BIOACCUMULATION OF
POLYCHLOROBIPHENYLS AND
RELATED HYDROPHOBIC CHEMICALS
IN FISH
W.A. Bruggeman
rijkswaterstaat
rijksinstituut voor zuivering van afvalwater
fl 85o>c?
BIOACCUMULATION OF
POLYCHLOROBIPHENYLS AND
RELATED HYDROPHOBIC CHEMICA
IN FISH
H!
RiJKSWAfERSTAAf
RIJKSWATERSTAAT
Dienst Binnenwateren RIZA
Maerlant 4-6
8224 A C
Postbus 17
8200 A A Lelystad
BIOACCUMULATION OF
POLYCHLOROBIPHENYLS AND
RELATED HYDROPHOBIC CHEMICALS
IN FISH
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van
doctor in de wiskunde en natuurwetenschappen
aan de Universiteit van Amsterdam,
op gezag van de rector magnificus
dr. D . W . Bresters
hoogleraar in de faculteit der
wiskunde en natuurwetenschappen,
in het openbaar te verdedigen
in de aula der universiteit
(tijdelijk in de Lutherse kerk,
ingang Singel 411, hoek Spui)
op woensdag 23 november 1983 te 16.00 uur
door
Willem Albert Bruggeman
geboren te's Gravenhage
1983
Offsetdrukkerij Kanters B . V . ,
Alblasserdam
PROMOTOR:
Uitgave:
PROF.
DR.
0.
HUTZINGER
Rijkswaterstaat
R i j k s i n s t i t u u t v o o r Z u i v e r i n g van
Postbus
17
8200 AA LELYSTAD - NL
Afvalwater
CONTENTS
Summary
7
I n l e i d i n g en s a m e n v a t t i n g
13
Chapter
27
1
Chapter 2
Hydrophobic
i n t e r a c t i o n s i n the a q u a t i c e n v i r o n m e n t
A c c u m u l a t i o n and e l i m i n a t i o n k i n e t i c s o f d i - , t r i - and
49
t e t r a c h l o r o b i p h e n y l s by g o l d f i s h a f t e r d i e t a r y and
aqueous
Chapter 3
exposure
Re v e r s e d - p h a s e
t h i n - l a y e r chromatography o f p o l y n u c l e a r
aromatic hydrocarbons
73
and c h l o r i n a t e d b i p h e n y l s :
R e l a t i o n s h i p w i t h h y d r o p h o b i c i t y as measured by aqueous
solubility
and o c t a n o l - w a t e r p a r t i t i o n
coefficient
Chapter 4
Bioaccumulation o f s u p e r - l i p o p h i l i c
Chapter 5
A b s o r p t i o n and r e t e n t i o n o f p o l y d i m e t h y l s i l o x a n e s
(silicones)
Chapter 6
i n f i s h : preliminary
by
7
pentachlorobenzene
p e n t a - 127
fish
Influence o f the c h l o r i n e s u b s t i t u t i o n p a t t e r n
dietary
i n tetra-
139
after
exposure
B i o a c c u m u l a t i o n and t r a n s f o r m a t i o n o f d i c h l o r o b i p h e n y l s
in
Dankwoord
111
and d e c a c h l o r o b i p h e n y l
c h l o r o b i p h e n y l s on t h e i r a c c u m u l a t i o n i n f i s h
Chapter 8
87
experiments
A b s o r p t i o n and e l i m i n a t i o n o f b r o m i n a t e d benzenes,
bromotoluene,
Chapter
chemicals i n f i s h
149
fish
159
5
SUMMARY
The
s u b j e c t of t h i s t h e s i s
biphenyls
was
(PCBs) and
i s the a c c u m u l a t i o n
r e l a t e d compounds i n f i s h .
the i d e n t i f i c a t i o n
Chapter
The
aim o f t h e
and q u a n t i f i c a t i o n o f t h e most
b i o l o g i c a l and p h y s i c o - c h e m i c a l
the b i o a c c u m u l a t i o n
of p o l y c h l o r i n a t e d
research
important
f a c t o r s and p r o c e s s e s t h a t i n f l u e n c e
of xenobiotic chemicals.
1 c o n t a i n s an i n t r o d u c t i o n i n the p h y s i c o - c h e m i c a l
partition
p r o c e s s e s which d e t e r m i n e t h e d i s t r i b u t i o n o f o r g a n i c c h e m i c a l s
a q u a t i c environment. Water s o l u b i l i t y ,
b i o s o r p t i o n and b i o a c c u m u l a t i o n
adsorption to organic m a t e r i a l ,
a r e r e l a t e d t o the h y d r o p h o b i c i t y
(measured as the o c t a n o l - w a t e r p a r t i t i o n
compound. In a d d i t i o n ,
may
several biological
coefficient)
o f the x e n o b i o t i c
f a c t o r s are i n t r o d u c e d t h a t
i n f l u e n c e the b i o a c c u m u l a t i o n k i n e t i c s i n f i s h ,
r a t e , membrane p e r m e a b i l i t y , b i o t r a n s f o r m a t i o n and
Emphasis i s on
i n the
the r e l a t i o n s h i p s between c h e m i c a l
such as
ventilation
food-chain
effects.
s t r u c t u r e and
p a r a m e t e r s , such as uptake and e l i m i n a t i o n r a t e s and
c o n s t a n t s , f o r the m o d e l l i n g o f the b i o a c c u m u l a t i o n
kinetic
equilibrium
process.
7
Chapter
2 d e s c r i b e s an e x p e r i m e n t a l
Uptake and e l i m i n a t i o n o f lower
c h l o r i n a t e d PCBs
c h l o r o b i p h e n y l ) a f t e r d i e t a r y and
s e p a r a t e l y . The
(di-, t r i -
and
f o o d was
very e f f i c i e n t ,
w i t h i n c r e a s i n g s o l u b i l i t y o f the
and b i o m a g n i f i c a t i o n f a c t o r s , h y d r o p h o b i c i t y and
tetra-
whereas
test
f a t content
are
o r d e r , m u l t i - compartment k i n e t i c model, by use
r e s i s t a n c e f a c t o r s and
a c t i v i t y c o e f f i c i e n t s f o r the d i f f e r e n t
ments. I t i s s u g g e s t e d
that food-chain
be
fish.
r e l a t i o n s h i p s between r a t e c o n s t a n t s , b i o c o n c e n t r a t i o n
explained i n a f i r s t
may
in
aqueous exposure were s t u d i e d
uptake from water and
clearance r a t e s decreased
compounds. The
approach o f b i o a c c u m u l a t i o n
an i m p o r t a n t p r o c e s s
accumulation
f o r extremely
of
compart-
(biomagnification)
hydrophobic
and
persistant
chemicals.
In c h a p t e r
3, v a r i o u s methods f o r measurement and
hydrophobicity o f apolar chemicals
a r e t e s t e d and
D i r e c t measurement o f o c t a n o l - w a t e r
partition
c a l c u l a t i o n o f the
compared.
coefficients
(P) i s
i m p r a c t i c a b l e f o r l o g -P > 5. E s t i m a t i o n o f h y d r o p h o b i c i t y v i a aqueous
solubility
(S) and m e l t i n g p o i n t i s r e s t r i c t e d t o compounds w i t h S -
values h i g h e r than
1
ug/1.
F o r PCBs, c a l c u l a t i o n o f l o g P from h y d r o p h o b i c
(tr-values) seems the most r e a s o n a b l e
n-values
The
substition
constants
approach, p r o v i d e d t h a t
different
a r e u s e d f o r c h l o r i n e atoms i n o r t h o , meta and p a r a
position.
r e t e n t i o n (Rm)
i n reversed-phase
t h i n - l a y e r chromatography shows a
good c o r r e l a t i o n w i t h l o g P o f d i f f e r e n t groups o f c h e m i c a l s
n u c l e a r aromatic
h y d r o c a r b o n s and
c o r r e l a t i o n s are observed
I t i s concluded
s u b s t i t u t e d benzenes).
(poly-
Deviating
for ortho-substituted chlorobiphenyls.
t h a t a reversed-phase
r e t e n t i o n i n d e x can be
a very
u s e f u l parameter f o r q u a n t i t a t i v e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s i n
environmental
Chapter
and
toxicological studies.
4 deals w i t h the bioaccumulation
chemicals
(log P >
Apparently,
p o t e n t i a l of
super-lipophilic
6.5).
t h e uptake e f f i c i e n c y o f h i g h e r c h l o r i n a t e d b i p h e n y l s
water i s d e c r e a s i n g , whereas t h e a b s o r p t i o n from i n g e s t e d f o o d
The
8
e l i m i n a t i o n o f hexa-, o c t a - and
decachlorobiphenyl
from
remains.
from f i s h i s
n e g l i g i b l e . This combination
accumulation
may r e s u l t i n a r e a l
( b i o m a g n i f i c a t i o n ) o f these
b i o c o n c e n t r a t i o n by l i p i d - w a t e r
S e v e r a l h i g h l y hydrophobic
food-chain
chemicals, i n s t e a d o f d i r e c t
partitioning.
compounds a r e n o t a c c u m u l a t e d
i n living
fish
(hexabromobenzene, o c t a c h l o r o d i b e n z o - p - d i o x i n , p e r c h l o r o t e r p h e n y l ) ,
which c a n be a t t r i b u t e d t o t h e i r m o l e c u l a r
penetration
into living
size hindering the
cells.
In c h a p t e r 5, a t t e n t i o n i s p a i d t o p o l y d i m e t h y l s i l o x a n e s (PDMS;
s i l i c o n e s ) . The h y d r o p h o b i c i t y o f o l i g o m e r i c PDMS (5-11 S i O - u n i t s ) , as
e s t i m a t e d by r e v e r s e d - p h a s e
l i q u i d chromatography, i s comparable t o
t h a t o f PCBs. However, r e s i d u e s o f PDMS i n f i s h
a r e v e r y low. P r o b a b l y ,
structural factors
bioaccumulation o f the c y c l i c oligomers
compounds c o n t a i n i n g more than
d e t a i l . A sharp d e c l i n e i s observed
tetra-
benzenes
t o penta-
(molecular size)
preclude
and o f t h e l i n e a r PDMS
s i z e i s i n v e s t i g a t e d i n more
i n the absorption e f f i c i e n c y o f
( a f t e r d i e t a r y exposure),
g o i n g from t r i - and
and hexabromobenzene. Comparison w i t h
b i p h e n y l i n t h i s experiment
exposure
ten SiO-units.
In c h a p t e r 6, t h e i n f l u e n c e o f m o l e c u l a r
brominated
after dietary
decachloro-
showed t h a t t h i s phenomenon cannot be
r e l a t e d t o the h y d r o p h o b i c i t y o r m o l e c u l a r w e i g h t o f t h e compounds.
The
b e s t parameter t o e x p l a i n the d i f f e r e n c e s
t o be t h e ' e f f e c t i v e d i a m e t e r '
Chapter
in
i n uptake k i n e t i c s
seems
o f the molecule.
7 describes the i n f l u e n c e o f the c h l o r o - s u b s t i t u t i o n pattern
t e t r a c h l o r o b i p h e n y l s on t h e i r a c c u m u l a t i o n
Most s t r i k i n g was t h e l a c k o f a c c u m u l a t i o n
i n fish.
o f 2,2',6,6'-tetrachloro-
b i p h e n y l and t h e r e l a t i v e l y h i g h c l e a r a n c e r a t e o f t h e 2,2'3,3'-isomer
(half l i f e
c a . 5 days) i n c o m p a r i s o n w i t h t h e o t h e r i s o m e r s
(tij 45 days).
These d i f f e r e n c e s a r e n o t c o m p l e t e l y e x p l a i n e d by t h e h y d r o p h o b i c i t y ,
b u t must be a t t r i b u t e d t o s t e r i c
f a c t o r s , t h a t may i n f l u e n c e a c t i v e
e x c r e t i o n o f t h e compounds.
In c h a p t e r 8, the i n f l u e n c e o f t r a n s f o r m a t i o n p r o c e s s e s on t h e t h e
b i o a c c u m u l a t i o n and e l i m i n a t i o n o f 2,5- and 4 , 4 ' - d i c h l o r o b i p h e n y l i s
9
q u a n t i f i e d . During
aqueous e x p o s u r e , a s i g n i f i c a n t amount o f b o t h
d i c h l o r o b i p h e n y l s was c o n v e r t e d
addition,
2,5-dichlorobenzoic
formation product
i n t o h y d r o x y l a t e d m e t a b o l i t e s . In
acid
(methyl
e s t e r ) was found
as a t r a n s -
2 , 5 - d i c h l o r o b i p h e n y l . The p r o d u c t i o n o f t h e s e
b o l i t e s , however, a c c o u n t e d
f o r l e s s than
meta-
30 p e r c e n t o f t h e t o t a l
e l i m i n a t i o n o f t h e d i c h l o r o b i p h e n y l s from t h e f i s h , and i s t h e r e f o r e
o f minor i m p o r t a n c e i n r e g u l a t i n g t h e b i o c o n c e n t r a t i o n o f t h e s e
chemicals.
The
f o l l o w i n g g e n e r a l c o n c l u s i o n s can be drawn from t h e e x p e r i m e n t a l
r e s u l t s and t h e o r e t i c a l c o n s i d e r a t i o n s .
1.
In p r i n c i p l e ,
chemicals
the b i o c o n c e n t r a t i o n f a c t o r o f apolar, p e r s i s t e n t
i n f i s h i s d i r e c t l y r e l a t e d t o the h y d r o p h o b i c i t y , as
measured by t h e o c t a n o l - w a t e r
The
partition
c o e f f i c i e n t P (up t o l o g P » 6 ) .
c l e a r a n c e r a t e i s most s e n s i t i v e t o the h y d r o p h o b i c i t y o f t h e
compounds and t h e l i p i d
content o f the f i s h .
2. The b i o c o n c e n t r a t i o n o f ' s u p e r - l i p o p h i l i c
is
r e s t r i c t e d by k i n e t i c
limitations,
1
chemicals
( l o g P>5.5)
i . e . , t h e maximum uptake r a t e
( r e l a t e d t o the v e n t i l a t i o n r a t e and t r a n s f e r e f f i c i e n c y )
and the
minimum ' c l e a r a n c e r a t e ' , as a r e s u l t o f d i l u t i o n by growth and
r e p r o d u c t i o n . I n a d d i t i o n , t h e a v a i l a b i l i t y o f the d i s s o l v e d
compounds i s d e c r e a s e d
3. F o o d - c h a i n
due t o the extreme h y d r o p h o b i c i t y .
accumulation
( b i o m a g n i f i c a t i o n ) can be t h e most
route f o r the bioaccumulation
fish,
of 'super-lipophilic'
depending on f e e d i n g h a b i t s and m e t a b o l i c
(e.g., t r o p h i c l e v e l ,
assimilation
important
chemicals i n
characteristics
efficiency).
4. The h y d r o p h o b i c i t y o f a p o l a r compounds can be e s t i m a t e d by d i r e c t
measurement o f o c t a n o l - w a t e r
partition
coefficients,
from aqueous
solubility
and m e l t i n g p o i n t d a t a , by c a l c u l a t i o n u s i n g
fragmental
o r s u b s t i t u t i o n constants
determination
o f reversed-phase
indices. For 'super-lipophilic'
hydrophobic
( T T - o r f - v a l u e s ) , o r by
l i q u i d chromatography r e t e n t i o n
chemicals,
only the l a s t
two methods
remain and may be used i n c o m b i n a t i o n .
5. M o l e c u l a r s i z e
(i.e.,
the e f f e c t i v e m o l e c u l a r
absorption o f chemicals
10
from water and f o o d .
diameter) can r e s t r i c t
6. B i o t r a n s f o r m a t i o n
can a c c e l e r a t e t h e e l i m i n a t i o n o f c e r t a i n
compounds, b u t i s p r o b a b l y
o f minor i m p o r t a n c e f o r t h e
organic
accumulation
o f most p o l y c h l o r o b i p h e n y l s i n f i s h .
7. B i o a c c u m u l a t i o n
tests should
be b a s e d on d i r e c t
a t l e a s t i n c l u d e a c l e a r a n c e p e r i o d and
comparison with
reference
compounds.
11
INLEIDING EN SAMENVATTING
Het
onderwerp van d i t p r o e f s c h r i f t i s de b i o - a c c u m u l a t i e
c h l o o r b i f e n y l e n en verwante h y d r o f o b e
v i s s e n . Het
de
onderzoek d a t h i e r i n wordt b e s c h r e v e n
fysisch-chemische
lijk
en b i o l o g i s c h e p r o c e s s e n
z i j n v o o r de b i o - a c c u m u l a t i e ,
v e r s c h i j n s e l bio-accumulatie
s c h i l l e n d e kanten
De
oudste
i s i n de
de
chemische
stoffen.
l o o p d e r j a r e n van
de b e s c h r i j v i n g d i e h e t
"voedselketenmet
i n d i e n afbraak- o f u i t s c h e i d i n g s m o g e l i j k h e d e n ontbreken,
z i c h op i n h e t o f g a n i s m e . Wanneer h e t d i e r op
g e g e t e n wordt, komen de
proces
het
voedsel;
hopen
zijn
de
beurt
s t o f f e n t e r e c h t i n de v o l g e n d e s c h a k e l
van
waar nog hogere g e h a l t e n opgebouwd worden. D i t
wordt wel b i o m a g n i f i c a t i e genoemd. V e r b i n d i n g e n
( g e b r u i k t a l s i n s e c t i c i d e ) en PCB's
schillende industriele
als
DDT
( p o l y c h l o o r b i f e n y l e n , met
toepassingen), die p e r s i s t e n t z i j n
a f b r e e k b a a r ) , en b o v e n d i e n l i p o f i e l
goed i n v e t )
ver-
belicht.
papieren heeft
de v o e d s e l k e t e n ,
op
d i e verantwoorde-
i n samenhang met
e f f e c t " benadrukt: d i e r e n k r i j g e n c h e m i c a l i e n binnen
stoffen
polyin
richt zich
s t r u c t u u r van de b e t r e f f e n d e m i l i e u v r e e m d e o r g a n i s c h e
Het
van
chemische v e r b i n d i n g e n
(slecht oplosbaar
ver-
(slecht
i n w a t e r , maar
zouden zo de h o o g s t e c o n c e n t r a t i e s b e r e i k e n i n h e t
l i c h a a m s v e t van
top-predatoren.
Voor h e t a q u a t i s c h m i l i e u ,
waarin
u i t e i n d e l i j k v e e l door de mens v e r s p r e i d e c h e m i c a l i e n t e r e c h t komen,
betekent
d i t d a t r e l a t i e f hoge g e h a l t e n worden gevonden i n v i s e t e n d e
v o g e l s en
zeezoogdieren.
B i j bovenstaande b e s c h r i j v i n g wordt nog
l a t e n op welke w i j z e de
van
een
voedselketen.
de v r a a g i n h e t midden
s t o f f e n t e r e c h t komen b i j de e e r s t e
De
slibdeeltjes,
door a d s o r p t i e en p a r t i t i e p r o c e s s e n zou h i e r b i j een
tweede b e n a d e r i n g
gaat geheel
schakels
u i t w i s s e l i n g tussen v e r s c h i l l e n d e "comparti-
menten" van h e t a q u a t i s c h m i l i e u (water,
De
organismen)
r o l kunnen s p e l e n .
van h e t v e r s c h i j n s e l b i o - a c c u m u l a t i e
(sinds
u i t van deze v e r d e l i n g s p r o c e s s e n . Lichaamsvreemde
zouden door v i s s e n g e l i j k met
opgenomen. Door m i d d e l
ge-
1970)
stoffen
z u u r s t o f u i t h e t w a t e r kunnen worden
van kieuwen en b l o e d zou u i t w i s s e l i n g
l i c h a a m s v e t en water kunnen p l a a t s v i n d e n , u i t e i n d e l i j k
tussen
leidend tot
13
een
e v e n w i c h t s s i t u a t i e . We
tor
lipofiliteit
Het
e e r s t e h o o f d s t u k van
nis
van
is hier
s p r e k e n dan
P,
de
i n het
lipofiliteit
de v e r d e l i n g s c o e f f i c i e n t i n h e t
i n water
d i e de v e r h o u d i n g t u s s e n
c
de
d i t p r o e f s c h r i f t g a a t nader i n op de
hydrofobe verdelingsprocessen
water) , de o p l o s b a a r h e i d
(K ),
bioconcentratie,-
fac-
allesbepalend.
s c h e t s t het verband tussen
als
van
aquatisch
van
een
stof
en
(uitgedrukt
twee-fasen systeem
( S ) ; en
beteke-
milieu
octanol-
de b i o c o n c e n t r a t i e f a c t o r
de c o n c e n t r a t i e s
i n v i s en w a t e r b i j
evenwicht weergeeft.
