studied.
ionisation
s o u r c e was 250"C.
internal
(99.95%, AGA,
Stockholm)
S p e c t r a w e r e scanned from -150 to -500 amu. The b i o l o g i c a l
s a m p l e s w e r e a n a l y s e d by m u l t i p l e molecular
in m e t h a n e
The e l e c t r o n e n e r g y was 70 eV and the t e m p e r a t u r e of the ion
ion d e t e c t i o n using two ions in the
ion c l u s t e r for the PCN c o n g e n e r s and the b r o m i d e ions for the standard
(i).
R E S U L T S AND D I S C U S S I O N
T h e i n v e s t i g a t e d product,
H a l o w a x 1014,
contains chlorinated naphthalene
c o n g e n e r s w i t h 4 to 8 c h l o r i n e atoms as the major c o n s t i t u e n t s as shown in F i g u r e IA. The PCN p a t t e r n in the animals one day after dosing was
rather similar to that of the t e c h n i c a l product,
of h e p t a - and o c t a c h l o r o n a p h t h a l e n e s w e r e
(Figure IB)
but the r e l a t i v e levels
lower in the animals.
This may very
w e l l be e x p l a i n e d by a less e f f e c t i v e a b s o r p t i o n of these c o m p o u n d s
(ii).
The h e x a c h l o r o n a p h t h a l e n e w i t h the s h o r t e s t GC r e t e n t i o n time was d o m i n a n t in intraperitoneal
fat I0 days after dosing.
T h i s p a t t e r n was further accen-
t u a t e d a f t e r 30 days and a f t e r 120 days the only peak d e t e c t e d c o r r e s p o n d e d to the h e x a c h l o r o n a p h t h a l e n e was
(Figure IC). The c o n c e n t r a t i o n of this c o m p o u n d
still after this p e r i o d a b o u t 50% of that m e a s u r e d after one day.
In the liver samples the stable h e x a c h l o r o n a p h t h a l e n e was d o m i n a n t a l r e a d y a f t e r one day. The liver c o n c e n t r a t i o n of this c o m p o u n d
(calculated on a
lipid w e i g h t basis) was in all samples h i g h e r than the c o r r e s p o n d i n g concentration compound
in the a d i p o s e tissue. The c o n c e n t r a t i o n ratio of the stable
in liver and a d i p o s e tissue was a b o u t 140 after ten days and 5 after
four months. H a l o w a x 1051 c o n t a i n s m a i n l y octa- and the two h e p t a c h l o r o n a p h t h a l e n e s but a l s o small amounts of h e x a c h l o r o n a p h t h a l e n e
isomers.
In the rat g i v e n this
m i x t u r e one h e x a - and one h e p t a c h l o r o n a p h t h a l e n e w e r e d e t e c t e d after 30 days. T h e r e w e r e no i n d i c a t i o n s of de novo f o r m a t i o n of h e x a c h l o r i n a t e d f r o m the h i g h e r c h l o r i n a t e d congeners.
isomers
622
CIs
CI s
CI7
CI4
ZL
F i g u r e i.
CI8
L
I
I
i
i
10
12
14
16
18
min
Gas c h r o m a t o g r a m s o b t a i n e d w i t h m u l t i p l e ion d e t e c t i o n of m o l e c u l a r ions for p o l y c h l o r i n a t e d naphthalenes. A: H a l o w a x 1014. B: Rat a d i p o s e t i s s u e one day a f t e r a H a l o w a x 1014 dose. C: Rat a d i p o s e t i s s u e 120 days after a H a l o w a x 1014 dose. C h r o m a t o g r a p h i c conditions: 25m x 0 . 2 2 m m id BPI (SGE, Australia), 70°(2min)-20°/min-170°-10°/min-280°C.
623
It has b e e n shown that the m o r e toxic c o n g e n e r s of PCDD/F are b i o a c c u m u l a t e d m o r e than the less toxic c o n g e n e r s found in h i g h e r c o n c e n t r a t i o n s
(12). Furthermore,
the toxic c o m p o u n d s are
in the liver than in a d i p o s e tissue
(13). The
r e s e m b l a n c e b e t w e e n these toxic c o m p o u n d s and the p e r s i s t e n t h e x a c h l o r o naphthalene
is s t r i k i n g and it is e s s e n t i a l to get more k n o w l e d g e about this
compound.
T h e p u r e PCN c o n g e n e r s a v a i l a b l e to us c o n t a i n one to five c h l o r i n e atoms. S e p a r a t i o n of the t e c h n i c a l c o m p l e x m i x t u r e s has earlier b e e n done by H P L C using both straight
(14,15)
and r e v e r s e d p h a s e columns
to i s o l a t e the p e r s i s t e n t H x C N by any of these m e t h o d s and a c h a r c o a l c o l u m n was tested. u s e d in the a n a l y s e s of P C D D / F
(16). The p o s s i b i l i t y seemed to be limited
The a d s o r b e n t used was of the type o f t e n
for c l e a n - u p purposes.
H a l o w a x 1014 was chroma-
t o g r a p h e d and the f r a c t i o n s a n a l y s e d by GC. The results are shown in F i g u r e 2. The c o m p o u n d w i t h the l a r g e s t r e t e n t i o n volume was the p e r s i s t e n t HpCN
(7a). The stable H x C N
(6a) a l s o had a large r e t e n t i o n v o l u m e but it was
not p o s s i b l e to isolate this compound. relatively
A possible explanation
small r e t e n t i o n v o l u m e of the o c t a c h l o r o n a p h t h a l e n e
c h l o r o s u b s t i t u t i o n in two p e r i p o s i t i o n s makes the m o l e c u l e retentions
less planar.
for the two H p C N
for the c o u l d be the
(i,8- and 4,5-) w h i c h p o s s i b l y
T h i s could a l s o e x p l a i n the d i f f e r e n t
isomers w i t h c h l o r i n e in one or two peri
positions.
CI 3
cl4 Cl 5 Cl 6
e
f Cl 7
a
b Cl 8
Toluene
I
I
i
,
I
,
100
200
300
400
500
600
F i g u r e 2.
R e t e n t i o n of c o m p o n e n t s
700
in H a l o w a x 1014 on a c h a r c o a l column.
ml
624
T a b l e i. G C / M S data of r e f e r e n c e compounds. in text and F i g u r e i.
Experimental
Structure
rel rt*
C11
2I-
0.85 0.86
C12
1,32,61,52,71,41,21,8-
1 03 1 04 1 04 1 04 1 05 1 06 1 .12
38 96 i00 I00 I00 14 i00
Cl 3
1,361,3, 71,4,61,2,51,2,62,3,71,2,71,2,31,3,8-
121 1.22 1.22 1.25 1.25 1.26 1.26 1.27 1.29
21 18 20 40 59 30 72
I00 i00 i00 i00 i00 i00
i00
0
,3 5,7,2 4,6,4 6,7,3 6,7,2 3,5,3 5,8,2 3,4,2 3,7,4,5,8-
1.37 1.41 1.44 1.44 1.45 1.47 1.47 1.47 1.54
8
1 1 1 1 2 74 2 1 I00
i00 I00 i00 i00 i00 i00 i00 i00 34
1,2,3,5,7-
1.61
2
2
i00
Cl 5
(M-35)-
described
n
C14
(M-69)-
conditions
2
(M-I)-
M-
i00 i00 66 62 93 i00 8
* r e t e n t i o n time r e l a t i v e to p e n t a c h l o r o b e n z e n e
The results
from the GC/MS a n a l y s e s of i n d i v i d u a l c h l o r o n a p h t h a l e n e s
are
c o m p a r e d w i t h those from the c o m p o n e n t s of the H a l o w a x 1014 in T a b l e s 1 and 2 As could be expected,
the lower c h l o r i n a t e d c o m p o u n d s
(CI 1 - C13)
s e n s i t i v i t y w i t h m e t h a n e as the r e a g e n t gas. N o or low m o l e c u l a r
gave low ion intensi-
ties w e r e o b s e r v e d for the lowest c h l o r i n a t i o n degrees and the f o r m a t i o n of chloride
ions due to d i s s o c i a t i v e e l e c t r o n capture is p r o b a b l y the d o m i n a t i n g
process.
The spectra of d i c h l o r o n a p h t h a l e n e s
while
show a d o m i n a t i n g
for the h i g h e r c h l o r i n a t e d h o m o l o g u e s M- is dominant.
(M-H)- ion
625
T a b l e 2. G C / M S data of c o m p o n e n t s in a t e c h n i c a l p o l y c h l o r o n a p h t h a l e n e p r o d u c t (Halowax 1014). E x p e r i m e n t a l conditions d e s c r i b e d in text and F i g u r e I.
rel rt*
(M-102)-
(M-70)-
(M-69)-
(M-68)-
1.37 1.41 1.43 1.45 1.49 1.50 1.53
Cl 4
cl 5
3
1.84 1.87 1.88 1.88 1.92 1.94
c d e f
(M-34)-
2 1
M-
I00 i00 i00 100
3 5 52 i00 i00 i00
1.61 1.64 1.65 1.68 1.69 1.70 1.71
C16 ~
(M-35)-
15
2
1 5 1 2
35
12 45
3 i00 3 i00 i00 i00 18
14
4 68 84 21 I00 4
i00 i00 i00 i00 51 i00
3 8 12 6 14
3
i00 75 I00 57 19 43 i00
C17
2.11
2
19
31
i00
Cl 8
2.41
5
32
30
i00
* r e t e n t i o n time r e l a t i v e to p e n t a c h l o r o b e n z e n e
F u r t h e r e x a m i n a t i o n s of the f r a g m e n t a t i o n p a t t e r n s r e v e a l e d an ion c o r r e s p o n d ing to (M-35)- for the di-, compounds common.
containing
tri- and t e t r a c h l o r i n a t e d naphthalenes.