Bij
v o o r k e u r z a l v e r d e r worden g e s p r o k e n van
van
lipofiliteit,
oplosbaarheid
d a a r de
de
eens verward wordt met
de
i n vet.
De h y d r o f o b i t e i t van
h e i d van
l a a t s t e term wel
"hydrofobiteit" in plaats
een
verbinding
i s a f h a n k e l i j k van
de
aanwezig-
a p o l a i r e , n i e t d i s s o c i e e r b a r e groepen i n h e t m o l e c u u l
afwezigheid
van
t e r a c t i e s tussen
en
p o l a i r e o f g e l a d e n g r o e p e n . Door de s t e r k e i n -
w a t e r m o l e c u l e n o n d e r l i n g worden n e u t r a l e ,
apolaire
s t r u c t u r e n a l s h e t ware u i t de w a t e r f a s e geduwd. Daardoor v e r z a m e l e n
h y d r o f o b e s t o f f e n z i c h i n a p o l a i r e media
oplosmiddelen, vet)
d e e l t j e s van
baarheid
en
aan
organische
i n water i s v a n z e l f s p r e k e n d
( a l s l o g P)
voor i e d e r e
een
des
In p r i n c i p e i s deze
t e b e r e k e n e n , u i t g a a n d e van
een
vaste
bijdrage
(de T T - o f f-waarde) .
a f z o n d e r l i j k e groep
r e l a t i e s tussen
lipofiliteit,
oplos
i n water en b i o c o n c e n t r a t i e f a c t o r door v e r s c h i l l e n d e o n d e r -
zoekers g l o b a a l bevestigd.
riaal
vaste
oplos-
g r o t e r a p o l a i r e groepen,
verbinding.
E x p e r i m e n t e e l i s h e t b e s t a a n van
baarheid
oppervlakken die binnen
laag.
meer en hoe
hoger de h y d r o f o b i t e i t van
grootheid
water mengbare
o o r s p r o n g gevonden kunnen worden. De
In h e t algemeen g e l d t : hoe
te
de n e u t r a l e
( n i e t met
i n water
verbindingen
(slib,
Ook
de
adsorptie
sedimenten) b l i j k t met
aan
n i e t - o p g e l o s t mate-
de h y d r o f o b i t e i t van
samen te hangen, wanneer de a d s o r p t i e c o e f f i c i e n t (K
de
)
oc
wordt u i t g e d r u k t
materiaal.
14
op b a s i s van
organisch
k o o l s t o f i n het
adsorberend
Door deze r e l a t i e s
in
zou de l i g g i n g van d i v e r s e
verdelingsevenwichten
h e t a q u a t i s c h m i l i e u i n p r i n c i p e kunnen worden b e r e k e n d ,
uitgaan-
de v a n de m o l e c u u l s t r u c t u u r v a n de s t o f . De weg n a a r m o d e l l e r i n g en v o o r s p e l l i n g van een b e l a n g r i j k a s p e c t van h e t g e d r a g van m i l i e u v r e e m d e v e r bindingen,
z e l f s van nog n i e t g e p r o d u c e e r d e , i s daarmee i n p r i n c i p e open.
In de p r a k t i j k b l i j k e n e r e c h t e r n o g a l wat haken en ogen aan d i e v o o r s p e l
ling
t e z i t t e n , met name a l s h e t g a a t om h e t v e r s c h i j n s e l
bio-accumulatie
K w a l i t a t i e f mag e r dan een en ander bekend z i j n o v e r de f a c t o r e n d i e een
rol
kunnen s p e l e n i n h e t b i o - a c c u m u l a t i e p r o c e s ,
afbreekbaarheid, k w a n t i t a t i e f l i g t
z o a l s l i p o f i l i t e i t en
d i t v e e l m o e i l i j k e r . S p e e l t op-
name v a n s t o f f e n v i a h e t v o e d s e l nu w e l o f n i e t een r o l van b e t e k e n i s ?
Is
de u i t w i s s e l i n g t u s s e n organisme en w a t e r w e l zo e f f e c t i e f ?
Wanneer g a a t de o m z e t t i n g s s n e l h e i d van een s t o f d o o r h e t organisme
meetellen?
Kunnen a l l e m o l e c u l e n
ongeacht de g r o o t t e nog worden op-
genomen d o o r h e t o r g a n i s m e , o f i s e r een k r i t i s c h e
Uitzonderingen
mulatie
op de g l o b a i e r e l a t i e
l i j k e n wel voor
grens
aan t e geven?
t u s s e n h y d r o f o b i t e i t en b i o - a c c u -
t e komen; deze kunnen de algemene r e g e l s l e c h t s
b e v e s t i g e n wanneer volkomen d u i d e l i j k i s waarom h e t u i t z o n d e r i n g e n
Kan
de h y d r o f o b i t e i t van de r e l e v a n t e s t o f f e n w e l goed genoeg
zijn.
vastge-
s t e l d worden?
Over deze v r a g e n ,
d i e a l l e b e t r e k k i n g hebben op de m o g e l i j k h e d e n
om
h e t g e d r a g van een s t o f i n h e t m i l i e u t e v o o r s p e l l e n aan de hand van
zijn
chemische s t r u c t u u r en f y s i s c h - c h e m i s c h e
volgende hoofdstukken
Door m i d d e l
e i g e n s c h a p p e n , gaan de
van d i t p r o e f s c h r i f t .
van l a b o r a t o r i u m e x p e r i m e n t e n
met v i s s e n en v e r s c h i l l e n d e
reeksen
hydrofobe,
milieuvreemde verbindingen
wordt
g e t r a c h t meer
inzicht
te verkrijgen i n het v e r s c h i j n s e l bio-accumulatie
en de f a c -
t o r e n d i e daarop van i n v l o e d z i j n . A l s t e s t v e r b i n d i n g e n z i j n
ste
i n eer-
i n s t a n t i e p o l y c h l o o r b i f e n y l e n (PCB's) g e k o z e n . Deze groep v e r b i n -
dingen
s t a a t bekend a l s z e e r s l e c h t a f b r e e k b a a r
sterk
bio-accumulerend.
( p e r s i s t e n t ) en
(De m o n d i a l e v e r s p r e i d i n g van PCB's i n h e t m i l i e u werd p a s bekend
doordat
ze werden aangetoond i n h e t v e t w e e f s e l van v i s s e n en v i s -
etende d i e r e n . O p z e t t e l i j k e v e r s p r e i d i n g d o o r de mens i n h e t m i l i e u ,
zoals dat b i j bestrijdingsmiddelen geschiedt, s p e e l t overigens b i j
PCB's geen r o l van b e t e k e n i s . Wel b e s t a a n
e r een a a n t a l
industriele
15
toepassingen.
fen
Door lekkage
en
a l s a f v a l na g e b r u i k
t o c h i n h e t water t e r e c h t k o m e n . Nog
e e n v o u d i g om
dingsgewijs
PCB's aan
zeer lage
steeds
kunnen deze
i s het echter
t e tonen i n de w a t e r f a s e
vanwege de
stof
niet
verhou-
concentraties.)
PCB's z i j n b e s l i s t n i e t r e p r e s e n t a t i e f v o o r a l l e k l a s s e n van m i l i e u vreemde o r g a n i s c h e
s t o f f e n ; wel
l a t i e b e l a n g r i j k e aspecten
dingen.
Door v e r s c h i l l e n i n mate van
molecuul ontstaan
die
hun
Vrijwel alle
deze groep v e r b i n -
c h l o r e r i n g van
u i t w e r k i n g kunnen hebben op h e t
V e r g e l i j k i n g met
a a n t a l v o o r bio-accumu-
worden met
v e r s c h i l l e n i n molecuulgrootte
het
i s n o d i g om
op v e r g e l i j k i n g van
menten i n h e t
testsysteem
tijd.
experimenten z i j n
l a g e c o n c e n t r a t i e s aan
comparti
en op b e p a l i n g en m o d e l l e r i n g van
het
t e s t s t o f f e n i n water en v i s , z i j n
onmisbaar. G a s c h r o m a t o g r a f i e
tie
(GC-ECD) i s i n h e t algemeen voldoende g e v o e l i g en
met
"electronen-invangst"
zo gekozen d a t h e t
gepakte kolommen v o o r de
scheidend
gaschromatografie
l a i r e kolommen, met
hun
de b e p a l i n g van
a a n t a l isomeren i n een
daardetec-
De
vermogen
testvan
voldoende i s . C a p i l -
grotere e f f i c i e n t i e ,
en b i j s t o f f e n waarvoor een
van
specifiek
c h l o o r - en broomhoudende v e r b i n d i n g e n .
z i j n meestal
z i j n gebruikt b i j
analysegang
(hst. 7),
s p e c i f i e k e detector ontbreekt
conen, h s t . 5 ) . De
combinatie
metrie
computer wordt t e n s l o t t e t e h u l p
(GC-MS) met
gebaseerd
Analysemethoden, g e s c h i k t voor h e t meten
bij
een
alge-
concentraties i n v e r s c h i l l e n d e fasen of
v e r l o o p i n de
stoffen
de
de gevonden verbanden te c o n t r o l e r e n .
beschreven bio-accumulatie
v o o r de g e b r u i k t e
bifenyl-
en h y d r o f o b i t e i t ,
bio-accumulatieproces.
andere k l a s s e n v e r b i n d i n g e n
mene g e l d i g h e i d van
de
kunnen een
bestudeerd
(sili-
gaschromatografie-massaspectrogeroepen
v o o r s t r u c t u u r - o p h e l d e r i n g en - b e v e s t i g i n g i n t w i j f e l g e v a l l e n .
V l o e i s t o f c h r o m a t o g r a f i s c h e methoden
en h o g e d r u k - v l o e i s t o f c h r o m a t o g r a f i e :
b r u i k t v o o r b e p a l i n g van
("reverse
RP-TLC en
de h y d r o f o b i t e i t van
De benodigde c h e m i c a l i e n van
16
gezuiverd.
RP-HPLC) z i j n
de
ge-
teststoffen.
voldoende z u i v e r h e i d z i j n
deels commercieel v e r k r i j g b a a r ; enkele
t h e t i s e e r d en/of
phase" dunne l a a g -
groten-
werden s p e c i a a l g e s y n -
De
b i o - a c c u m u l a t i e - e x p e r i m e n t e n b e s c h r e v e n i n h o o f d s t u k 2 hebben
betrekking
op
bifenylen).
l a a g g e c h l o r e e r d e PCB's
De
nadruk l i g t
( d i - , t r i - en
h i e r b i j op
de
ontwikkeling
methoden, zowel p r a k t i s c h a l s t h e o r e t i s c h , om
p r o c e s t e b e s t u d e r e n . Dat
van
begint
met
een
toediening
v i s s e n v i a h e t water v e r g e l e k e n met
Goudvissen
Uit
(Carassius
deze e e r s t e
di-,
t r i - en
de
het
(welhaast een
van
de
v i s s e n . Het
tor,
K
d o o r de
kan
continu
ververst
aan
voedsel.
de
gebruikt.
onderzochte
n i v e a u van
(de
het
uiteinde-
bioconcentratiefacvoer
(de
biomagni-
e x p o s i t i e worden b e i d e s t e r k
eliminatie-snelheidsconstante
v o o r de
verschillende
bepaald
stoffen
a f z o n d e r l i j k worden gemeten i n een
experiment, waarbij v i s s e n die
in
v i a het
c o n c e n t r a t i e v e r h o u d i n g t u s s e n v i s en
Deze l a a t s t e g r o o t h e i d
principieel
zowel u i t v o e d s e l a l s u i t w a t e r z e e r
t e b e r e i k e n e v e n w i c h t t u s s e n v i s en w a t e r
langdurige
oplossing
als proefdieren
lijk
f i c a t i e f a c t o r , K ) na
m
geschikte
teststoffen
toediening
auratus) z i j n h i e r b i j
tetrachloorbifenylen
de
een
experimenten v a l t te concluderen dat
e f f i c i e n t worden opgenomen d o o r de
) en
van
bio-accumulatie-
methode om
hydrofobe s t o f f e n i n water te k r i j g e n
p r o b l e e m ) ; v e r v o l g e n s wordt de
tetrachloor-
" o p g e l a d e n " z i j n met
de
"schoon" water worden gehouden om
hoe
s n e l ze de
van
10 dagen v o o r 2 , 5 - d i c h l o o r b i f e n y l ,
s t o f f e n weer k w i j t r a k e n . De
2
testverbindingen
te
bepalen
halfwaarde-tijd
t o t 60
(k )
eliminatie
varieerde
dagen v o o r 2,3',4*,5-
tetrachloorbifenyl.
Voor een
eenvoudige b e s c h r i j v i n g van
( s n e l h e i d en e v e n w i c h t s l i g g i n g
lingsmodel i n principe geschikt.
en w a t e r e l k a l s een
binding
z i c h van
r e d i g g e a c h t met
Dit
extra
De
c o m p a r t i m e n t ; de
concentratie
model werd v o o r de
ratuur
bio-accumulatieproces
eerste
orde
uitwisse-
H i e r b i j wordt u i t g e g a a n van
water n a a r v i s en
de
het
) b l i j k t een
s n e l h e i d waarmee een
vis
ver-
omgekeerd v e r p l a a t s t wordt e v e n -
i n het betreffende
u i t w i s s e l i n g met
b e s c h r e v e n ; t o e g e v o e g d wordt nu
compartiment.
w a t e r a l e e r d e r i n de
de
opname van
lite-
voedsel a l s
bron.
individuele variatie
b e l a n g r i j k deel
verschillende
v o l d o e n d e om
heerbare
vissen
de
i n de
te v e r k l a r e n
"op
een
g e h a l t e n i n de
u i t een
lijn
vissen
i s voor
v e r s c h i l i n vetgehalte.
te brengen" i s het
echter
g e h a l t e n i n v i s u i t t e drukken op b a s i s van
een
Om
niet
extra-
vetten.
17
Wanneer aangenomen wordt d a t h e t
verbindingen
f u n g e e r t , kan
lichaamsvet
op g r o n d van
a l s o p s l a g voor
t h e o r e t i s c h e overwegingen
a f g e l e i d worden d a t de a f g i f t e s n e l h e i d van een
evenredig
z a l z i j n met
f o b i t e i t van
h e t v e t g e h a l t e van
s t o f omgekeerd
de v i s en de
hydro-
de v e r b i n d i n g .
In h e t v o o r g e s t e l d e k i n e t i s c h e model wordt de u i t w i s s e l i n g
verschillende
( v i s ) compartimenten b e p a a l d
door
f a c t o r e n voor het passeren
van
tussen
activiteitscoeffi-
c i e n t e n v o o r de v e r s c h i l l e n d e f a s e n i n c o m b i n a t i e
De
de
met
de g r e n z e n t u s s e n de
weerstands-
compartimenten.
v e r v e r s i n g s s n e l h e i d van h e t water d a t l a n g s de kieuwen
i s mede b e p a l e n d
v o o r de u i t w i s s e l i n g s s n e l h e i d van
PCB's; de z u u r s t o f b e h o e f t e van
de
stroomt
onderzochte
de v i s b e l n v l o e d t d a a r d o o r
direct
de o p n a m e s n e l h e i d u i t h e t w a t e r , maar n i e t de e v e n w i c h t s l i g g i n g
(bioconcentratiefactor) .
Zolang
de e f f i c i e n t i e
van
r e l a t i e f hoog b l i j f t
opname van
(zoals voor
een
PCB's), z a l een hogere h y d r o f o b i t e i t ook
f i c a t i e veroorzaken,
doordat
stoffen
een
laaggechloreerde
s t e r k e r e biomagni-
de a f g i f t e s n e l h e i d l a g e r i s . Daardoor
zou de b i j d r a g e v i a de v o e d s e l k e t e n
hydrofobe
verbinding u i t voedsel
de o n d e r z o c h t e
i n de a c c u m u l a t i e
(log P octanol-water
van
> 5) h e t a a n d e e l
d i r e c t e b i o c o n c e n t r a t i e ( v i a u i t w i s s e l i n g met
zeer
van
water) kunnen
de
over-
t r e f fen.
Cp g r o n d van
s c h a t t i n g e n aan
worden d a t de l i p o f i l i t e i t
en met
de hand l i t e r a t u u r g e g e v e n s mag
van h o o g g e c h l o r e e r d e
b i f e n y l e n (hexa- t o t
d e c a c h l o o r b l f e n y l ) , u i t g e d r u k t a l s de o c t a n o l - w a t e r
coefficient
b i e d (max.
(P) z a l o p l o p e n
l o g P = 4-5).
aangeduid a l s
verwacht
partitie-
t o t v e r b u i t e n h e t d i r e c t meetbare
ge-
D e r g e l i j k e s t o f f e n worden i n d i t p r o e f s c h r i f t
"super-lipofiel".
Betrouwbare k w a n t i t a t i e v e gegevens b e t r e f f e n d e de h y d r o f o b i t e i t
de b i o - a c c u m u l a t i e - k i n e t i e k van
Wel
worden nog
dergelijke verbindingen
s t e e d s hoge g e h a l t e n
aan v e r s c h i l l e n d e hexa- en
chloorbifenylen aangetroffen i n waterdieren
iandse wateren, geanalyseerd
onderzoek).
18
en
ontbreken
(o.a. a a l i n de
door h e t R i j k s i n s t i t u u t v o o r
echter
hepta-
Neder-
Visserij-
In h o o f d s t u k 3 worden v e r s c h i l l e n d e methoden v e r g e l e k e n om
a a n v a a r d b a r e s c h a t t i n g van
verbindingen
h y d r o f o b i t e i t van s u p e r - l i p o f i e l e
m e t i n g van
de
v e r s c h i l l e n d e mono- en
substitutie-constante
stellen
een
te komen.
Door r e c h t s t r e e k s e
(P) van
de
tot
voor c h l o o r
(TT) , waarmee de
O p v a l l e n d i s het
0-tX/lO-positie
octanol-water
(2 o f 6)
i n h e t b i f e n y l systeem v a s t
l o g P van
afwijkend
partitiecoefficient
d i c h l o o r b i f e n y l e n wordt g e t r a c h t
hogere PCB's b e r e k e n d kan
g e d r a g van
ten opzichte
b i f e n y l e n met
van
tussen
te
worden.
chloor
de b i n d i n g
een
in
de
de
fenyl-
ringen.
De
berekende l o g P-waarden van
zienlijk
l a g e r u i t dan
b i f e n y l met
een
de hogere PCB's v a l l e n d a a r d o o r aan-
v e r w a c h t ; t o c h kan
berekende l o g P van
^9.6
een
met
stof als
recht
decachloor-
"super-lipofiel"
genoemd worden.
V e r v o l g e n s wordt de waarde van
grafie a l s referentiesysteem
u i t w i s s e l i n g tussen
en
de
de
s t a t i o n a i r e fase
tuur) bepaalt
" r e v e r s e d - p h a s e " dunnelaag-chromato-
v o o r de h y d r o f o b i t e i t b e p r o e f d .
loopvloeistof
(C ,
d a a r b i j de
een
1 8
(een m e t h a n o l - w a t e r mengsel)
a p o l a i r e , c h e m i s c h gebonden s t r u c -
loopsnelheid
van
s t a a t model voor de h y d r o f o b i t e i t . Een
w a a r b i n n e n de h y d r o f o b i t e i t en
lopen,
r e t e n t i e - i n d e x kan
Er b l i j k t
en
de
wordt a l s r e f e r e n t i e - r e e k s
s t o f een
een
De
de
testverbinding
en
s e r i e n-alkyl-benzenen,
r e t e n t i e f a c t o r regelmatig
gebruikt,
z o d a t van
op-
iedere
test-
worden b e p a a l d .
d u i d e l i j k verband te z i j n
de berekende l i p o f i l i t e i t
( l o g P)
tussen
de
retentie-index
voor v e r s c h i l l e n d e
groepen
s t o f f e n , waaronder p o l y c y c l i s c h e a r o m a t i s c h e k o o l w a t e r s t o f f e n
c h l o o r b e n z e n e n . De
dan
men
op
r e t e n t i e - i n d e x van
grond van
chloor; het
PCB's i s e c h t e r
e f f e c t i s nog
de
geringe bijdrage
s t e r k e r dan
voor
lijkt
OUtho-
van
b i j octanol-water
i s d a a r d o o r n i e t z o n d e r meer m o g e l i j k om
s t o f f e n een
lager
de berekende l o g P zou v e r w a c h t e n . D i t
weer v o o r a l samen t e hangen met
Het
de
en
partitie.
willekeurige
octanol-water p a r t i t i e c o e f f i c i e n t te berekenen u i t
de
reversed-phase r e t e n t i e - i n d e x . Er i s echter
om
b i n n e n een
bepaalde k l a s s e
verbindingen
de
w e i n i g op
tegen
retentie-index
z e l f s t a n d i g te gebruiken voor k w a n t i t a t i e v e s t r u c t u u r - a c t i v i teitsrelaties
(QSAR). Omgekeerd zou een
b e r e k e n i n g van
de
lipo-
19
filiteit
leiden
( l o g P) v o o r een bekende k l a s s e
t o t v o o r s p e l l i n g van de r e t e n t i e i n een
chromatografie
Tot
van v e r b i n d i n g e n kunnen
"reversed-phase"
systeem.
op z e k e r e hoogte
kan ook
aan de hand van de o p l o s b a a r h e i d
i n water een betrouwbare s c h a t t i n g van de h y d r o f o b i t e i t worden
gemaakt, i n d i e n ook met
h e t s m e l t p u n t van d i e s t o f r e k e n i n g wordt
gehouden. De o p l o s b a a r h e i d van
s u p e r - l i p o f i e l e verbindingen i n
(beneden 1 ug/1)
water i s e c h t e r dermate l a a g
d a t aan de m o g e l i j k -
h e i d van een betrouwbare en r e p r o d u c e e r b a r e m e t i n g moet worden
t w i j f e l d . Voor de h o o g g e c h l o r e e r d e
b i f e n y l e n wordt daarom
ge-
voorlopig
de nieuwe berekende l o g P aangehouden a l s b e s t e s c h a t t i n g van
de
hydrofobiteit.
De b i o - a c c u m u l a t i e van de hogere
aantal
andere
aromaten),
komt aan de orde
i s v e r g e l i j k b a a r met
v i s s e n maar guppen
dieren.
PCB's, i n v e r g e l i j k i n g met
hydrofobe milieuvreemde
i n hoofdstuk
die u i t hoofdstuk
4. De e x p e r i m e n t e l e
v a r i a t i e wordt v e r m i n d e r d .
opzet
2, z i j h e t d a t nu geen goudals proef-
t e b e s c h i k k e n o v e r een
b i n n e n de b e p e r k i n g e n
waardoor de s p r e i d i n g i n de metingen
een
(gehalogeneerde
( P o e c i l i a r e t i c u l a t a ) gekozen z i j n
D i t maakt h e t m o g e l i j k om
a a n t a l exemplaren
verbindingen
groter
van h e t t e s t s y s t e e m ,
t e n g e v o l g e van
individuele
Door g e b r u i k van r e f e r e n t i e s t o f f e n
(pentachloorbenzeen, 2 , 5 - d i c h l o o r b i f e n y l
b i f e n y l ) wordt de v e r g e l i j k b a a r h e i d
en 2 , 2 ' , 5 , 5 ' - t e t r a c h l o o r -
van v e r s c h i l l e n d e
experimenten
bevorderd.
Z o a l s verwacht
i s de i n v l o e d van de g r o t e h y d r o f o b i t e i t van hoog-
gechloreerde bifenylen vooral
terug
t e v i n d e n i n een s t e r k
l a a g d e e l i m i n a t i e s n e l h e i d . B i j o c t a - en d e c a c h l o o r b i f e n y l
veris zelfs
van een meetbare e l i m i n a t i e geen s p r a k e meer; de d a l i n g van de
halten
i n de v i s s e n na h e t s t o p p e n van de e x p o s i t i e
t e s c h r i j v e n aan
aan de
ook
"verdunning"
r a a k t e n deze PCB's dan
k w i j t dan m a n n e l i j k e . U i t w i s s e l i n g met
s p e e l t v o o r deze v e r b i n d i n g e n n a u w e l i j k s een
20
i s geheel toe
t e n g e v o l g e van g r o e i en o v e r d r a c h t
jongen. V r o u w e l i j k e d i e r e n
minder langzaam
ge-
water
r o l ; er i s slechts
sprake
van
e e n r i c h t i n g s v e r k e e r , doordat
nog w e l
opname u i t water
en
v o e d s e l m o g e l i j k i s . V o o r a l de opname u i t w a t e r l o o p t e c h t e r s t e r k
terug, vermoedelijk
hydrofobe
doordat
maar a l s " m i c r o - a g g r e g a a t "
De
combinatie
een
een
b e l a n g r i j k d e e l van
stoffen niet werkelijk v r i j
dalende
van
de
deze e x t r e e m
i n o p l o s s i n g aanwezig i s ,
( c o l l o i d a a l of i n m i c e l l e n ) i n het water.
( a l t i j d optredende) verdunning
door g r o e i
opname e f f i c i e n t i e h e e f t t o t g e v o l g d a t de
t r a t i e f a c t o r b i j e x t r e e m hoge h y d r o f o b i t e i t weer l a g e r w o r d t .
scheidene
onderzoekers
c u l e e r d . De
hebben r e e d s op een
r e s u l t a t e n van
dergelijke daling
de e x p e r i m e n t e n met
aan
d a t de t h e o r e t i s c h maximale b i o c o n c e n t r a t i e f a c t o r i n de
10^
(gebaseerd
w o r d t benaderd d o o r
De
op h e t n a t g e w i c h t
van
echter v r i j
c l u d e e r d moet worden d a t de b i o - a c c u m u l a t i e
k l a s s i e k e o p h o p i n g van
s e l k e t e n . De w i j z e van
nog
efficient,
van
zodat
gecon-
super-lipofiele
ver-
i n v i s n i e t b e p a a l d wordt door e v e n w i c h t s - p a r t i t i e p r o c e s s e n ,
maar e e r d e r door een
milieu
buurt
de v i s ) ; deze waarde
2,2',4,4',5,5'-hexachloorbifenyl.
opname v i a h e t v o e d s e l b l i j f t
bindingen
Vergespe-
PCB's en guppen geven
van
ligt
met
bioconcen-
(meer aan
deze s t o f f e n v i a de
v e r s p r e i d i n g en h e t voorkomen i n h e t
d e e l t j e s gebonden dan
voed-
aquatisch
o p g e l o s t i n water) z u l l e n
toe b i j d r a g e n .
Opvallend
g e l i j k met
b i j deze e x p e r i m e n t e n i s d a t een
de PCB's z i j n
wordt opgenomen
toegediend
(voor z o v e r
aantal testverbindingen die
noch u i t v o e d s e l , noch u i t w a t e r
meetbaar).
D i t g e l d t v o o r hexabroombenzeen, o c t a c h l o o r d i b e n z o - p - d i o x i n e en
c h l o o r - p - t e r f e n y l . De
v e r k l a r i n g h i e r v a n moet n i e t
worden i n extreme h y d r o f o b i t e i t , a l s wel
een
hier
gezocht
i n de m o l e c u u l g r o o t t e ,
e f f e c t i e v e opname d o o r de v i s i n de weg
w o r d t h i e r d i e p e r op
zozeer
per-
die
s t a a t . In h o o f d s t u k
6
ingegaan.
T o t nu t o e z i j n k w a n t i t a t i e v e gegevens o v e r de b i o - a c c u m u l a t i e
milieuvreemde v e r b i n d i n g e n voor h e t merendeel a f k o m s t i g
van
van
chloor-
en broomhoudende k o o l w a t e r s t o f f e n . E n e r z i j d s komt d i t d o o r d a t d i t
type
stoffen
( v o o r a l de m e e r v o u d i g g e h a l o g e n e e r d e aromaten) een
a f b r e e k b a a r h e i d p a a r t aan
een hoge h y d r o f o b i t e i t ,
geen s t e r k p o l a i r e g r o e p e n aanwezig z i j n ,
zolang
a n d e r z i j d s kan
lage
althans
het
ook
21
t e maken hebben met
zoekers
of
de m o g e l i j k h e d e n
en de
i n t e r e s s e n van
Door de o n t w i k k e l i n g van
de g a s c h r o m a t o g r a f i e
"electron capture"
z i j n a p o l a i r e organo-halogeen
(ECD)
met
de
i n h e t algemeen a l i n z e e r l a g e c o n c e n t r a t i e s aan
dien z i j n gechloreerde
liteit
en
ducten
van
de
k o o l w a t e r s t o f f e n vanwege hun
fysisch-chemische
Zo i s h e t ook
met
verbindingen
Boven-
chemische
i n v o e r i n g van
geschikte vervan
de
mogelijk-
kostprijs.
PCB's. De o n g u n s t i g e
milieu-eigenschappen
van
i n d u s t r i e l e PCB-mengsels z i j n v o l d o e n d e bekend: t o x i c i t e i t ,
s i s t e n t i e en
per-
bio-accumulatie.
Over gedrag en p o t e n t i e l e e f f e c t e n van
mogelijke
PCB-vervangers
i n h e t m i l i e u z i j n v e e l minder gegevens b e s c h i k b a a r .
resterende
stabi-
eigenschappen u i t e r s t b e l a n g r i j k e p r o -
de chemische i n d u s t r i e . De
heden o f op een hogere
gevoelige
te tonen.
vangende s t o f f e n s t u i t v e e l a l op gebrek aan k e n n i s
s p e c i f i e k e toepassingen
v l o e i s t o f i n transformatoren,
systemen, v e r g e n
product;
onder-
opdrachtgevers.
van
i s d a a r een
thans
en h y d r a u l i s c h e
van.
e i g e n s c h a p p e n van
PCB-vervangers t e v i n d e n b u i t e n de k l a s s e van
no-halogeen
verbindingen.
u i t z o n d e r i n g . Deze p o l y m e r e n z i j n opgebouwd u i t een
gunstige
keten
komen v o o r .
vanging
toepassingen
van
orga-
van
afwis-
m e t h y l g r o e p e n daaraan g e k o p p e l d ;
zowel c y c l i s c h e s t r u c t u r e n a l s r e c h t e k e t e n s
i s s l e c h t s een
de
(PDMS; s i l i c o n e n ) vormen daarop een
s e l e n d z u u r s t o f en s i l i c i u m , met
het
D i t v o o r a l maakt h e t
m o e i l i j k om
Polydimethylsiloxanen
nog
PCB's, onder andere a l s
condensatoren
bepaalde fysisch-chemische
onbrandbaarheid
De
de v e l e m o g e l i j k e
g r o e p s t o f f e n ; de w e r e l d - j a a r p r o d u c t i e wordt g e s c h a t
PCB-vervan
deze
op
50 m i l j o e n
Commerciele " s i l i c o n e n o l i e " b e s t a a t u i t een mengsel van
polymeren.
De
gemiddelde k e t e n l e n g t e , en daarmee de v i s c o s i t e i t
is
a f h a n k e l i j k van
van h e t mengsel,
de beoogde t o e p a s s i n g . U i t m i l i e u c h e m i s c h
z i j n v o o r a l de r e l a t i e f k o r t e k e t e n s
van
belang
(max.
20
oogpunt
dimethyl-
s i l o x y - u n i t s ) , d i e e c h t e r s l e c h t s een b e t r e k k e l i j k g e r i n g d e e l
de
van
t o t a l e p r o d u c t i e uitmaken.
V e r g e l i j k i n g van h e t m i l i e u g e d r a g van PCB's met
geheel afwijkende
22
kg.
groep van
d a t van
de
chemisch
de p o l y d i m e t h y l s i l o x a n e n b i e d t een moge-
lijkheid
om v e r o n d e r s t e l d e r e l a t i e s t u s s e n h y d r o f o b i t e i t ,
molecuul-
g r o o t t e en b i o - a c c u m u l a t i e o n a f h a n k e l i j k t e t o e t s e n . D a a r b i j moet
e c h t e r w e l e e r s t een a a n t a l h i n d e r n i s s e n worden overwonnen d i e
op h e t g e b i e d " h a n t e e r b a a r h e i d " ,
liggen
analysemethode en m e t i n g van h y d r o -
fobiteit.
Hoofdstuk 5 b e s c h r i j f t een e x p e r i m e n t e l e / t h e o r e t i s c h e benadering
voor
deze problemen en g e e f t de e e r s t e r e s u l t a t e n v a n een b i o - a c c u m u l a t i e o n d e r z o e k . De o n d e r z o c h t e
polydimethylsiloxanen z i j n
meren met een k e t e n l e n g t e
t o t c a . 16 S i O - u n i t s e n e e n a a n t a l c y c l i s c h e
( 4 , 5 , 6 en 9 u n i t s g r o o t ) . De h y d r o f o b i t e i t
PDMS-oligomeren
van
deze v e r b i n d i n g e n i s g e s c h a t m.b.v. " r e v e r s e d - p h a s e "
chromatografie
index t.o.v.
alkylbenzenen;
( a l s l o g P)
vloeistof-
de methode i s v e r g e l i j k b a a r met de RP-TLC
3 . D a a r u i t kan opgemaakt worden d a t
i n hoofdstuk
PDMS-oligomeren met v i e r
t o t e l f SiO-units dezelfde hydrofobiteits-
range b e s t r i j k e n a l s de PCB's
van
oligo-
( i n d i t g e v a l RP-HPLC) door b e p a l i n g van de r e t e n t i e -
methode b e s c h r e v e n
( d i - t o t d e c a c h l o o r b i f e n y l ) . Een mengsel
deze s i l i c o n e n h e e f t "meegelopen" met de b i o - a c c u m u l a t i e - e x p e r i -
menten b e s c h r e v e n
i n hoofdstuk
4
( s u p e r - l i p o f i e l e verbindingen)
d i r e c t e v e r g e l i j k i n g t u s s e n PCB en PDMS m o g e l i j k
van
om
t e maken. De a n a l y s e
de PDMS-verbindingen s t e l t e c h t e r andere e i s e n en werd
lijk
afzonder-
u i t g e v o e r d op een a a n t a l g e s e l e c t e e r d e v i s - , v o e r - en watermon-
s t e r s d i e t i j d e n s dat experiment
In
lineaire
waren genomen.
t e g e n s t e l l i n g t o t de PCB's van v e r g e l i j k b a r e h y d r o f o b i t e i t worden
de g e v o e r d e PDMS-oligomeren n i e t o f s l e c h t s i n z e e r g e r i n g e
hoeveel-
heden teruggevonden i n de v i s s e n . Op grond v a n deze waarnemingen
lijkt
hetwaarschijnlijk
d a t de m o l e c u u l g r o o t t e
van de
b e l a n g r i j k e ) hogere PDMS-oligomeren een e f f e c t i e v e
accumulatie
oligomeren
n i e t worden
Bij
(commercieel
opname en b i o -
i n v i s voorkomt. Opname van g e r i n g e h o e v e e l h e d e n
kleinere
(max. 10 S i O - u n i t s ) u i t water o f v o e d s e l mag v o o r a l s n o g
uitgesloten.
vervolgexperimenten
z o u moeten worden g e t r a c h t meer i n z i c h t t e
v e r k r i j g e n i n de a c c u m u l a t i e - k i n e t i e k , v o o r a l i n de opname u i t w a t e r
en e l i m i n a t i e s n e l h e i d v a n PDMS-oligomeren. Ook o v e r de o p l o s b a a r h e i d
van
de i n d i v i d u e l e v e r b i n d i n g e n
i n water i s nog t e w e i n i g bekend.
23
De e x p e r i m e n t e n
beschreven
i n h o o f d s t u k 6 werpen meer l i c h t op
de
m o g e l i j k e i n v l o e d van de m o l e c u u l g r o o t t e op de opname van c h e m i c a l i e n
door v i s s e n . In h o o f d s t u k 4 was
a l g e b l e k e n d a t b i j de hogere
PCB's
nog v r i j w e l geen sprake i s van een door m o l e c u u l g r o o t e
gelimiteerde
opname u i t v o e d s e l . Het v e r s c h i l
i n de a c c u m u l a t i e van
octachloor-
d i b e n z o - p - d i o x i n e v e r g e l e k e n met
decachloorbifenyl i s niet
i n v e r b a n d t e brengen met
( r e s p . 460
h e t m o l e c u u l g e w i c h t van b e i d e
499).
en
Een v e r k l a r i n g van h e t v e r s c h i l v a n u i t h e t molecuulvolume
" e f f e c t i e v e doorsnede"
hand. Ook
latie
van h e t m o l e c u u l
e e r d e r e o n d e r z o e k e r s beschouwden h e t gebrek
onder e x p e r i m e n t e l e omstandigheden
c a t i e van een
door. Ook
de
aan accumutetrabroom-
a l s een m o g e l i j k e
l i m i t e r e n d e m o l e c u u l g r o o t t e . De e x p e r i m e n t e n
broombenzenen en verwante
den. Een
o f de
l i g t e c h t e r meer v o o r
i n hexabroombenzeen, i n t e g e n s t e l l i n g t o t t r i - en
benzeen,
rechtstreeks
stoffen
indi-
met
v e r b i n d i n g e n i n h o o f d s t u k 6 gaan h i e r o p
h i e r b l i j k t geen hexabroombenzeen i n v i s t e worden gevons c h e r p e overgang
i s t e z i e n t u s s e n 1 , 2 , 4 , 5 - t e t r a - en p e n t a -
broombenzeen; de o p n a m e - e f f i c i e n t i e van pentabroombenzeen i s meer
dan
t i e n k e e r zo l a a g ; p e n t a c h l o o r b e n z e e n wordt evenwel goed opge-
nomen, e v e n a l s d e c a c h l o o r b i f e n y l .
Het wel o f n i e t opgenomen worden van s t o f f e n d i e een
vergelijkbare
h y d r o f o b i t e i t hebben zou h e t b e s t v e r k l a a r d kunnen worden door
v e r s c h i l i n " e f f e c t i e v e doorsnede":
een c i r k e l
p o s i t i e nog
h i e r o n d e r wordt de d i a m e t e r
een
van
v e r s t a a n waar h e t b e t r e f f e n d e m o l e c u u l i n de g u n s t i g s t e
j u i s t doorheen
zou kunnen. De k r i t i s c h e d o o r s n e d e ,
l i g g e n t u s s e n d i e van h e x a c h l o o r b e n z e e n ,
1,2,4,5-tetrabroombenzeen enerzijds
decachloorbifenyl
(stoffen die a l l e
nog
zou
en
duidelijk
worden opgenomen) en pentabroombenzeen en o c t a c h l o o r d i b e n z o - p - d i o x i n e
a n d e r z i j d s . V e r m o e d e l i j k z a l e r s p r a k e z i j n van een o v e r g a n g s t r a j e c t .
( I n d i e n deze h y p o t h e s e
wege z i j n
juist
i s , zou
1,2,3,5-tetrabroombenzeen
ongunstige c o n f i g u r a t i e beduidend
den opgenomen dan de
1 , 2 , 4 , 5 - i s o m e e r i n een
minder
het
24
een g e l i j k a a n t a l chlooratomen)
gevolg z i j n
goed moeten wor-
vergelijkingsexperiment.)
E v e n t u e l e a c c u m u l a t i e v e r s c h i l l e n t u s s e n isomere
(PCB's met
van-
polychloorbifenylen
kunnen n a u w e l i j k s d i r e c t
van o v e r s c h r i j d i n g van een k r i t i s c h e
diameter, gezien
het f e i t
d a t d e c a c h l o o r b i f e n y l wel d u i d e l i j k d o o r v i s s e n wordt opge-
nomen. Toch v e r t o n e n
schillen
stuk
t e t r a c h l o o r b i f e n y l e n o n d e r l i n g wel
i n mate van opname en r e t e n t i e ,
degelijk
z o a l s beschreven
ver-
i n hoofd-
7. O p v a l l e n d i s b i j v o o r b e e l d h e t gebrek aan b i o - a c c u m u l a t i e
van
2 , 2 ' , 6 , 6 ' - t e t r a c h l o o r b i f e n y l . V e r s c h i l l e n i n h y d r o f o b i t e i t kunnen
dit
v e r s c h i j n s e l ook
tieel kritische
baarheid
maar t e n d e l e v e r k l a r e n . O v e r b l i j v e n d e
f a c t o r e n z i j n h e t dipoolmoment en de
(mogelijke omzettingssnelheid)
van
poten-
metaboliseer-
de v e r s c h i l l e n d e v e r -
bindingen.