For the
six to eight c h l o r i n e atoms the (M-34)- was more
The p e n t a c h l o r o n a p h t h a l e n e s
gave a m i x e d p a t t e r n in this region.
The
loss of 34 mass units in spectra f r o m p o l y c h l o r i n a t e d c o m p o u n d s has earlier been reported
(17) and is p r e s u m e d to c o r r e s p o n d to loss of a c h l o r i n e
r a d i c a l and a d d i t i o n of a h y d r o g e n radical. suggested 1,8-
for this p r o c e s s
(and/or 4,5-) p o s i t i o n s gave m o r e
chlorine
Several m e c h a n i s m s h a v e been
(18). The c o m p o u n d s w i t h c h l o r o s u b s t i t u e n t s
in
intense ions c o r r e s p o n d i n g to a
loss than their isomers.
The relative retentions
in T a b l e 1 i n d i c a t e that the closer the c h l o r i n e
a t o m s are p o s i t i o n e d in the molecule, a homologous
the longer is the r e t e n t i o n time w i t h i n
group. The c o m p o u n d s w i t h 1,8-
s e e m to h a v e extra long retention.
(and 4,5-)
chloro s u b s t i t u t i o n
626
B o t h r e t e n t i o n time and m a s s s p e c t r u m of the stable c o m p o u n d cate that no 1 , 8 - ( a n d / o r 4 , 5 - ) c h l o r o possible hexachloronaphthalenes f u l f i l l e d in two, (IV and III,
substitution
O f the ten
(Figure 3) this s u b s t i t u t i o n p a t t e r n
namely 1,2,3,5,6,7-
is
and 1 , 2 , 3 , 4 , 6 , 7 - h e x a c h l o r o n a p h t h a l e n e
respectively).
The c h l o r i n a t i o n of the 1 , 5 - d i a m i n o n a p h t h a l e n e hexa-,
(6a) thus indi-
is present.
gave three PCN congeners:
( r e t e n t i o n time r e l a t i v e to p e n t a c h l o r o b e n z e n e
1.84;
one
I M- = I00,
I(M_34 )- = 5, one hepta- and o c t a c h l o r o n a p h t h a l e n e . The y i e l d was low and no a t t e m p t s to isolate the p r o d u c t s w e r e made. The GC and MS p r o p e r t i e s of the o b t a i n e d HxCN, w h i c h on the b a s i s of the s y n t h e t i c p a t h w a y s
should have
the s t r u c t u r e IV, agreed v e r y well w i t h the c o r r e s p o n d i n g data f r o m the bioaccumulating
~
c,
c,
c,
c,
Cl
c o m p o n e n t in the PCN products.
c, y
Cl
y
Cl
I
c,
a
c,
a- ~
Cl
II
CI Cl Vl
F i g u r e 3.
" K ='
Cl
c,
c,- y
v
Q Cl VIII
c,
c,
a
c,-~,
Cl
III
CI Cl Vll
The ten p o s s i b l e
c,
CI
IV
V
Cl IX
X
s t r u c t u r e s of h e x a c h l o r o n a p h t h a l e n e .
The data p r e s e n t e d h e r e thus i n d i c a t e that the s t r u c t u r e of the p e r s i s t e n t compound
is 1 , 2 , 3 , 5 , 6 , 7 - H x C N .
This is in a n a l o g y w i t h the s t r u c t u r e s of the
P C D D / F s w h i c h s e e m to be m o s t p e r s i s t e n t 1,2,3,6,7,8-HxCDD a n i m a l s of p r e y
and 1 , 2 , 3 , 6 , 7 , 8 - H x C D F
in the environment.
Both
are d o m i n a n t among the P C D D / F s in
(12).
A m o n g the PBN found in the p o l y b r o m i n a t e d b i p h e n y l s one p e r s i s t e n t c o n g e n e r has b e e n i d e n t i f i e d as 2 , 3 , 4 , 5 , 6 , 7 - h e x a b r o m o n a p h t h a l e n e 1 , 2 , 3 , 5 , 6 , 7 - H x B N has p r e v i o u s l y b e e n s y n t h e s i s e d know,
(8). The
(15) but has,
not b e e n t e s t e d for p e r s i s t e n c e and b i o l o g i c a l activity.
as far as we
627
The similarities between the stable HxCN and the most potent congeners of the PCDD and PCDF families are alarming.
Even if 2,3,7,8-TeCDD was a thousand
times more potent than the HxCN the latter could be a major cause of the total effect due to much higher concentrations to synthesise 1,2,3,5,6,7-hexachloronaphthalene
in the environment.
Attempts
in amounts large enough for
biological testing are therefore underway.
ACKNOWLEDGEMENT
Parts of this study were supported by the Research Committee of the National Environmental
Protection Board.
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(Received in Germany 20 February 1986; accepted 5 March 1986)