Dit
l a a t s t e a s p e c t wordt i n h o o f d s t u k
mag
worden dat een
e l k a a r i n tenminste
3,4
en
aantal v r i j e
een
(ongesubstitueerde)
plaatsen naast
5 p l a a t s ) een p o s i t i e f e f f e c t op de m e t a b o l i s e e r b a a r h e i d
zou hebben; door o x i d a t i e
polaire
zouden deze p l a a t s e n met
( k o p p e l i n g van
hydroxylgroepen
- meestal
groepen) zou v e r v o l g e n s de o p l o s b a a r h e i d i n w a t e r en
de u i t s c h e i d i n g b e v o r d e r d
worden, b o v e n d i e n
( o n g e l a b e l d ) n i e t meer a l s PCB
De
twee o n d e r z o c h t e
mulatie-experiment.
tionaire
Het
(2,5- en 4 , 4 ' - d i c h l o o r b i f e n y l )
d e e l t e worden omgezet t i j d e n s een
t o t a l e omzettingspercentage
s i t u a t i e b e h o o r l i j k oplopen;
s t o f i n ongewijzigde
Verschillen
daar-
worden m e t a b o l i e t e n
v o o r de
kan
bio-accu-
i n een
sta-
eliminatiesnelheid
en de a c c u m u l a t i e f a c t o r i s de d i r e c t e u i t s c h e i d i n g van
kelijke
-
gemeten.
dichloorbifenylen
b l i j k e n b e i d e v o o r een
de
oorspron-
vorm e c h t e r van meer b e l a n g .
t u s s e n de d i c h l o o r b i f e n y l e n komen meer t o t u i t i n g i n
h e t a a n t a l v e r s c h i l l e n d e typen
totale
Verwacht
d e r b e i d e f e n y l r i n g e n (met name de
b e z e t kunnen worden. Door c o n j u g a t i e
mee
8 nader o n d e r z o c h t .
omzettings-
t r a n s f o r m a t i e - p r o d u c t e n dan
i n de
en e l i m i n a t i e s n e l h e i d . Het meest o p v a l l e n d
n a a s t de vorming van mono- en d i h y d r o x y d e r i v a t e n i s de p r o d u c t i e ,
i n n i e t o n b e l a n g r i j k e h o e v e e l h e d e n , van
(of
d e r i v a t e n daarvan), een
2,5-dichloorbenzoezuur
r i n g - s p l i t s i n g s p r o d u c t van
2,5-dichloor-
bifenyl.
25
U i t de i n d i t p r o e f s c h r i f t b e s c h r e v e n
experimenten en t h e o r e t i s c h e
overwegingen aangaande de b i o - a c c u m u l a t i e i n m i l i e u v r e e m d e
d i n g e n komt i n h o o f d l i j n e n h e t volgende
B i o - a c c u m u l a t i e van h y d r o f o b e
b e e l d naar
e f f i c i e n t e opname i n c o m b i n a t i e met
de s t o f f e n . I n t e r p r e t a t i e van
lijk
voren.
v e r b i n d i n g e n i n v i s s e n i s t e beschouwen
a l s een o p h o p i n g i n h e t v e t w e e f s e l t e n gevolge
een
verbin-
een
van
t r a g e v e r w i j d e r i n g van
de b i o - a c c u m u l a t i e i s s l e c h t s moge-
door b e s t u d e r i n g van de k i n e t i e k , h e t g e b r u i k van
referentie-
s t o f f e n i s d a a r b i j gewenst.
De b e l a n g r i j k s t e
f a c t o r i n het proces
v e r b i n d i n g , i n c o m b i n a t i e met
Extreem hoge h y d r o f o b i t e i t kan
i s de h y d r o f o b i t e i t van
h e t v e t g e h a l t e van h e t o r g a n i s m e .
e c h t e r s l e c h t s i n d i r e c t gemeten o f
b e r e k e n d worden. Extreem h y d r o f o b e
v e r b i n d i n g e n worden n i e t meer
merkbaar u i t g e s c h e i d e n door de v i s . De opname u i t h e t
wordt dan b e l a n g r i j k e r dan
de
voedsel
de opname d i r e c t u i t h e t water;
de
b i o c o n c e n t r a t i e i s aan een maximum gebonden. B i j v o o r s p e l l i n g
van
de b i o - a c c u m u l a t i e van m i l i e u v r e e m d e
moet eveneens t e r d e g e
ten gevolge
van
organische
r e k e n i n g gehouden worden met
de m o l e c u u l g r o o t t e
of mogelijke
verbindingen
beperkingen
omzettingen
door o r g a n i s m e n . Voor de meeste PCB's z i j n deze l a a t s t e
f a c t o r e n o v e r i g e n s van o n d e r g e s c h i k t e
26
betekenis.
twee
CHAPTER
1
HYDROPHOBIC
INTERACTIONS
IN
THE AQUATIC
ENVIRONMENT
27
Reprint from
T h e H a n d b o o k o f E n v i r o n m e n t a l Chemistry, Volume 2/Part B
Edited by O. Hutzinger
CD Springer-Verlag Berlin Heidelberg 1982
Printed in Germany.
Hydrophobic Interactions in the Aquatic Environment
W.A.
Bruggeman
Laboratory of Environmental and Toxicological Chemistry, University of Amsterdam
Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands
Introduction
The fate of organic chemicals in the aquatic environment depends on physical
transport, physico-chemical distribution, and transformation processes. Characteristics of the aquatic ecosystem, as well as the properties of the chemical in question will determine the rate and extent of general processes like transport by air and
water flows, evaporation, sorption by sedimenting particles or organisms, and biological or (photo)-chemical degradation [1].
Some important parameters in aquatic ecosystems are temperature, biomass
production and organic matter content. One of the main factors is depth, which
influences the amount of sunlight penetrating per unit volume, the oxygenation of
the water layers and the exchange with bottom sediments and atmosphere. Enormous differences exist between tidal zones and open ocean, and in fresh water between shallow ponds, brooks and deep lakes (See also Van der Ploeg [2]).
In principle, it is possible to investigate fate and effect of a chemical in one particular water body by imitating the natural system on laboratory scale; the so called
model ecosystem. However, several interactions will be too complex to allow representation in a single "microcosm", and many limitations of the method are inherent to the micro character of the laboratory system [3].
For a more general and complete picture it is necessary to analyze environmental processes by a combination of laboratory and field studies. Important interactions can be studied in detail in the laboratory and the findings eventually be extrapolated to various natural systems: especially comparison of new chemicals with
well known ones can be useful.
Modeling and prediction of the behavior of chemicals is possible when sufficient kinetic information about the relevant processes is available. A n example of
such a model is given by Branson [4]. It includes transport, distribution and transformation parameters in terms of first order rate constants (Fig. 1).
29
Evaporation
"ki
Water
Hydrolysis
Input
Uptake
Binding
k
Release
Depuration
5
Sediment
Material Balance Equation
k - k, ACw - k VCw - k FCw + k F C + k SCw + k SC
0
2
3
4
Fish
Depurati
Fish
Uptake
Degradat
(Hydroly:
Evaporat ion
(SIS
dt '
f
5
6
S
n 2
o
UOI
, dCw
3
2
Fig. 1. Pond model. (Branson, 1978 [4]). V = Volume of water, ml; A = Surface area, cm ; F = Fish
mass, gm; S = Sediment mass, gm; Cw = Concentration of chemical in water; k = Rate constant;
C = Concentrations of chemical in fish; C = Concentration of chemical in sediment
f
s
Distribution Equilibria
For a chemist, the representation of the environment as a complex chromatographic system may be attractive: a combination of various flow (air and water)
and distribution processes (gas/water/solid exchange). In that case, the main quantifying parameters are the flow rates, which will be different for each aquatic system, and the distribution coefficients, which are functions of the chemical and the
phase composition only. Energetically, water is the least favourable environment
for hydrophobic chemicals, such as alkanes, polynuclear aromatic and halogenated hydrocarbons, and silicones. This finds expression in a low aqueous solubility,
the tendency to partition into organic phases, and relatively fast evaporation from
aqueous solution, even for high boiling compounds. The latter phenomenon is often referred to as "codistillation", which is a rather confusing term, since simultaneous evaporation of water is not necessary.
In Fig. 2 a scheme is given of the general equilibrium distribution of various
classes of chemicals over the gas, water and solid organic phase, as related to
aqueous solubility and vapour pressure. Chemicals with a high vapour pressure
will be found in the gas phase (atmosphere). Those with high aqueous solubility
will be dissolved in water, whereas hydrophobic chemicals with low vapour pressure tend to be adsorbed on particles, or to accumulate in living organisms. Loss
of hydrophobic chemicals from solution by evaporation can be important if their
30
Fig. 2. Water solubility and vapour pressure of organic chemicals
fugacity is high enough. This parameter is directly related to the ratio between vapour pressure and aqueous solubility; its theory and use in environmental chemistry has been discussed by Mackay [5].
A distribution equilibrium can be described by a partition coefficient: which is
defined as the ratio of the concentrations in the phases concerned. Usually, this is
done by relating weight/weight or weight/volume units, however, for thermodynamic reasons mole fractions are preferred. In these terms, aqueous solubility, defined as the concentration of a solute in water in equilibrium with its pure phase
(mole fraction =1), is one of the partition coefficients used in environmental
chemistry. Other examples are bioconcentration factors (organism/water) and soil
sorption coefficients (soil/water). As a model parameter for the lipophilicity of organic compounds, the octanol/water partition coefficient, P, or K , is most often
used in pharmacology and drug design; it is correlated with the biological activity
of drugs. A compilation of lipophilicity data (as partition coefficients) is found in
a review by Leo et al. [6].
Hydrophobicity is the unifying principle for phenomena like lipophilicity and
low aqueous solubility. The driving force is found in the energy (aG), needed to
bring the compound into solution. A hydrophobic molecule is "pulled out" of an
aqueous phase, due to the strong attractions between H 0 molecules, that are diso w
2
31
turbed by the presence of an apolar molecule. The effect is dependent on the volume occupied by the apolar moiety of that molecule, against its capacity to interact
with water molecules by hydrogen bonding and polar or ionizable groups [7].
Methyl and phenyl groups for example increase the hydrophobicity, while O H - ,
C O O - and NH -groups give negative contributions to l o g K . In apolar organic
phases, only the relatively week Van der Waals forces remain, therefore differences
in attraction between solvent and solute molecules are small.
The group contributions are relatively independent and additive. These linear
free energy relationships are well documented for octanol/water partition coefficients, and calculation of l o g K is possible for many compounds by summation
of simple fragmental constants [8-10]. Therefore, l o g K is often used for correlation with biological and environmental equilibrium partition coefficients and even
in partition related kinetic studies, e.g., bioaccumulation in fish. The empirical
relation with aqueous solubility (as log S) is described by Chiou [11]. Important
improvements in the correlation are obtained for solid compounds when a correction term for crystal energy is introduced [12]. For rigid molecules like polynuclear
aromatic hydrocarbons, the melting point gives sufficient quantitative information
for this refinement:
2
ow
o w
o w
l o g K = -logS-0.01 x m p + 0.50.
(1)
o w
The thermodynamic basis of all correlations between parameters describing hydrophobic interactions is the aqueous phase activity coefficient of the substance, y [5].
Adsorption
Aqueous concentrations of environmental chemicals can be regulated by reversible
adsorption on aquatic sediments. The relationship between the concentration of a
compound in the solid phase and the concentration in the aqueous phase is then
given by adsorption isotherms. In the laboratory, they can be determined by equilibrating known amounts of dissolved chemical, water and sediment in suspension
and measuring the concentration in at least one of the phases. Different points of
the isotherm are obtained by varying the amounts of sediment or chemical in the
water.
In principle, three different regions in adsorption isotherms can be distinguished (see also Huang [13]):
1. linear partition between water and adsorbent at sufficiently low concentrations,
2. saturation of the adsorbent at higher concentrations,
3. saturation of the aqueous phase at concentrations close to the solubility of the
compound: precipitation.
Usually, parts of the curve can be described by the Freundlich isotherm, an empirical equation which is used when theory is not available to predict the shape:
C =KC
s
n
n>0.
w
(2)
This parabolic relationship results in a straight line in double log plots of C versus
C :
logC = n l o g C + logK.
(3)
s
w
s
32
w
In special cases, this isotherm may cover a large concentration range, i.e., several
orders of magnitude.
Saturation of the sorbent at high dissolved concentrations is indicated by n > 1.
This phenomenon is typical for specific interactions between solute molecules and
free sites on the adsorbent, e.g., ion exchange processes. This type of adsorption,
often called chemisorption, is better described by the Langmuir isotherm and its
derivatives [13]:
C /C = K ( X - C ) .
S
W
(4)
S
The maximum adsorbable concentration, X , is equivalent to the total number of
adsorption sites per unit adsorbent (C = X , when C -*oo).
At C = 1 / K , one half of these sites is occupied ( C = X / 2 , when C = 1 / K ) . Only
at very low concentrations, the amount adsorbed is proportional to the aqueous
concentration (C = K . X . C when C < < X ) .
Typical for hydrophobic sorption processes is a range of linear partitioning at
low concentrations, and apparent saturation of the water phase at aqueous concentrations close to the solubility of the compound (Fig. 3). The best fit in the Freundlich equation is then obtained with n > 1.
The vertical part of the curve may be considered as an artifact of the laboratory
procedure: initial concentrations of the chemical have been close to or even exceeding the solubility in water [14].
The adsorption of hydrophobic chemicals at lower concentrations is best described by a linear isotherm, which is equivalent to n = 1 in the Freundlich equation:
S
w
w
s
S
W
w
s
C = KC .
S
W
For most apolar compounds, the adsorption coefficient, K , ist strongly related to
the organic carbon content of the soil or sediment. Correlation with particle size
33
8
log K
0 C
= 1.00 log P-0.21
7-
6-
5
0
0
J
1_
1
2
3
log K
4
5
6
7
Ow
Fig. 4. Correlation between organic carbon adsorption coefficient (K ) and octanol-water partition
coefficient of organic chemicals (K„J. (After Karickhoff, 1979 [15])
oc
is less obvious [15, 16]. On the other hand, the strength of adsorption is directly
related to the lipophilicity of the chemical. The relationship between the organic
carbon partition coefficient ( K = concentration per gram organic carbon, divided
by the concentration in water) and lipophilicity, as predicted by Briggs [17], was
shown by Karickhoff et al. [15] (Fig. 4).
Originally, this correlation was based on data from ten chemicals, mainly
polyaromatic hydrocarbons, but it proved to be valid for many other apolar compounds with l o g K ranging from 2 to 7, and applicable on soils and aquatic sediments with widely differing organic carbon content and particle size. As expected,
a similar relationship was found between soil sorption and aqueous solubility, provided that the appropriate correction for the crystal energy was applied [16].
Deviations were observed for "super-hydrophobic" chemicals ( l o g K > 7 ) .
This may be due to difficulties in preparing true aqueous solutions or in determining the dissolved concentrations, since these chemicals tend to form micro-aggregates or micelles in the aqueous environment, especially when other organic compounds are present which can act as surfactants. For the same reasons, direct measurement of octanol-water partition coefficients above log K = 5 will produce too
low values.
Chemicals containing highly polar or charged groups, especially cationic ones,
such as the herbicide paraquat, can be adsorbed by surfaces with ion exchange capacity, e.g., clays and humic substances. Adsorption of these rather hydrophilic
o c
o w
ow
o w
34
compounds will be underestimated when only the hydrophobic sorption is taken
into account.
The partitioning into organic matter has but a few similarities with true surface
adsorption. Organic particles in aquatic sediments, originating from decaying organisms, will be completely porous, non-rigid structures. This may explain why the
correlation with between sorption and organic carbon content is relatively independent of particle size. In general, equilibrium sorption of organic solutes by suspended particles is completed for more than ninety percent within hours, and is
rapidly reversible. On the other hand, exchange of molecules sorbed in the interior
may need more time than desorption of molecules on the external surface of the
particle. Thorough investigation of sorption and desorption kinetics may eventually lead to multi-compartment exchange models [18].
In nature, exchange between non-suspended sediments and the overlaying water may be limited by diffusion through sediments layers, but sedimenting and resuspended organic particles will provide an important storage mechanism for hydrophobic pollutants.
Biosorption
Adsorption of hydrophobic chemicals is not reserved to soil particles or detritus:
all organic particles with high surface to mass ratio, including microorganisms,
show this phenomenon. In addition, microorganisms can absorb and excrete
chemicals via a mechanism of direct exchange with their environment (e.g., nutrients and waste products). Bigger organisms, with their relatively small exposed
surface area, need additional absorption and transport systems - gastro-intestinal
tract, respiratory organs, circulation of body fluids - to supply all cells.
Uptake and partitioning of lipophilic compounds into lipid compartments of
the cell (membranes, fat or wax droplets) can proceed via the same direct and rapid
exchange mechanism. This uptake by microorganisms, whether it is true absorption or only adsorption on the outer surface, is sometimes called biosorption.
For most essential compounds, efficient regulatory mechanisms exist that are
able to maintain internal steady state levels independent of the external concentrations. In contrast, equilibrium concentrations of persistent, hydrophobic, xenobiotic chemicals are proportional to the exposure concentration, as a result of passive exchange mechanisms. N o enzyme system is involved in the uptake, and no
energy is required to maintain a certain level in the cell: even killing does not necessarily change the equilibrium.
Bioaccumulation
Bioaccumulation in higher animals is a more complex distribution process, and in
this respect is resembles the distribution in aquatic ecosystems: to predict concentrations in the different compartments at different times, knowledge is required
about:
1. input concentrations and rates
2. flows
35
water
food
faeces
Fig. 5. Fish model: possible routes for uptake and excretion of lipophilic chemicals.
compound,
metabolites
original
3. partitioning (coefficients; rates)
4. chemical transformations, output rates.
For lipophilic organics in aquatic organisms the scheme of pathways in Fig. 5
might be valid.
Accumulation of persistent lipophilic chemicals (e.g. D D T ) from food, which
is the most important source for terrestial (air-breathing) animals, is now recognized as only a minor pathway in aquatic organisms [19, 20]. Because of the low
oxygen content of water relative to air, fish and many other aquatic organisms
must pass large quantities of water, and the equilibration of chemicals between
body and water by passive transport can be very fast. This is true especially for lipophilic compounds, which can easily permeate through biological membranes,
the barriers surrounding all living cells and cell organelles like chloroplasts,
mitochondria and nuclei.
Biomembranes
In principle, these membranes consist of a bi-molecular phospholipid layer (Fig. 6),
the hydrophobic "tails" of the fatty acid groups from both sides forming a fluid
lipid layer in the middle, and the hydrophilic polar phosphatidylcholine "heads"
(Fig. 7) facing outward.
Besides phospholipids, also steroids, like cholesterol, and glycolipids are found
in cellular membranes, in varying proportions. Proteins are normaly associated
36
Fig. 6. The fluid mosaic model of the structure of cell membranes. Proteins are embedded in a lipid
matrix. (Singer and Nicolson: Science 175, 720,1972)
• CH,
CH,
• CH, • • • • CH,
• Hydrophobic tails
Lecithin
Charged groups
Fig. 7. Molecular structure of lecithin, one of the phospholipids found in cell membranes
with the hydrophilic surface on both sides of the membrane, but can also penetrate
into the lipid layer by their hydrophobic groups, if in the right configuration. They
can thus act as active transport agents or form pores to allow the passage of ionic
other hydrophilic compounds.
Lipophilic compounds however are easily partitioned into the lipid fraction of
the membrane, and they can pass it by simple diffusion, instead of active transport
mechanisms.
37
The membrane model described above is called the "fluid mosaic" model, and,
as the name indicates, the lipid double layer of the membrane is not a rigid, "built"
structure, but can be considered as a natural association and orientation of lipids
in an aqueous environment. (The liquid film separating the interior of a soap
bubble from its environment is probably the best macroscopic phenomenon to
keep in mind, when thinking of a biomembrane). Phospholipid "vesicles", consisting of a globular lipid double layer around an aqueous interior, can be prepared
by ultra-sonic treatment of aqueous lecithin suspensions, and have already been
used for the investigation of the uptake of chlorinated hydrocarbons into model
membranes [21]. Usually, a complete layer of cells has to be passed to enter an organism, whereas a membrane is only the barrier separating a cell from its environment.
Besides by diffusion through membranes, hydrophobic substances can penetrate into cells by formation of micelles, which are able to fuse with biomembranes.
This mechanism is especially important for the uptake of lipids from the intestine,
where conjugated bile acids provide the surfactant activity which is needed for the
association of lipids into micelles.
In the intestinal cell, triglycerides are converted together with proteins into l i poprotein droplets, called chylomicra, which are excreted by membrane fusion and
enter the lymphatic system, through which they are carried to the blood stream.
Fat droplets, presumably surrounded by a single phospholipid layer, are responsible for storage of lipids in cells. It is thought that absorption of lipophilic xenobiotic chemicals from the intestine and their transport to body cells proceed via the
same route.
Partitioning of chemicals by passive transport processes from ambient water into storage fat and lipoid tissue will proceed via a number of membranes, and both
rate and ultimate equilibrium partition factor will be dependent on the lipophilicity
of the xenobiotic. The absolute amounts accumulated are normally proportional
to the fat content of the organism, which is dependent on species (see Table 1), sex,
age and condition.
Table 1. Fat content of animal species
Species
Terrestrial
Wild living mammals and birds
Domestic animals and man
Marine mammals
Fish:
Cod and haddock
Herring
Eel
Perch and pike
Carp
Salmon
Trout
Oyster
Lobster
Insects (average)
38
% Fat
1-3
13-25
>15
0-1
5-20
15-30
1-2
1-10
2-15
1
1.2
3
10
Bioaccumulation Tests
Bioaccumulation is considered to be an important factor in the overall assessment
of potential pollutants for the various "toxic substances laws", since it enhances
the probability of long term toxic effects in the target species and adds to the contamination of human food sources, either directly or via animal feed. Therefore,
attention has to be paid to proper definition, description and testing of the phenomena with various potential pollutants. For this purpose, laboratory model systems are developed that have to be tested with chemicals which are known for their
bioaccumulation under field conditions. A summary of definitions and parameters
for the description of the aquatic accumulation process is given in the appendix.
A useful model for the bioaccumulation and elimination process in fish (trout)
muscle, after exposure to a constant concentration of the pollutant in water, was
developed by Neely, Branson and coworkers [22, 23]. A graphic representation of
the process for 2,2',4,4'-tetrachlorobiphenyl is given in Fig. 8.
The bioaccumulation process can be described kinetically as concurrent first
order uptake and elimination processes:
k,
C
^ C
f
and
dQ
dt
C
C
ki
k
f
2
w
=
=
=
=
:
kiC —k2C
w
(5)
f
concentration in fish
concentration in water
1 st order uptake rate constant
1 st order elimination (clearance) rate constant.
100
j
1
1
1
T — C = 14 jjg/l
w
1
uptake^,
s t e a d y state
~ - - _ _ clearance
3-10
~C
0.1 I
i
i
10
i
20
30
W
= 1.5 yg/\
40
days
Fig. 8. Bioconcentration and clearance of 2,2,4,4 -tetrachlorobiphenyl in trout muscle. (After
Branson et al., 1975 [22])
39
At the start of the bioaccumulation experiment, the concentration of pollutant
in the fish is negligible, and the uptake rate is proportional to the concentration
in the water:
Q = 0^^-
f
= k C .
1
(6)
w
During the clearance time, the concentration in the water is virtually zero. The
elimination rate is then proportional to the concentration in the fish:
C =0 - ^ = - k
w
C
2
(7)
f
or
^=-k dt
(8)
2
after integration:
taQ-lnQ-kjfe-U).
(9)
Single log plotting of clearance data (log C vs t) will result in a straight line,
with slope — k , the clearance (depuration) rate constant. The uptake rate
constant k can be calculated from the slope at t->0 in a linear graph of C vs
time.
A plateau value is reached when the rate of elimination equals the uptake
rate: At equilibrium:
f
2
t
f
^£=0:k C =k C
1
w
2
(10)
f
or
K
- = r
t
=
n
k T
<>
where K represents the bioconcentration factor.
A prediction of C during the accumulation process can be made by
combining eqs. (5) and (11), when C is constant, in continuous flow systems:
b c
f
w
1
Cf=C oo - ( l - e - ^ ) ,
(12)
f
where C oo = K ^ C ^ , , the concentration in the fish at equilibrium.
Confirmation of the assumed two compartment first order kinetics is found in:
- the proportionally lower concentrations in the fish at the lower water concentration (Fig. 8: constant vertical distance between the two curves)
- the straight line during the clearance process.
This model has to be adjusted if more than two rate determining compartments
are involved; not only ambient water and storage fat, but also other pools, which
are subject to a slower or more rapid exchange, e.g., blood. In this case two
(or more) stages will be observed in the clearance process, with different half lives
and slopes [24, 25].
It follows that a true and constant clearance half life for bioaccumulated
pollutant is only defined in the case of two compartment first order kinetics:
f
(9):
In&^kife-ti)
half life: t - t i = t 4
2
i f Q =2C
40
f 2
so
_ln2_0.69
~ k - k •
2
(13)
2
The exchange rate will depend on the amount of water passing through the gills.
This ventilation rate is coupled to the oxygen concentration in the water in relation
to the oxygen need of the organism, which will vary according to temperature, size,
species and activity [26]. It is possible to estimate an absorption efficiency, E , for
a chemical, if the ventilation rate unter the test conditions can be determined:
k,=Er,
(14)
1
-1
where r represents the weight specific ventilation rate (ml water x g fish" x time ).
Neely [27] compared the absorption efficiencies of various chemicals, and found
a correlation with lipophilicity (as l o g K ) . For certain chlorinated biphenyls, the
efficiency approaches the value estimated for oxygen (80%). Apparently, membrane permeability is no longer the limiting factor for the uptake of these highly
lipophilic compounds in fish [25].
ow
Food Chain Accumulation
When a chemical is fed to a fish, it will be partly or completely absorbed from the
intestine, and eventually stored in the body fat. Its excretion will again be dependent on the concentration in the blood, and thus on the amount in the fish as a
whole. A plateau level in the fish will be reached when the amount daily excreted
equals the amount daily fed. So the steady-state concentration in the fish will be
proportional to:
1. the concentration in the food
2. the feeding rate
3. the absorption efficiency for that particular chemical, and
4. inversely proportional to the elimination rate.
This is similar to bioaccumulation directly from water, and the same type of
steady-state factor can be defined; the (bio) magnification factor:
K
m
= ^ H at equilibrium,
(15)
'-food
which is also dependent on the lipophilicity
factor is usually small (<10) compared to
( D D T : 100,000), but can be important under
amount of lipophilic chemical accumulated
(K'bc x C ) :
of the chemical. The magnification
the direct bioaccumulation factor
natural conditions, due to the high
from water by the food organisms
w
Theoretical concentration via food organisms: K
Theoretical concentration from water: K x C
b c
Kb,, and
m
x K' x C
b c
w
w
are comparable, and dependent on the fat content of the organisms.
41
log
k
ow
Fig. 9. Correlation between bioconcentration factor (K )and octanol-water partition coefficient (K ).
(After Neely et al., 1974 [23]). 1. 1,1,2,2-tetrachloroethylene; 2. carbon tetrachloride; 3. p-dichlorobenzene; 4. diphenyloxide; 5. diphenyl; 6. 2-biphenyl phenyl ether.; 7. hexachlorobenzene;
8. 2,2 ,4,4 -tetrachlorodiphenyloxide
bc
ow
Bioaccumulation and Lipophilicity Correlation
Bioaccumulation, resulting from equilibration of concentrations in water and fish
fat, can be compared to the partitioning of a chemical between n-octanol and water; the bioconcentration factor, K , is then similar to the n-octanol/water partition coefficient, K . Neely et al. [23] have shown that the linear relationship between l o g K and log K in Eq. [17], Fig. 9, can be a fair approximation for many
chemicals with l o g K between 3 and 7.
b c
o w
o w
b c
o w
l o g K = 0.5421ogK + 0.124.
b c
(17)
ow
The "ecological magnification" (EM) defined by Metcalf et al. [28], is in fact a
bioconcentration factor obtained in a "model ecosystem", and shows the same
type of correlation with K .
Important deviations from the predicted "ecological magnification" or bioconcentration factor will be observed when a compound is easily metabolized by the
organism.
Striking examples are the D D T analogs in Table 2, which show greatly different biodegradability indexes (BI). Methiochlor (R = C H S - ) is not accumulated
in fish since it is easily oxidized to a polar metabolite, in contrast to D D T itself
(R = C 1 - ) [29].
Molecular size might be also a limiting factor to bioaccumulation: penetration
through the biological membranes will be inhibited if the "fluid mosaic" is
disturbed by the bulky nature of the molecule. Indications of this phenomenon are
the preferential absorption of low-molecular weight n-alkanes by rats, after oral
administration [31], and the retention/size relation found by Hardy et al. [32]: when
o w
3
42
rNOvr-~00(NO —
O O O — ——• o" — —
-.1 E
X S &
OOO
XXX
Tt Ov rr,
OO — ON
K r> fN
o
X
QU u
oo o r-\c — «n
Tf o
o o o
I I
o
I
M
rsi o
v> © <n"
I
h
D
D
i/i o m
r - <N Tt
fN
a
Ej
2
8
3
8
f*-i
(N
—
4
2
i?
"8
o o o
—o o
^ ^ 't
•a
—
X
o
—
q
X
x
o
X
u
o o
o
o o
o o
o
X
u
q
X
oi os a.
X
o
X
o
X X _
43
35
A
15
17
19
21
23
25 27
Carbon no.
29
31
33
Fig. 10. Percentage retention of n-alkanes by the codling liver after 6 months feeding with oil as a
function of carbon number. (Hardy et al., 1974 [32])
feeding fish a diet contaminated with n-alkanes, a maximal retention (absorption
minus excretion) was found at n = 26 (Fig. 10). Presumably this optimum chain
length is the result of balancing increasing lipophilicity against size limitations.
However, it must be kept in mind that the large cholesterol molecule is a membrane
constituent itself, and that high molecular "crown ethers" are known for their capability of carrying cations through biomembranes. The membrane is not a simple
molecular sieve, and more structural parameters, like the presence of a polar
"head" together with an apolar"tail". will influence the association with biomembranes.
The kinetic approach, i.e., the use of rate constant as proposed by Neely, Branson and coworkers [4, 22, 23, 27] has the advantage over the static equilibrium approach, that the contributions of simultaneously occurring processes can be taken
into account under dynamic conditions. A static situation (constant water concentration, equilibrium) does not exist in nature; however, a steady state might be approached.
Neely's log K / l o g K correlation [23] can be useful to predict the bioaccumulation potential of persistent environmental chemicals with l o g K < 7 . Higher
logK values cannot practically be measured in the classical way (n-octanol/water
equilibration); they can be estimated by extrapolation via calculation, but then
there is no experimental method to check the calculated values. A more recent,
promising method is the determination of hydrophobicity by comparison of retention times in reversed phase high performance liquid chromatography, using methanol-water mixtures as eluent [33]. The use of aqueous solubility for correlation
with bioconcentration will meet the same difficulties as described in the adsorption
section. On the other hand, new experiments have to show whether Neely's linear
relationship holds for "superlipophilics".
o w
b c
o w
44
Bioaccumulation rates, as well as aqueous solubilities, are expected to decrease
rapidly at higher logK values; so the absolute concentration in fish might be low,
even with very high equilibrium bioconcentration factors.
Summary
Although bioaccumulation and adsorption of organic pollutants in the aquatic environment can be considered, in principle, as partition processes that are closely
related to the lipophilicity of a given chemical compound, it is not easy to predict
concentrations in water, sediments and biota of natural ecosystems. Equilibrium
concentrations ratios, i.e., bioaccumulation factors and adsorption coefficients,
can be estimated either by structure activity correlations [34, 35] (via calculation
or direct determination of n-octanol/water partition coefficients, aqueous solubilities, or reversed-phase liquid chromatographic retention), or by direct measurements in aquatic systems at steady state.
The most useful information about the behaviour of new compounds however
will be obtained from kinetic parameters, i.e., uptake and elimination rate constants, by direct comparison with "well known" chemicals in various simple, well
understood, laboratory model systems.
Appendix
From: Working paper on bio-accumulation test, drafted by the Japanese delegation, O E C D - Chemicals testing programme, Expert group C (Degradation/Accumulation), 2nd meeting, Berlin, 1978
I. Definitions of Terms and Indicators Relating to Bio-accumulation
(1) Definitions
• Accumulation
The phenomenon that an environmental element accumulates a certain chemical substance as a residue in a greater concentration than the environmental element normally holds, for a long time,
through its contact with a surrounding medium or its life activities.
• Bioaccumulation - bioconcentration
= biomagnification
The ability of living organisms to concentrate, accumulate and magnificate a chemical substance in
it, either directly from a surrounding medium or indirectly through the food chain.
• Direct bioaccumulation — bioconcentration
The phenomenon that a chemical substance accumulates in species by direct contact with a surrounding medium through oral, percutaneous, or sometimes respiratory courses.
• Indirect bioaccumulation = biomagnification
The phenomenon that a chemical substance accumulates in species through different trophic levels
in a food chain.
• Concentration factor = bioconcentration factor
= accumulation coefficient
The quotient of the test chemical substance concentration in the test organisms divided by the concentration in the test water, when the rate of uptake and clearance are equal,
(the plateau value)
• Uptake
The process of sorbing test chemical substance into and/or onto the test organisms.
• Clearance — depuration
= elimination
= bioelimination
The process of losing test chemical substance from test organism.
45
• Steady state = plateau
The condition in which uptake and clearance of test chemical by test organism are equal at a given
condition.
• Biological half life = excretion half life
= depuration half-concentration
The time needed to decrease the concentration of test chemical in the organism to one half of the
initial concentration.
(2) Indicators and Significance of
(i) Primary
Bio-Accumulation
Indicators
Indicators of accumulation in a living organism
Concentration
factor
Ecological
magnification
factor
i) Quotient obtained when the concentration of a chemical substance in a
living organism is divided by the concentration of the chemical substance
in water
ii) Quotient obtained when the concentration of a chemical substance in a
living organism is divided by the concentration of the chemical substance
in feeds
Quotient obtained when the concentration of a chemical substance in an aquatic
organism is divided by the concentration of the chemical substance in water.
... Ecosystem
Retention
Quotient obtained when the absolute amount of a chemical substance in a
living organism is divided by the absolute amount of the chemical
substance which the organism has ingested
Absolute
concentration
Concentration of a chemical substance in a living organism
Absolute
amount
Absolute amount of a chemical substance contained in a living organism
(ii) Secondary
Indicators
Indication of speed of accumulation in a living organism
Uptake rate
Rate at which a living organism directly takes into or onto a chemical
substance from water
Clearance rate
Rate at which a living organism excretes a chemical substance which has been
accumulated inside it
Biological
half-life
The time taken for the amount of a chemical substance accumulated in a
living organism to drop to half of the initial amount
(iii) Tertiary
Indicators
Indicators of parameters which predict or suggest the bio-accumulation
Partition
coefficient
Indicates the property of a chemical substance to dissolve in lipid. Normally
measured in terms of the value in water-octanol
Solubility
Solubility in water is important
Molecular size
Size of largest molecule which can be absorbed by a living organism
46
II. Various Parameters in a Bio-accumulation
Test
(1) Physical and Chemical Properties of a Chemical Substance
Solubility
Generally, a compound which easily dissolves in water does not easily
concentrate in a living organism
Partition
coefficient
Generally, if the partition coefficient in octanol-water is large, the ability to
concentrate in a living organism is also large
Molecular weight
There is presumably a limit molecular size of a chemical substance which can
be absorbed by a living organism
Membrane
permeability
The smaller the molecule, the greater is the permeability
Absorption
coefficient
Permeability of neutral or non-charged chemical substance, or of lipophilic
compounds is normally large
References
1. Baughman, G.L., Burns, L.A.: Transport and transformation of chemicals: a perspective, in: The
handbook of environmental chemistry (Hutzinger, O., ed.), Springer-Verlag, Heidelberg, 1980,
Vol. 2 part A, p. 1
2. Van der Ploeg, S.W.F.: Basic concepts of ecology, ibid., this Vol.
3. Isensee, A.R.: Laboratory microecosystems, ibid., Vol.2 part A, p.231
4. Branson, D.R.: Predicting fate of chemicals in aquatic environment from laboratory data, in: Estimating the hazard of chemical substances to aquatic life, A S T M STP 657,(Cairns, J., Dickson,
K . L . , Maki, A.W., eds.), Amer. Soc. Testing Materials, 1978, p. 55
5. Mackay, D.: Solubility, partition coefficients, volatility and evaporation rates, this handbook,
Vol.2 part A, p.31
6. Leo, A., Hansch, C , Elkins, D.: Chem. Rev. 71, 525 (1971)
7. Horvath, C , Melander, W.: J. Chromatog. Sci. 15, 393 (1977)
8. Hansch, C , Quinlan, J.E., Lawrence, G.L.: J. Org. Chem. 33, 347 (1968)
9. Rekker, R.F.: The hydrophobic fragmental constant, Elsevier, Amsterdam, 1977
10. Hansch, C , Leo, A.: Substitution constants for correlation analysis in chemistry and biology,
Wiley, New York, 1979
11. Chiou, C.T., et al.: Environ. Sci. Technol. / / , 475 (1977)
12. Yalkowski, S.H., Valvani, S.C.: J. Chem. Eng. Data 24, 127 (1979)
13. Huang, P.M.: Adsorption processes in soil, in: The handbook of environmental chemistry (Hutzinger, O. ed.), Springer-Verlag, Heidelberg, 1980, Vol.2 part B, p.47
14. Shin, Y.-O., Chodan, J.J., Wolcott, A.R.: J. Agr. Food Chem. 18, 1129 (1970)
15. Karickhoff, S.W., Brown, D.S., Scott, T.A.: Water Res. 13, 241 (1979)
16. Karickhoff, S.W.: Chemosphere 10, 833 (1981)
17. Briggs, G.G.: A simple relationship between soil adsorption of organic chemicals and their octanol/
water partition coefficients, in: Proc. 7th British Insecticide Fungicide Conf., 1973, p. 83
18. Karickhoff, S.W.: Sorption kinetics of hydrophobic pollutants in natural sediments, in: Processes
involving contaminants and sediments, Proc. Amer. Chem. Soc. Nat. Meet. Honolulu, april, 1979
19. Hamelink, J.L., Spacie, A.: Ann. Rev. Pharmacol. Toxicol. 17, 167 (1977)
20. Rosenberg, D.M.: Quaest. Entomol. / / , 97 (1975)
21. Lakowicz, J.R.: Biochim. Biophys. Acta 471, 401 (1977)
22. Branson, D.R., et al.: Trans. Amer. Fish. Soc. 104, 785 (1975)
23. Neely, W.B., Branson, D.R., Blau, G.E.: Environ. Sci. Technol. 8, 1113 (1974)
24. Konemann, H., Van Leeuwen, K.: Chemosphere °, 3 (1980)
25. Bruggeman, W.A., et al.: Chemosphere 10, 811, (1981)
26. Norstrom, R.J., McKinnon, A . E . , deFreitas, A.S.W.: J. Fish. Res. Board Can. 33, 248 (1976)
47
27.
28.
29.
30.
31.
32.
33.
34.
35.
48
Neely, W.B.: Environ. Sci. Technol. 13, 1506 (1979)
Metcalf, R.L., Sangha, G.K., Kapoor, LP.: ibid. 5, 709 (1971)
Kapoor, LP., et al.: J. Agr. Food Chem. 21, 310 (1973)
Zitko, V.: Metabolism and distribution by aquatic animals, in: The handbook of environmental
chemistry (Hutzinger, O. ed.), Springer-Verlag, Heidelberg, 1980, Vol. 2 part A, p. 221
Albro, P.W., Fishbein, L.: Biochim. Biophys. Acta 219, 437 (1970)
Hardy, R., et al.: Nature 252, 577 (1974)
Veith, G.D., Austin, N.M., Morris, R.T.: Water Res. 13, 43 (1979)
Kenaga, E.E., Goring, C.A.I.: Relationship between water solubility, soil-sorption, octanol-water
partitioning, and bioconcentration of chemicals in biota, in: Aquatic Toxicology, A S T M STP 707
(Eaton, J.C., Parrish, P.R., Hendricks, A.C., eds.), Amer. Soc. Testing Materials, 1980
Kenaga, E.E.: Ecotoxicol. Environ. Safety 4, 26 (1980)
CHAPTER
2
A C C U M U L A T I O N AND
ELIMINATION
TETRACHLOROBIPHENYLS
AQUEOUS
BY
KINETICS
GOLDFISH
OF
DI-,
AFTER DIETARY
TRI-
AND
AND
EXPOSURE
49
Chemosphere, V o l . 1 0 , No.8, pp 811 - 832, 1981
Printed i n Great B r i t a i n
OO45-6535/8l/O80811-22£o2.OO/O
© 1 9 8 1 Pergamon P r e s s L t d .
ACCUMULATION AND ELIMINATION KINETICS OF DI-, TRI- AND TETRA CHLOROBIPHENYLS
BY GOLDFISH AFTER DIETARY AND AQUEOUS EXPOSURE
W.A. Bruggeman, L . B . J . M . Martron, D. Kooiman and 0. Hutzinger
Laboratory of Environmental and Toxicological Chemistry
University of Amsterdam, Nieuwe Achtergracht 166
1018 WV Amsterdam, The Netherlands
ABSTRACT
Bioaccumulation kinetics of f i v e d i - , t r i - and tetrachlorobiphenyls from water and food were
studied in laboratory experiments with g o l d f i s h {Carassius auratus).
F i r s t order rate constants
for uptake from water and clearance were determined a f t e r simultaneous administration of the
f i v e compounds in constant concentration, and were related to bioconcentration factors obtained
in a s t a t i c fish-water e q u i l i b r a t i o n system. Biomagnification by retention of the PCB's from food
was studied in a separate experiment.
The difference in clearance rates f o r the chlorobiphenyls is the main reason for the d i f f e r e n t
bioconcentration and biomagnification factoi-s.
Absorption e f f i c i e n c i e s from water and food are higher than 402. Clearance half l i v e s vary from
10 days f o r 2,5-dichlorobiphenyl to 60 days for 2 , 3 ' , 4 ' , 5 - t e t r a c h l o r o b i p h e n y l , which i s correlalated with the decreasing aqueous s o l u b i l i t i e s of the compounds. Bioconcentration factors are
between 0.4 x 10 and 1.5 x 10°, biomagnification factors between 0.2 and 1.7, based on extractable l i p i d s . Substitution of chlorine in the position para to the phenyl-phenyl bond influences
hydrophobicity and bioaccumulation of the PCB's more strongly than substitution in ortho position.
6
A k i n e t i c model is developed which accounts for the influence of the l i p i d content of the f i s h
on the clearance rate of a chemical. Reproducible determination of the bioconcentration potential
of environmental chemicals i s possible by use of an "internal bioaccumulation standard" in a k i netic test system. Food chain accumulation in f i s h i s l i k e l y to be an important process only f o r
persistent chemicals with extremely low water s o l u b i l i t y .
INTRODUCTION
Environmental chemicals that are present in low concentrations in a i r and water may bioaccumulate
to high levels in l i v i n g organisms.
Contaminated food is usually the main source of a l l except very v o l a t i l e pollutants f o r a i r
breathing animals such as birds and mammals. Non-degradable, l i p o p h i l i c compounds accumulate in
fatty tissue, since e f f e c t i v e transformation and excretion mechanisms do not exist f o r such chemicals; they may thus be transferred through a food chain and reach top predators in unexpected
high concentration, a process called biomagnification.
For aquatic organisms, direct uptake of chemicals from water is probably much more important than
bioaccumulation from food. Fish extract l i p o p h i l i c compounds from water via their g i l l s together
with oxygen (Norstrom et a l . , 1976) and so very high concentrations in the body fat can eventua l l y be reached by equilibrium p a r t i t i o n i n g . Although bioaccumulation i s not a toxic e f f e c t in
i t s e l f , long term effects may be observed when tissue concentrations of certain chemicals are
steadily increasing in organisms by bioaccumulation.
High concentration factors in f i s h cause high exposure concentration of environmental chemicals
f o r fish-eating animals and man. Therefore, estimation of the bioaccumulation potential in f i s h
is important for the evaluation of possible environmental hazards from synthetic chemicals.
Adequate prediction and testing of the accumulation behaviour of chemicals requires a thorough
understanding of the b i o l o g i c a l , physico-chemical and structural factors influencing the kinetics
of the bioaccumulation process. An accelerated bioconcentration test procedure based on the
1
51
2
kinetics of uptake from water and clearance has been develooed by Branson et a l . (1975) . using
2,2',4,4'-tetrachlorobiphenyl as a model compound. This test procedure, carried out with d i f f e r ent chemicals was the basis of the correlation between loq P (the octanol/water p a r t i t i o n coeff i c i e n t ) and log K (the bioconcentration factor) in f i s h (Neely, 1974) .
The accuracy of such a correlation is highly dependent on the reproducibility of the tests. Experimental conditions (oxygen concentration, temperature) and individual differences between
test organisms ( s i z e , a c t i v i t y , fat content) can cause differences in bioaccumulation rate and,
to a certain extent, also in steady state l e v e l . Therefore, i t seems advisable not only to standardize test conditions and organisms, but also to add a suitable chemical as an internal
standard in each test and to measure i t s bioaccumulation behaviour simultaneously, which w i l l allow
a direct comparison between d i f f e r e n t chemicals.
Information about the kinetics of bioaccumulation a f t e r dietary exposure of aquatic organisms i s
r e l a t i v e l y scarce. Recently, Macek et a l . (1979)1 pointed out that food chain transfer was i n s i g n i f i c a n t compared to direct bioconcentration bv f i s h f o r six out of seven bioaccumulating pollutants. DDT was the only exception, producing a 30-502 increase over direct exposure steady state body burden of fathead minnows when their food organisms had also been exposed to the same
aqueous concentration. DDT also showed the highest bioconcentration factor of the seven chemicals
studied.
Retention and accumulation of chemicals administered via food w i l l be dependent on the uptake eff i c i e n c y on one hand and on the clearance rate of the compound on the other. A low clearance rate
proved to be the main factor responsible f o r a high bioconcentration factor of a chemical (Neely,
1974, KBnemann, 1980)3'=> and is therefore expected to play a key role in the bioaccumulation process of chemicals in f i s h .
In this study, polychlorinated biphenyls (PCB's) are used as model compounds for bioaccumulation
experiments. Five PCB's with the same chlorine substitution pattern in one ring of the biphenyl
nucleus ( i . e . 2,5-dichloro-), but d i f f e r i n g in the second phenyl r i n g , were studied simultaneously in bioaccumulation experiments with goldfish (Carassius auratus).
In this way, relationships
between chemical structure, aqueous s o l u b i l i t y and accumulation parameters could be determined
using simple f i r s t order kinetics for uptake from water and food and f o r clearance of the chemicals.
3
D
MATERIALS AND METHODS
Fish. 45 Fish with lengths between 4.5 and 5.2 cm were selected from a batch of 100 one year
old g o l d f i s h .
Chemicals. PCB's (2,5-dichloro-, 2 , 2 ' , 5 - t r i c h l o r o - , 2 , 4 ' , 5 - t r i c h l o r o - , 2 , 2 ' , 5 , 5 ' - t e t r a c h l o r o and 2,3',4',5-tetrachlorobiphenyl) were synthesized according to Sundstrom (1973) by the reaction of chlorinated a n i l i n e with iso-amylnitrite in the presence of excess para-dichlorobenzene.
They were p u r i f i e d by recrystal1ization and adsorption chromatography until a purity of more
than 97% was achieved, as determined by gas chromatography.
Solvents: Analytical grade n-hexane and toluene were r e d i s t i l l e d before use.
Exposure. PCB-saturated water was prepared according to the method described by Veith et a l .
(1975)/ with some modifications: The f i v e PCB's (0.5 g each) were dissolved in 500 ml hexane.
This solution was mixed with 250 g p u r i f i e d sea sand (Merck) and the solvent slowly removed in a
rotary evaporator. The resulting PCB-coated sand (0.2%) was poured into a funnel shaped column.
Aquarium water (2/3 Amsterdam tap water + 1/3 deionized water) was saturated with PCB's by rec i r c u l a t i o n through the PCB-coated sand column.
6
Exp.
1: Constant
concentration
Twelve goldfish were exposed to PCB saturated water in a 30 1 glass covered aquarium. Plants
[Elodea sp.) were present f o r additional oxygen supply and uptake of excess mineral nutrients
which otherwise would cause algal blooms in long term experiments. The water was continuously recirculated through the PCB column and a small sedimentation tank ( F i g . 1).
Except f o r the c i r c u l a t i o n pump and teflon stopcocks, contact between water and material other
than glass was avoided in the c i r c u l a t i o n system. Glass tubes were t i g h t l y connected with the aid
of a small pieces of PVC-tubing. To prevent evaporation of the chemicals, the system was kept
closed and not aerated. The oxygen content of the water was maintained at about 7 mg O2/I by a
very small stream of oxygen gas. Water temperature was 23°C. Fish were sampled a f t e r 1,2,4,8 and
13 days.
52
F i g . I. Exp. 1: Continuous exposure to PCB saturated water.
oxygen inlet
r
I
water flow
glass wool plug
0
coated sand column
V
circulation pump
tap
Exp.
2: Equilibration
and
clearance
Twelve goldfish were placed in PCB-contaminated water, which was prepared with the PCB-coated
column that had previously been used for experiment 1. The same system was used for c i r c u l a t i o n
and oxygen supply, but without the PCB column, to allow the PCB concentrations in the water to
decrease during the e q u i l i b r a t i o n experiment. Fish samples were taken at day 1,2,3,6,8 and 23.
When PCB concentrations in the water had equilibrated, the six remaining f i s h were transferred
to clean water in a 30 1 aquarium to study clearance of the chemicals.
The aquarium water was vigorously aerated and f i l t e r e d over active carbon to remove PCB's released by the f i s h . During this clearance study, f i s h were fed sparingly only to orevent loss of
weight. Time intervals between f i s h sampling increased from one week in the beginning to 8 weeks
at the end of the experiment. The last f i s h was s a c r i f i e d at the 116th clearance day.
Exp.
3:
Food
F o r t i f i e d food, containing 10 gg of each compound per gram was prepared by mixing 100 g commerc i a l dry f i s h food (Tetramin) with 50 ml of a 20 mg/l solution of the PCB's in toluene. The s o l vent was evaporated under vacuum. Twenty goldfish were kept in a "clearance" system as described
for experiment 2. They were fed the f o r t i f i e d food at a rate of ca. \% of the f i s h weight per
day, in small portions to prevent water contamination by feed rests. Every ten days one f i s h was
s a c r i f i c e d and analyzed, and a f t e r 150 days the experiment was terminated.
Analysis: Sampling,
extraction
and clean
up
Fish: For each sample, one f i s h was netted and k i l l e d in l i q u i d nitrogen, measured and weighed
and kept frozen or immediately thawed and homogenized in a porcelain mortar. The homogenate was
extracted by s t i r r i n g with 100-150 ml toluene at reflux temperature f o r 100 minutes. The extract
was c l a r i f i e d by centrifugation or by gravity s e t t l i n g during a few hours. Half the volume was
used f o r determination of extractable
lipid Height: toluene was removed by rotary evaporation,
the o i l y residue was transferred to a "small v i a l in 2 ml of hexane and l i p i d weight determined
a f t e r 60 minutes evaporation under a stream of nitrogen.
FCH's in the original extracts were detected and quantified by gas chromatography with electron-capture detection (GC-ECD, see below). When PCB-concentrations were low compared to background
signals, the extract was prepurified by concentrated s u l f u r i c acid treatment and preparative
thin-layer chromatography (si 1ica/hexane) and concentrated by partial evaporation of the solvent.
A recovery of 80% by this clean up procedure was measured.
Samples of food and collected faeces were ground and extracted as described f o r f i s h . Each water
sample (0.1-3 1) was shaken three times with at least 30 ml fresh toluene each time, layers were
separated by centrifugation, i f necessary, and the extracts combined. PCB concentrations were
determined by GC-ECD (see below), a f t e r volume adjustment by d i l u t i o n or evaporation.
Biotransformation
products: Final water and f i s h samples of experiment 1 were investigated f o r
PCB transformation products as described by Tulp et a l . (1977)°. The procedure included acid
53
hydrolysis, extraction and methylation of free metabolites, fractionation by s i l i c a adsorption
chromatography and investigation by combined gas chromatography - mass spectrometry.
Gas chromatography. Quantitative gas chromatographic analysis was performed by peak height determination and comparison with standard mixtures of the f i v e PCB's. Volumes of 0.1-3 ul were i n jected on one of the following gas chromatographic systems, both equipped with electron-capture
detector (Table 1).
TABLE 1
Gas chromatography
1
system
gas chromatograph
Varian 1400, adapted f o r
2a,b
Tracor 550
c a p i l l a r y GC
detector
H on Sc
63
glass,27m x 0.3 mm ( i . d . )
glass, 1.80 m x 2mm i . d .
3
column
stationary phase
N i , DC or pulse mode
WCOT
packed with:
0V-101, 15% dynamic coating
a 3% SE-30 on Chromosorb W,
acid washed, 100-120 mesh
b 0.2% Carbowax 20 M on
Chromosorb W,
acid washed, 100-120 mesh,
prepared according to
9
Aue et a l . (1973) .
c a r r i e r gas
N , 1 kgf/cnT
N£, 25 ml/min
detector make up
45 ml/min
75 nl/min
temperatures: i n j e c t o r
240°C
275°C
2
oven
200°C iso or
160°C iso (packing a)
145°C iso (packing b)
progr., 8°/min, 150-250°C
detector
outlet 240°C
285°C
quantification range
250°C
2-100 pg injected
injection
0.2 - 20 ng injected
0.1 - 3 ul
0.5 - 3 w l , s p l i t 1:50
Each system separated the f i v e PCB's within ten minutes, however, f o r PCB concentrations below
200 ng/ml, system 2b (*>3Ni. "Aue"-packing, isothermal) was the most e f f i c i e n t .
54
RESULTS AND CONCLUSIONS
Water saturation.
Within one week a f t e r the start of the c i r c u l a t i o n through the PCB-coated column, the aqueous concentrations of the PCB reached their plateau l e v e l . At least 75% of this
level was obtained already within the f i r s t two days. The saturation concentration was maintained during the f i r s t aqueous experiment with g o l d f i s h . Values are given in Table 2 as mean
aqueous concentrations, they were i n the same order of magnitude as the aqueous s o l u b i l i t i e s of
various d i , t r i and tetrachlorobiphenyls determined by Weil et a l . (1974)10. After prolonged
running, small white clots were observed in the sand column, which a f t e r analysis appeared to
consist mainly of the f i v e PCB's stripped o f f the sand. This phenomenon did not disturb the plateau levels of the chemicals dissolved i n the water. Only minor fluctuations (+ 20%) were observed that were attributed to adsorption of the PCB's on suspended d e t r i t a l material.
PCB-concentrations measured i n f i s h and water at d i f f e r e n t time intervals during experiment 1, 2
and 3 are l i s t e d in Tables 2-4, and 6.
Kinetic model Measured PCB concentrations in f i s h and water were interpreted in terms of the
f i r s t order two compartment exchange mode as proposed bv Branson et a l . (1975) , to compare uptake and clearance rates and concentration factors of the d i f f e r e n t chemicals. Similar equations
were used to describe bioaccumulation a f t e r dietary exposure ( F i g . 2, Eq. 1).
2
C»
Cfd
cf
FOOD
22l
dt
FISH
WATER
= e.f.Cw + k C
v
w
- k .C,
2
F i g . 2. Bioaccumulation k i n e t i c s .
Cfd. C f , C = concentrations of chemical i n food, f i s h and water
f
= feeding rate (food weight x f i s h weight
x time )
w
£
• absorption e f f i c i e n c y f o r ingested chemical
ki, kj
= rate constants f o r uptake from water and clearance (time
)
Exp. 1. For a constant water concentration
C , without dietary exposure, the concentration i n
the f i s h at time t after the start is given by Eq. 2 (from Eq.lby integration):
k,
M *
F • w • (l-exp<-k t))
(>
w
1
=
C
2
2
From this equation, ki can be calculated when k i s known (exp. 2b) and the f i s h concentration
at time t i s measured. At equilibrium, the r a t i o between f i s h and water concentrations i s represented by the bioconcentration factor
(Eq. 3)
2
Cf(-)
k,
w
2
Exp. 2a. When fish and water are equilibrated
without further addition of the chemical, the concentration i n the water decreases according to Eq. 4:
C (t) = C (o) - p . C
w
w
f
(1)
p = g f i s h exposed per gram aquarium water
whereas the f i s h concentration increases according to Eq. 5 and 6:
55
(from Eqs. 1 + 4 ) :
dC
f
JJ! • k C ( o ) - ( k j . p
r
w
k ).C
+
2
(5)
f
by integration:
k
l
f
k p + k
• w
" ( l - e x p f - t k j . p + k ).t>)
Exp. 2b. Clearance of the chemical in clean water (C = 0 = C
decay curve (Eqs. 7,8)
C
( t )
=
C
r
dC
( 0 )
2
2
f H
(6)
; Eq. 1) follows an exponential
f
~aT~
=
k
C
" 2' f
(7)
a f t e r integration:
C ( t ) = C (o) exp ( - k t )
f
f
(b)
2
t = time a f t e r transfer to clean water.
A straight l i n e w i l l be observed in a logarithmic plot of the f i s h concentration versus time,
when f i r s t order kinetics for release from one compartment i s followed. The slope corresponds to
the clearance rate constant k , which is used for calculation of the uptake rate constant, bioconcentration factor and accumulation curves by Eqs.2, 3, 6, and in a similar way for calculation
of the biomagnification parameters in the food experiment (exp. 3). The biological half l i f e
( t j ) of the chemical in the f i s h i s calculated by Eq. 9:
2
Exp. 3. Constant dietary exposure results in increasing f i s h concentrations u n t i l a plateau l e vel is reached when the clearance rate equals the uptake rate as shown by Eq. 10:
C (t) = | ^
f
. C .(l-exp (-k .tl)
f d
(10)
2
By this equation, the absorption e f f i c i e n c y i s calculated using k (from exp. 2b) and measured
f i s h concentrations. The ratio between the concentrations in f i s h and in the food at steady state is given by the biomagnification factor IC,:
2
m
c
f d
k
2
Conclusions
Experiment
1: Exposure
to constant
water
concentration
(Table 2)
F i f t y percent of the exposed goldfish died within seven days. They a l l showed very low a c t i v i t y ,
except during the last few hours before death, when severe loss of balance and orientation and
high v e n t i l a t i o n frequency were observed. Fish that were found dead contained somewhat higher
amounts of PCB's than those s t i l l a l i v e at that time of sampling. Data from dead f i s h were not
included in Table 2.
An almost linear increase of the PCB concentrations in the f i s h i s observed, with minor i n d i v i dual deviations and without indications of a plateau level during the thirteen days exposure.
Since the time needed to reach 50% of the plateau level in a constant exposure experiment i s
equal to the h a l f - l i f e in a clearance study (Eqs. 2,9), i t is clear that the clearance rates of
the PCB's are very low, corresponding to biological h a l f - l i v e s of more than one week. After 13
days, PCB concentrations in extractable l i p i d s were extremely high, e.g. 2.7% f o r 2,5-dichlorobiphenyl; concentration factors were higher than 80 x 10^. A faster e q u i l i b r a t i o n at lower PCB
concentrations was obtained in the second aqueous exposure experiment, where the water was not
56
continuously resaturated.
TABLE 2
Exp
day
1. Concentration of f i v e PCB's in goldfish duri nq exposure to PCB-saturated water
wet weight
PCB- concentration, pg/g wet
2,4',52,2',52,5di
tri
tri
extr. l i p .
w
(g)
0
5.5
4.1
1
1
4.5
7.4
120
weight
2,2',5,5'tetra
2,3' , 4 ' , 5 tetra
-
-
-
-
60
60
40
10
2
4.3
6.4
290
160
110
50
10
4
4.2
5.6
460
270
190
130
30
8
5.4
5.1
1120
770
490
320
70
2510
1820
1140
720
170
190
110
75
55
22
13
9.2
3.2
mean aqueous concentration:
(ug/1: + 20%)
Experiment
2: Equilibration
and clearance
(Tables 3,4,
Fig.
3)
TABLE 3
Exp. 2. E q u i l i b r a t i o n . PCB concentrations
PCB concentration, ug/1 water
2,4' ,52,2',5,5'2, 52,2',5tri
tri
tetra
di
day
saturation
f i s h added:
equi1ibration
in water
7. 3
9 7
9 7
9
7
1
2
3
4
7
1
2
3
6
7
8
10
13
14
20
22
23
1
0
0
0
0
0
0
0
0
0
0
0
24
48
30
24
20
29
33
27
31
29
29
76
130
185
150
130
137
50
26
16
10
9.5
12.0
10.6
9.4
9.5
18.7
9.5
8.3
90
135
91
79
84
39
22
15
9.5
8.5
10.5
9.0
6.8
6.2
14.6
5.0
4.5
74
117
69
57
65
38
23
24
11
8.5
11.0
8.8
6.4
5.2
15.2
4.1
3.8
2,3',4',5tetra
16.4
24
12.5
12
12
7.9
5.5
4.3
3.0
2.0
4.5
6.3
2.8
4.6
3.1
1.4
1.1
58
n
-I
c
o
XI -r•i-
59
TABLE 4
Exp. 2. Equilibration and clearance. PCB concentrations in goldfish
day
wet
% extr.
PCB concentration, pg/g wet weight
weight
lip.
2,5-
LD
equi1ibration
clearance
1
2
3
6
8
23;
0
8
15
27
39
62
116
2,2',5-
di
3.6
3.6
4.1
3.4
3.9
3.8
4.7
4.7
5.2
4.6
5.5
5.2
4
4
4
3
3
3
2
3
2
3
5
2
4
4
9
5
6
2
2
8
5
0
1
9
2
2
3
2
2
3
1
1
0
0
0
0
tri
23
36
29
85
74
79
034
115
148
163
145
001
88
101
130
145
125
177
92
89
21
23
23
0
2,4',5tri
3
4
5
6
6
3
9
2
42
69
75
97
119
102
144
91
88
43
53
54
9
2
6
6
1
1
8
3
7
0
2,2',5,5'-
2,3',4',5-
tetra
tetra
43
58
80
104
97
161
124
106
76
83
74
22
4
6
14
12
16
44
33
29
25
24
23
11
6
9
0
7
4
7
0
31
50
05
62
28
2
8
1
4
8
6
7
Exhaustion of the PCB coating in the sand column during experiment 1 was the main cause for aqueous start concentrations deviating from the previous saturation l e v e l . This resulted in a 2,5-dichlorobiphenyl start concentration of 7 ug/1, even lower than the concentration of 2 , 3 ' , 4 ' , 5 -tetrachlorobiphenyl.
A rapid i n i t i a l uptake by f i s h is coupled with a rapid decrease in the aqueous concentrations.
The time needed f o r equilibration was longer f o r PCB's containing more chlorine ( F i g . 3). After
23 days, concentration factors of the PCB's in extractable l i p i d s were higher than 300 x 10 .
3
In the clearance experiment, PCB concentrations in water were below the detection l i m i t , i . e . ,
1 ng/l (ppt).
Striking differences were observed in the clearance rates of the f i v e PCB's in f i s h ( F i g . 3). Typical gas chromatograms in F i g . 4 show the r e l a t i v e enrichment of the higher chlorinated biphenyls in f i s h tissue during e q u i l i b r a t i o n and clearance. Due to individual differences between
f i s h , linear regression analysis was required for determination of f i r s t order clearance rate
constants of the PCB's (k in Table 5, straight line slopes in F i g . 3).
Clearance rate constants were also calculated for a l l possible sample pairs separately according
to Eq. 12 (from Eq. 8):
2
In C ( t j ) - ln C ( t )
f
"2
'
f
t- - t.
U
t.
2
'
-
(12)
Here, k i s the slope of the line that can be drawn to connect two sample points in F i g . 3. This
is useful only i f :
2
The possible r e l a t i v e error in k w i l l become too large at lower concentration r a t i o s . Nine pairs
of samples from Table 4 meet this requirement. The results of these calculations are given in
Table 5, together with the ko's determined by linear regression analysis according to Eq. 8.
A variation of a factor two i s found in the absolute values for each chlorobiphenyl when d i f f e r ent sample pairs are compared.
The differences between the compounds are most c l e a r l y demonstrated by the relative
clearance
rate constants, which do not overlap when calculated for d i f f e r e n t sample pairs (Table 5).
This is also demonstrated by the p a r a l l e l lines in F i g . 5, which gives the relationships between
k ' s calculated f o r selected sample pairs and k ' s determined by linear regression analysis for
a l l samples.
2
2
60
2
TABLE 5
PCB clearance rate constants ( k , ) , absolute and r e l a t i v e
Sample pair
0 - 27
0 - 39
0 - 62
0 - 116
8 - 116
15 - 116
27 - 116
39 - 116
62-116
all
3
samples '
2,5
di
abs
rel
120
81
53
71
64
69
56
66
92
4
4
4
4
4
4
4
3
4
66
2,2'5
trj
abs
rel
2,4',5
tri
abs
rel
2,2',5,5'tetra
abs
rel
29
76
42
18
00
31
00
88
00
78
51
33
52
50
53
44
52
74
2
3
2
3
3
3
3
3
3
79
00
75
06
13
31
14
06
22
44
25
16
24
21
23
18
23
33
1
1
1
1
1
1
1
1
1
57
47
35
41
31
44
29
35
43
28
17
12
17
16
16
14
17
23
1
1
1
1
1
1
1
1
1
00
00
00
00
00
00
00
00
00
4 40
48
3 20
21
1 40
15
1 00
2,3',4',5tetra
abs
rel
21
15
10
11
9
9
8
9
13
10
8
0
7
8
0.75
0.88
0.83
0.65
0.61
0.56
0.62
0.58
0.57
0.67
A t
a L
days
27
39
62
116
108
101
89
77
54
Clearance rate constant of f i v e PCB's calculated from d i f f e r e n t sample pairs,
abs: Value calculated from the concentration ratio in two f i s h samples,
r e l : Value relative to 2,2 ,5,5'-tetrachlorobiphenyl.
1
a
' v a l u e s obtained by linear regression analysis, represented by the straight l i n e slopes
in F i g . 3.
t,
t
2
*2
sample pairs
10
k
2
by
linear r e g r e s s i o n
HO
- 2
1
day' )
F i g . 5. Clearance rate constants (kj) of f i v e PCB's, calculated from d i f f e r e n t
f i s h samples. Letters A-E correspond to compounds indicated in f i g . 4.
Parallel lines show constant ratios between clearance rate constants.
Experiment
3.
Food.
PCB concentrations in f i s h during the 150 days feeding study are l i s t e d in Table 6.
TABLE 6
Exp. 3. Dietary exposure: 10 ug/g food. PCB concentrations in g o l d f i s h .
day
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
wet weight
% extr.lin.
2.8
3.5
7.3
6.2
3.2
4.2
3.3
2.2
5.3
3.4
4.9
3.6
6.1
4.9
3.8
1.8
2.7
1.9
2.4
3.3
3.5
3.4
4.4
2.4
3.4
3.0
2.4
2.8
3.3
2.5
PCB-concentration, ug/g wet weight
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
321
514
274
500
313
857
879
50
434
706
469
278
393
367
553
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
536
857
274
677
469
929
485
73
472
529
367
361
459
510
658
0
1
0
1
0
1
1
2
1
2
1
1
1
1
1
714
23
411
52
563
52
45
50
51
59
43
11
41
29
97
0
1
0
1
0
1
1
2
1
2
1
1
1
1
2
643
29
521
55
719
86
15
86
70
29
39
39
57
39
13
0.821
0.943
0.548
1.42
0.625
1.67
1.24
2.64
1.79
2.65
1.63
1.67
1.72
2.00
2.79
The maximum PCB level was reached a f t e r 80 days exposure. Due to individual differences between
the f i s h , no smooth curves are obtained when the PCB concentrations are plotted against time
(Fig. 6a). The f i v e PCB's are d i r e c t l y compared by concentration ratios in F i g . 6b, where the
concentration of 2,2',5,5'-tetrachlorobiphenyl i s set to 1.00 (as an internal standard) and the
concentrations of the other PCB's are scaled proportionally.
The concentrations of the f i v e PCB's i n i t i a l l y increased at about the same rate (concentration
ratios = 1), indicating comparable absorption e f f i c i e n c e s . After 150 days exposure however, a
difference of a factor f i v e was found between the extremes of this series of compounds, which
apparently was caused by the d i f f e r e n t clearance rates.
Determination
of bioaccumulation
parameters
and theoretical
curves.
Basis f o r a l l calculated parameters l i s t e d in Table 7 was the clearance rate constant k j , directly determined in the clearance experiment (2b). Using the parameters k i , k2 and c and measured
exposure concentrations in water and food, theoretical PCB concentrations in f i s h at d i f f e r e n t
times in the experiments were calculated. The resulting curves are drawn in Figs 3 and 6.
Bio
transformation.
Small amounts of d i - and trichloromethoxybiphenyls were detected by GC-MS in a methylated extract
of 3 1 hydrolyzed aquarium water, taken d i r e c t l y a f t e r termination of the f i r s t experiment ( f i s h
in PCB saturated water). Careful analysis of another sample of 2.5 1 water from the same aquarium, a few months l a t e r , showed the presence of a large amount of a dichlorobenzoic acid, presumably formed from 2,5-dichlorobiphenyl, as well as a variety of hydroxylated d i - and t r i c h l o r o biphenyls (as their methylated derivatives). Configurations of these metabolites w i l l be reported elsewhere in a more detailed aquatic metabolism study. The p o s s i b i l i t y that micro-organisms
were responsible f o r the biotransformations cannot be excluded, and is very probable f o r the d i chlorobenzoic acid (Ballschmiter et a l . , 1977 , Tulp et a l . , 1978)1 • 12. s i nee the amounts of
hydroxylated PCB's detected were very small . compared with the parent compounds, i t seems reasonable to assume that metabolism does not play an important role in the excretion of PCB's from
fish.
1
62
§ tj
OOO
~H
o o — <-*
O
<U
••- CT>
*-> re
CU
O
•<
T3 TD
CL CL
o o o o o
CU
O
LO
LO
LO
« LO •» •
CM
CM n
C M C M C M CM C M
64
41
10
(TJ
HJ
CU
DISCUSSION
For the f i v e PCB's studied, i t can be concluded (Table 7) that the differences in the apparent
aqueous s o l u b i l i t i e s (S) are reflected only by the d i f f e r e n t clearance rate constants (l<2). Subs t i t u t i o n of chlorine in the position para to the phenyl-phenyl bond had a much stronger i n f l u ence on the clearance rate than substitution in the ortho p o s i t i o n . These differences could not
be explained by any of the known calculation methods for the l i p o p h i l i c i t y of organic compounds.
Only the use of the substitution constants d i r e c t l y derived from aqueous s o l u b i l i t i e s of chlorobiphenyl isomers (Tulp & Hutzinger, 1978) resulted in a good correlation with the clearance
rate constants.
13
Much less variation was observed in the intestinal absorption e f f i c i e n c e s ( e ) and the uptake rate constants (ki) of the compounds. Thus, the variation in the biomagnification and bioconcentration factors (Km and K ) was mainly dependent on differences in the clearance rate constants.
c
In order to get an insight in the g i l l membrane permeability for the PCB's studied here, the effiency transfer c o e f f i c i e n t s (E) were calculated from the uptake rate constants ( k i ) , using the
equations and c o e f f i c i e n t s discussed by Neely (1979)^.
These calculations are based on the determination of the amount of water flowing past the g i l l
surface (the ventilation volumetric rate: R ) of the experimental animals, from their oxygen
need, the oxygen concentration in the water and the e f f i c i e n c y transfer c o e f f i c i e n t f o r oxygen.
v
TABLE 8
Ca1cu1ation of the e f f i c i e n c y transfer c o e f f i c i e n t E
TaTter Neely, 1979)14
E . k.
1
kj
F x 0.75 x 82 x T(K) x mg of 0, l "
(j-a F
x 32
1
x 10"
6
2
(13)
= uptake constant in h 1
F
• mean f i s h weight : 4.5 g
0.75
= e f f i c i e n c y transfer c o e f f i c i e n t f o r oxygen
T
= temperature : 295 K
oxygen concentration
a
in water
:
7 mg 1
= metabolic rate c o e f f i c i e n t f o r g o l d f i s h
at 23°C : standard : 0.15
active
: 0.32
ml 0
2
h"
1
Assuming a semi-active metabolic rate here, the c o e f f i c i e n t
a is taken as 0.24 ml 0
2
h" .
The formula contains a conversion factor for oxygen from mg to ml
3
Result: E • 0.934 x 10" x kj
1
(kj in d" )
(14)
1
1
For the d i - and trichlorobiphenyls studied the k j was > 0.9 x lO^d" or 37.5 h " . This resulted
in an estimated e f f i c i e n c y transfer c o e f f i c i e n t of 0.84, which i s even higher than the c o e f f i cient f o r oxygen (0.75), and must be very close to the real absolute maximum. The theoretical l i mit, being 1.00, corresponds to the case that a l l water passing through the g i l l s is completely
cleared of the dissolved chemical.
For chemicals that approach this condition, the uptake rate is a function of the v e n t i l a t i o n rate and the water concentration only, membrane permeability no longer being a l i m i t i n g factor.
65
This i s an explanation f o r the fact that no increase in uptake rate constants was found f o r the
PCB's containing more chlorine atoms, that are expected to be more l i p o p h i l i c . In contrast to the
correlation between e f f i c i e n c y transfer c o e f f i c i e n t and log P (P = the octanol/water p a r t i t i o n
c o e f f i c i e n t ) described by Neely (1979) , even lower uptake rate constants (ki) were calculated
for the tetrachlorobiphenyls, especially f o r the least water soluble compound, 2 , 3 ' , 4 ' , 5 - t e t r a chlorobiphenyl. It i s not certain at the present time whether this is due to a real decrease in
membrane permeability, or to binding of the compound to organic colloids in the aquarium water,
r e s t r i c t i n g the a v a i l a b i l i t y of "dissolved" chemical. However, i t is l i k e l y that addition of bulky apolar substituents w i l l not i n f i n i t e l y stimulate the membrane permeability f o r organic molecules, since the l i p i d double layer w i l l have i t s own size l i m i t a t i o n s .
14
The e f f i c i e n c y transfer c o e f f i c i e n t , E, has limited v a l i d i t y as a bioaccumulation parameter for
comparison of d i f f e r e n t chemicals, since i t i s dependent on the r a t i o between the rate of transfer through the g i l l membrane and the ventilation rate. A l l compounds which have transfer rates
much higher than the ventilation rate w i l l show an e f f i c i e n c y close to 1.00. On the other hand,
very low transfer rates w i l l dominate the whole uptake process: the uptake rate constant, k j , w i l l
be independent of the ventilation rate, R. Thus, at d i f f e r e n t ventilation rates, d i f f e r e n t
" e f f i c i e n c i e s " w i l l be determined, since the e f f i c i e n c y transfer c o e f f i c i e n t , E i s defined as:
(15)
Therefore, measured uptake and clearance rates should ultimately be converted into transfer or
permeation c o e f f i c i e n t s , giving the ratio between transfer rate and concentration gradient over
an exchange barrier between two compartments.
Clearance rate constants tend to decrease during a clearance experiment i f two or more compartments are involved which are cleared at d i f f e r e n t rates (Konemann, 1980)'. However, some authors
tried to describe this phenomenon by use of concentration dependent rate constants or second order kinetics (Ellgehausen et a l . , 1980)15.
This implies that a lower dose of the chemical would result in a smaller clearance rate constant.
On the contrary, f i r s t order multicompartment kinetics makes the rate constant in clearance experiments time dependent. Assuming passive transport mechanisms for persistent, apolar, l i p o p h i l i c chemicals, only f i r s t order kinetics is suitable.
For some f i s h sampled in the clearance period, measured concentrations of individual PCB's were
ca. 30% higher or lower than the concentrations calculated from "average" clearance rate constants (k->), determined by linear regression analysis of a l l samples. The question arose whether
these individual deviations could be the result of measurable differences between the f i s h e s ,
i . e . , the l i p i d content, varying from 2.2 to 5.1% of the fresh body weight.
Kinetic model II
Lipophilic chemicals accumulate in f a t t y tissue. Individual variations in l i p i d content w i l l i n fluence bioaccumulation factors and clearance rates, therefore, i t is r e a l i s t i c to take the body l i p i d s as the storage compartment in a kinetic model. The blood may serve as a transport compartment, whereas the most e f f i c i e n t exchange of compounds occurs via the g i l l s , the gate compartment, through which water is pumped at a rate R (Fig. 7).
A l l compartments are considered nomogeneous, as far as the concentration of the bioaccumulating
chemical is concerned. The transfer rate from one compartment to another w i l l be proportional to
the difference in thermodynamic a c t i v i t i e s (y x C) and w i l l be dependent on surface properties
and area of the exchange barrier (e.g., biomembrane), expressed together as the transfer c o e f f i cient k.
At equilibrium, thermodynamic a c t i v i t i e s in a l l compartments are equal. The concentration ratio
is the reciprocal of the ratio of a c t i v i t y c o e f f i c i e n t s in both phases: (Mackay, 1977)'°
equi1ibriurn: ^ =
(16)
The model is characterized by the following set of rate equations:
net uptake in f i s h :
66
F.
= R (C - C )
w
(17)
FISH
WATER
1
R
G
T
gill
blood
lipid
1
k
1
c
Yt
g t
k,|
Ci
c,
g
1 J
Y
|
w
k„
kgt
Y,
1
I
gastro-intestinal
f L.
tract
FOOD
Fig. 7. Uptake and clearance of l i p o p h i l i c chemicals in f i s h .
Kinetic model.
F
G,T,L
-1
Cfd
:
:
:
:
:
:
:
tl
:
:
:
R
f
f i s h mass
mass of gate, transport or l i p i d compartment
concentration of chemical in each compartment
concentration in ambient water
concentration in food
a c t i v i t y c o e f f i c i e n t of chemical in the
d i f f e r e n t compartments
transfer c o e f f i c i e n t s for exchange between
two compartments
v e n t i l a t i o n rate
absorption e f f i c i e n c y from food
feeding rate
dC
net increase in gate compartment:
« (C -C )-k (Y C -Y C )
R
G-
w
dC
net increase in transport compartment:
dC.
L--j^ =
increase i n l i p i d compartment:
T-
g
G T
G
w
t
(18)
t
t
= gt(Y C -Y C )-k
K
w
G
t
t
t l
( Y ^ - Y ^ )
(19)
( Y ^ - Y ^ )
(20)
A true transport compartment has a low storage capacity, compared to the storage compartment, and
a high turnover rate. A f t e r a certain time, needed f o r adaption, the transport compartment w i l l
be i n a kind of slowly changing steady state. The net exchange with the neighbouring compartments i s then much higher than the net increase i n the transport compartment i t s e l f .
R
c
C
( w- g'
:
k
(
9
t W
Y
t
C
t> = " t l ^ t W l l
(
2
1
)
The smaller the storage capacity of a compartment, the better this "transport" condition i s f u l f i l l e d . The storage capacity of a compartment can be defined as the ratio of the total amount
and the a c t i v i t y of a compound i n that compartment.
67
X C
-x
X
storage capacity:
—*- = —
(22)
x x
x
(where X • mass of compartment X)
Thus, low storage capacities for l i p o p h i l i c chemicals are characteristic for aqueous compartments of limited volume, e . g . , g i l l s , blood.
From Eq. 21
Y
k
< gt
+
k
Y
C
=
t1>*t t
or:
C
y
Y
FT
TT^
c
^ t
•
Cg
•
"
.
V
k—
gt
+
K
k
C
—
(">
K
y^
c
TTTT " g
« W
Y
" V
C
<wg -
"
+
c
TTT " l
1
k—
tl
thus:
+
F->
"
g t
Y
,w
<r
+
r~
*
1
+
C
t l
q " <Fg
K
+
tl
c
<w - V
Y
C
r-) w
gt
»•
TT"
+
V
t—
tl
K
•
C
' l
T
gt
1
"
(*)
K
26
l
< )
1
Y
1
FT
gt • FT>
*tl
v C - ,C,
f
li—
gt
K
'rr
»
< )
C,1
thus (from Eq. 21):
R
23
tl>l l
,
FT
J —
*
.
"Ic—
tl
+
Vw g
w
- rr • w c
TY
i
(27
R
The rate equation f o r uptake and clearance in f i s h i s then:
R
*gt
k
tl
This expression can be understood as:
the rate of transport of a chemical from water to the storage compartment is equal to the a c t i vity gradient, divided by the sum of the resistance f a c t o r s . A resistance factor is here defined as the reciprocal of the transfer c o e f f i c i e n t .
Thus f a r , the model can be called the "direct current" model, using thermodynamic instead of
e l e c t r i c potentials.
The model is equivalent to the original 1st order bioconcentration model (Branson, Neely)
if:
=
k
C
k
C
l w " 2 f
1
w
l'-T-y
~
Zw
1
gt
( 2 9 )
a n d
k
l
=
E
' f
<
N e e l
>' i ^ )
1
4
(30)
Y
k
31
<)
1
+
R
68
k
t
_ L
tl
k
:
1 ^ 1
R
k
k
gt
t
K
i
Y
Kc = r— • —— • 1
'1
1 • l i p i d content of the f i s h (g l i p i d / g f i s h weight) = t
W
(34)
The capacity of the transport compartment w i l l play an important role a f t e r a sudden change in
the aqueous concentration ( C ) , e . g . , at the start of an uptake or clearance experiment. The
loading or deloading of the transport compartment w i l l dominate the flow, and Eq. 2 8 i s then reduced to:
W
dC^
Y
C -
Y-IC,
w
r-ar-/
,
«s + —
R
k
1
35
<>
gt
A two phase clearance curve i s now the r e s u l t . In theory, the equilibration between l i p i d and
blood may be much faster than the equilibration between blood and the g i l l compartment (kgt < <
k ) , in that case, a second phase is lacking since the l i p i d - b l o o d barrier has no s i g n i f i c a n t
transfer resistance.
For very l i p o p h i l i c compounds, the f i r s t phase w i l l be of less importance: the capacity of the
transport compartment w i l l be low compared to the l i p i d compartment. Also high 1 i p o p h i 1 i c i t i e s ,
characterized by high aqueous a c t i v i t y c o e f f i c i e n t s (YW) w i l l cause a faster e q u i l i b r a t i o n of the
f i s h compartments. A l l dissolved chemical i s immediately extracted from the g i l l water, the eff i c i e n c y transfer c o e f f i c i e n t (E) w i l l approach unity, and the v e n t i l a t i o n rate w i l l be the l i miting factor f o r the exchange process:
t l
i - < < k _ k,,
Y
gt' t l
»
W
£
dt
or E -
• i < C - H . It)
F
w Y
I
v
(36)
w
1.00
Transport of "super-permeating" chemicals w i l l be determined by flow, not by d i f f u s i o n l i m i t a tions.
The kinetic model discussed above allows correction of the clearance data f o r individual d i f f e r ences in l i p i d content.
For each f i s h , the clearance i s described by:
ln C (t) • In C (0) - - 1 . k. . 4
(37)
w
In p r i n c i p l e , the biological parameters determining k j are weight s p e c i f i c (R/F, kgf/F and
k t l / F ) and w i l l be v i r t u a l l y constant f o r a narrow range of f i s h weights (remember: R » F ° - ° ,
Table 8 ) .
If clearance starts from equilibrium, a l l f i s h had the same concentration at t = 0. The clearance
rate constant is then inversely proportional to the l i p i d content of the f i s h .
Therefore , s i g n i f i c a n t improvements in the correlations are expected when PCB concentrations (Cf)
in the f i s h during the clearance period are plotted against the clearance time, divided by the
l i p i d content of the f i s h sample ( t / 1 ) . The resulting graphs are shown in F i g . 8 .
f
f
69
As predicted, two phases can be discerned now: a rapid phase during less than 17 days (for a
goldfish containing 3% extractable l i p i d ) , and a slower one f o r the rest of the time. Individual
deviations from the curve are n e g l i g i b l e now, as compared with the estimated r e l a t i v e inaccuracy.
During the f i r s t phase, only a twofold reduction of the i n i t i a l concentrations was achieved for
the PCB's studied, with the possible exception of 2,5-dichlorobiphenyl which exhibited a 5-10
f o l d reduction during the f i r s t 17-24 days+ However, the small number of measurements in this
region did not allow precise determinations of rate and duration of the f i r s t clearance phase.
Also, the differences in concentration decrease in both phases were small compared with the poss i b l e inaccuracy of 20% in the concentration measurements.
The discussed relationship between clearance rates and l i p i d content of the f i s h may explain the
individual variations between the f i s h and is an indication of the use of an "internal standard"
procedure in bioaccumulation tests f o r automatic correction of individual differences, as shown
by the results in this paper.
Correction of the PCB concentrations
f o r the l i p i d content of the sample, i . e . , by plotting ug
PCB per gram of l i p i d instead of g PCB per gram of f i s h fresh weight, did not improve the correlations.
Equilibrium bioaccumulation factors are affected by the l i p i d content, since they are inversely
proportional to the clearance rate constants:
from Eq.34:
M
K . J . — •
C
F,
Y}
1
'
(38)
1
where K-| represents the lipid/water p a r t i t i o n c o e f f i c i e n t . For l i p o p h i l i c chemicals, this seems
to be the only absolute bioconcentration parameter, r e l a t i v e l y independent of the test organism.
Ratios between l i p i d concentration factors of d i f f e r e n t compounds w i l l be reflected by the relaclearance rate constant.
tive
The clearance rate constant (k2) in i t s e l f is an important parameter for the description of the
process of biomagnification of chemicals in food, in combination with the intestinal absorption
efficiency (t).
In this paper, i t is shown that the absorption e f f i c i e n c i e s f o r a l l the PCB s studied are high,
in the order of 50% or above.
The accuracy of the determinations of the intestinal absorption e f f i c i e n c i e s is affected by the
large individual differences between the f i s h e s , as a result of the way of food administration:
the amount of food ingested by each f i s h individually could not be determined, and small losses
to the bottom or to the f i l t e r were unavoidable.
The magnitude of the biomagnification factor i s given by:
" . • ^ • t ' V F - ' r
1
( 3 9 )
(see Eqs. 11,30,38)
Thus, the biomagnification factor is dependent on:
1) the r a t i o of the intestinal and g i l l absorption e f f i c i e n c i e s (-|), which are expected to be
interrelated and in the same order of magnitude,
.
2) the r a t i o between the weight-specific feeding rate, f, (g food x g f i s h x day" ) and the
weight s p e c i f i c v e n t i l a t i o n volumetric rate, R / F , (ml water x g f i s h " ! x day-1); the l a t t e r
parameter i s related to the s p e c i f i c oxygen need (metabolic a c t i v i t y : Q/F) and inversely
proportional to the oxygen content of the water,
3) the l i p i d concentration f a c t o r , K j , times the l i p i d content of the f i s h , 1.
- 1
1
v
It seems reasonable to assume that the second f a c t o r , j^-Lr in Eq. 32 is r e l a t i v e l y independent
of the organism and i t s a c t i v i t y , since 1) the amount v' of oxygen needed w i l l be related to
the amount of food consumed: t y p i c a l l y , the second factor w i l l be in the order of 10- -10" .
(In the experiments described here, ca. 10 mg of food was consumed per gram f i s h , against 1 1 of
water extracted d a i l y ) .
Therefore i t can be stated that the l i p i d concentration factor is also the most important parameter determining the biomagnification of an organic chemical: minor influences are expected from
the oxygen content of the water in proportion to the c a l o r i c value of the food, and of the r a t i o
b
D
71
of the intestinal and g i l l absorption e f f i c i e n c i e s . Thus, biomagnification factors > 1 can be expected only f o r very l i p o p h i l i c chemicals: log P > 5. This is in agreement with the conclusions
of Macek (1980) * from experiments with DDT and less l i p o p h i l i c chemicals.
Under natural conditions, a biomagnification factor equal to unity may imply a s i g n i f i c a n t contribution of the ingested food to the total bioaccumulation of a compound, since the aquatic food
organisms are subject to the same bioconcentration phenomena (K,) as the feeders
Thus:
1
c
V
fd
K..C
c w
'fd
(40)
(41)
c w
where Cf = steady state concentration in f i s h
fd "
"
" food
Cw
"
" water
c
=
The factor (<„, + 1).K may be called the ecological bioaccumulation f a c t o r , representing the actual fish/water concentration ratio at steady state conditions. Our present bioaccumulation research focusses on the behaviour of "superlipophi1ic" and high molecular weight compounds
With higher biomagnifiation factors even a real food chain bioaccumulation e f f e c t might be poss i b l e in aquatic organisms.
C
REFERENCES
1
2
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
U976)°
r S t r 0 m
°792( 1975)°"'
G
E
' '
E
'
M c K i n n o n
B l d J
'
H
S
'
a n d
A
s
w
- - - deFreitas. J . F i s h . Res. Board. Can., 33, 248-267
A l e x a n d e r
a n d
W
B
' -
N e e
' y - Trans. Amer. Fish. S o c , 104, 785-
W.B. Neely, D.R. Branson and G.E. Blau. Environ. S c i . Technol., 8, 1113-1115(1974)
K . J . Macek, S.R. P e t r o c e l l i and B.H. Sleight I I I . i n : "Aquatic Toxicology", ASTM STP 667,
L . L . Marking and R.A. Kimerle (Eds.). American Society for Testing and Materials, 1979, pp.
251-268.
H. Konemann and K. van Leeuwen. Chemosphere, 9, 3-19(1980).
G. Sundstrom. Acta Chem. Scand., II, 600-604(79731.
G.D. Veith and V.M. Comstock. J . FTsh. Res. Board Can., 32, 1849-1851(1975).
M.Th.M. Tulp, K. Olie and 0. Hutzinger. Biomed. Mass Spectrom., 4, 310-316(1977).
W.A. Aue, C R . Hastings and S. Kapila. J . Chromatography, 777 29^-307(1973).
L. Weil, G. Dure and K.E. Quentin. Wasser- und Abwasser^Forschung, 7, 169-175(1974).
M.Th.M. Tulp, R. Schmitz and 0. Hutzinger. Chemosphere, 7, 101^108(7978).
K. Ballschmiter, Ch. Unglert and H . J . Neu. Chemosphere, 6% 51-56(1977).
M.Th.M. Tulp and 0. Hutzinger. Chemosphere, 7, 849-860(lf78).
W.B. Neely. Environ. S c i . Technol., 13, 1506-1510(1979).
H. Ellgehausenl J . A . Guth and H.0. r i s e r . Ecotoxicol. Environ. Safety, 4, 134-157(1980)
D. Mackay. Environ. S c i . Technol., U, 1219(1977)
(Received
72
A
'
i n The N e t h e r l a n d s 9 J u l y
1981)
CHAPTER
3
REVERSED-PHASE
AROMATIC
THIN-LAYER
HYDROCARBONS
RELATIONSHIP
SOLUBILITY
WITH
AND
AND
CHROMATOGRAPHY O F
CHLORINATED
HYDROPHOBICITY
AS
BIPHENYLS:
MEASURED
OCTANOL-WATER PARTITION
POLYNUCLEAR
BY
AQUEOUS
COEFFICIENT
Journal of Chromatography,
238 (1982) 335-346
Elsevier Scientific Publishing Company, Amsterdam — Printed in The Netherlands
C H R O M . 14,636
REVERSED-PHASE THIN-LAYER C H R O M A T O G R A P H Y OF POLYNUCLEAR AROMATIC HYDROCARBONS A N D CHLORINATED BIPHENYLS
RELATIONSHIP WITH HYDROPHOBICITY AS M E A S U R E D BY AQUEOUS
SOLUBILITY A N D O C T A N O L - W A T E R PARTITION COEFFICIENT
W. A. B R U G G E M A N , J. V A N D E R STEEN and O. H U T Z I N G E R *
Laboratory of Environmental and Toxicological Chemistry, University of Amsterdam, Nieuwe
166, 1018 WV Amsterdam
(The
Achtergracht
Netherlands)
(Received December 10th, 1981)
SUMMARY
Reversed-phase thin-layer chromatography with methanol-water as eluent
was used to study relationships between retention and hydrophobicity of non-polar
aromatic compounds. For polynuclear aromatic hydrocarbons, n-alkyl- and chlorosubstituted benzenes, good correlation was observed between R values and log
P
. Significantly different correlations were obtained for polychlorinated biphenyls (PCBs) containing chlorine atoms in the positions ortho to the phenyl-phenyl
bond. This effect could not be explained by the measured lower 7r
values for
ortho chlorine in PCBs. Estimation of hydrophobicity from aqueous solubility, S,
combined with melting point data is restricted to S values higher than 1 ng/l. A
group-contribution approach, including a retention index scale, will be very useful for
correlation and prediction of reversed-phase retention, hydrophobicity and environmental and toxicological properties of chemicals.
m
o c t a n o l
oclanol
INTRODUCTION
Hydrophobicity is now recognized as the driving force for the typical distribution processes of non-polar organic chemicals in aqueous environments, such as
solubility in water, octanol-water partitioning, bioconcentration in aquatic animals
and soil sorption phenomena . It is often directly related to toxicological and pharmacokinetic properties of drugs and other chemicals, as expressed by correlations
between the parameter studied and the octanol-water partition coefficient (as log P )
of structurally related compounds . The abundance of octanol-water partitioning
data in the literature and the existence of extensive data compilations have led to
methods of calculating partition coefficients, based on the additivity of structural
group contributions to the hydrophobicity of a molecule . Hydrophobicity may be
expressed as an increase in the free energy (AG), which is related to log y , the activity coefficient in aqueous solution.
1
ow
2
3,4
w
75
Correlations between octanol-water partition coefficient and aqueous solubility have been described by several authors ^ . Recently, Yalkowsky and Valv a n i and Mackay and co-workers
discussed an important improvement in such
correlations, made by introduction of a crystal energy term based on the melting
point of the substance.
The retention mechanism in reversed-phase liquid chromatography (RP-LC) is
also attributed to solvophobic effects . In "classical" liquid-liquid chromatography, the partition coefficients between the apolar stationary phase and the polar
eluent are related to the capacity factors
5
10
9
11,12
13,14
p
x
= T
r
(1
*' =
>
r
s
s
where P = partition coefficient, V = retention volume, V = volume of mobile
phase in column, K = volume of stationary phase and k' = capacity factor. Tomlins o n has discussed the use of these chromatographic hydrophobic parameters for
correlation analysis of structure-activity relationships.
In modern R P - L C , however, the stationary phase is not a viscous liquid coating, but is chemically bonded to an inorganic support. This has made the method
accessible to high-performance liquid chromatographic (HPLC) techniques, but, at
the same time, it reduces eqn. 1 to a rather theoretical expression without much
practical use for the determination of partition coefficients from retention data (and
vice versa), since partitioning of solutes is now restricted to a very thin "brush" layer
of apolar molecular tails (e.g., octadecyl groups) . Direct comparison of R P - L C
capacity factors with other, "pure", hydrophobic parameters is also precluded by the
use of eluents which often contain water only as a minor constituent.
The first choice for any attempt to correlate R P - L C retention with hydrophobicity now seems to be a methanol-water eluent system, since methanol has nearly the
same type of proton donor and acceptor properties as water, which makes it useful as
a kind of "organic diluent". A linear increase in log k', often observed when the
percentage of methanol in solvent mixtures used for isocratic elution is decreased, is
in accordance with this approach. Several authors have found a linear relationship
between log k' and alkyl chain length for a homologous series of solutes, and substituents such as carboxylic acid, hydroxyl and phenyl groups also give constant
contributions to log k', analogous to the Hansch constants (71) in the octanol-water
partitioning system' . Direct relations between log k' (R ) and log / ° have been
described for chlorinated benzene derivatives in C - H P L C and for a number of
aromatic and organochlorine compounds in C
thin-layer chromatography
(TLC)
, whereas H P L C gradient elution ( 2 2 - 7 5 % methanol) resulted in a nonlinear relationship .
The main purpose of these correlations was to find an alternative to the direct
measurement of octanol-water partition coefficients. This is especially useful for
highly lipophilic and impure compounds, for which detection and phase separation
may become problematic when the classical method is applied, since the concentrations to be measured in the two phases can differ by a factor of 10 or more. Several
compounds of environmental interest, such as certain polyaromatic hydrocarbons,
polychlorinated benzenes, biphenyls and terphenyls, and non-polar polymeric strucr
0
s
15
16
7
2 3
M
o w
2 4
1 8
1 8
2 5 , 2 6
27
5
76
tures, will fall into this category. A reliable estimation of their hydrophobicity will
greatly facilitate prediction and determination of their environmental mobility and
bioaccumulation potential . The advantage of hydrophobicity determinations from
R P - L C retention parameters alone is clear: compounds with different hydrophobicities would be separated and quantification would not be required. At the same time,
a systematic and quantitative approach using linear free energy relationships might
provide tools for prediction of the R P - L C retention behaviour of chemicals with
known structures .
In this study, C reversed-phase thin-layer chromatography (RP-TLC), using
methanol-water mixtures as eluent, was chosen as a relatively simple and inexpensive,
neutral reversed-phase system to compare the retention of polynuclear aromatic
hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) with the octanol-water
partition coefficients, and with aqueous solubility as an important physico-chemical
property related to hydrophobicity.
28
23
1 8
EXPERIMENTAL
Measurement
of octanol-water
partition
coefficients
Approximately 5 mg of the test chemical were dissolved in 2.5 ml of watersaturated 1-octanol (Baker, analytical grade). Of this solution, 1 ml was equilibrated
with 500 ml octanol-saturated distilled water in a volumetric flask by magnetic stirring during 30 min. Layers were separated by centrifugation at ca. 1000 g for 10 min,
and the octanol layer was then pipetted from the water layer. A 100-ml sample of the
water was extracted with ca. 100 ml distilled toluene in three portions. Care was taken
to prevent contamination of the water sample by octanol droplets. The combined
toluene extracts were evaporated to 1 ml to enable quantitation of the chlorinated
compounds by gas chromatography with electron-capture detection, whereas a 0.25ml sample of the octanol phase was sufficiently diluted {e.g., x 400) with distilled
toluene to obtain nearly the same concentration.
Partition coefficients were then determined from concentration ratios,
measured by successive injections of the concentrated water extract and the diluted
sample of the octanol layer on a Tracor 550 G C system equipped with a linearized
N i electron-capture detector (ECD) and Spectra-Physics 4100 computing integrator. G C conditions: glass column (1 m x 2 mm I.D.) packed with 2% Dexsil 300
G C on Chromosorb 750 (100-120 mesh); carrier gas, argon-methane (95:5), flowrate 25 ml/min; detector make-up, 75 ml/min; isothermal operation at temperatures
between 90 and 150°C (injector and outlet, 250°C; detector, 325°C).
For a direct comparison, partition coefficients of isomers were determined
simultaneously in one partition experiment, if the G C conditions allowed separate
detection and quantification of the compounds.
6 3
Reversed-phase
thin-layer
chromatography
Chemicals were spotted with the aid of glass capillaries on Whatman K.C-18
R P - T L C plates (20 x 20 cm, thickness 0.2 mm), with fluorescent indicator. Chromatograms were obtained by ascending elution with mixtures of methanol (Baker, analytical grade) and de-ionized water in a closed 2-1 glass tank system, after equilibration with the aid of wet filter-paper, or by horizontal "sandwich" elution (Camag;
77
Varo-KS-Kammer). Aromatic, UV-quenching substances were detected as dark
spots on fluorescent plates in 254 nm UV-light. The most important retention parameter, R , was calculated for each spot
M
R
(2)
= log k' = log(l//c - 1)
M
f
where R
front.
= distance travelled by spot divided by distance travelled by the eluent
F
Chemicals
Polyaromatic hydrocarbons (PAHs), 1,2,3,4-tetra- and pentachlorobenzene
were obtained from Aldrich, and «-alkylbenzenes from Poly Science Corporation.
Polychlorinated biphenyls (PCBs) were purchased from Analabs, except 2,2',5-triand 2,2',5,5'-tetrachlorobiphenyl, which were synthesized according to Sundstrom ,
and decachlorobiphenyl which was prepared by perchlorinated . Each chemical was
at least 97% pure.
29
30
RESULTS
The octanol-water partition coefficients (as log P) of chlorinated biphenyls and
of 1,2,3,4-tetrachlorobenzene, measured in this study (Table I), show that substitution of chlorine in the position ortho to the phenyl-phenyl bond has a less pronounced effect on log P than substitution in the meta or para position. n values
derived from these measurements are given at the bottom of Table I. They are somewhat lower than the n value (0.66) derived from log P of tetrachlorobenzene, and the
values for substitution of chlorine in an aromatic ring given by Rekker and Hansch
and Leo (0.71-0.74).Log/ values for higher chlorinated biphenyls were calculated
using these new differential substitution constants, which resulted in considerably
lower values than those estimated previously.
cl
3
4
5
TABLE 1
M E A S U R E D log P
PHENYLS
oyl
V A L U E S FOR T E T R A C H L O R O B E N Z E N E , MONO- A N D DICHLOROBI-
The reproducibility and the accuracy of G C determinations were better than 0.10 log P units.
Compound
log P.
1,2,3,4-Tetrachlorobenzene
2- Chlorobiphenyl
3- Chlorobiphenyl
4- Chlorobiphenyl
2,2'-Dichlorobiphenyl
3,5-Dichlorobiphenyl
3,3-Dichlorobiphenyl
4,4 -Dichlorobiphenyl
4.75
4.59
4.71
4.61
5.00
5.37
5.30
5.36
Calculated n
ct
78
+ 0.1
+ 0.1
+0.1
± 0.1
± 0.1
± 0.1
+ 0.1
± 0.1
values for PCBs (log P biphenyl =
4.10)
Correlation
with aqueous
solubility
11
As discussed by Mackay et al. , octanol-water partition coefficients can be
correlated with aqueous solubilities of solids if a melting point correction is included.
For aqueous solubilities measured at 25°C, the simplest form of the correlation equation is
log P
ovl
= -
log 5 -
0.01
x m.p. + a
(3)
where S = aqueous solubility (mol/1) 'and m.p. = melting point (°C). The term a,
related to the logarithm of the activity coefficient in octanol, y , is only slightly
dependent on the type of compounds in the correlation. Mackay et al. found a
mean y„ value of 4.8 for 45 organic, mainly aromatic, compounds, which is in agreement with the value of 0.55 for the term a in eqn. 3 calculated from the P A H data of
Yalkowsky and Valvani . The relationship between the calculated log P values of
PCBs and log S + 0.01 x m.p. (Table Ha) was fitted to eqn. 3, as shown in Fig. 1
where it is compared with the same correlation for PAHs, mentioned above. The
solubility data of PCBs in Table Ha were taken from Weil et al. , who gave the most
complete set obtained by one, reliable method which is also suitable for other almost
insoluble compounds. The melting point data in Table Ha were collected by Hutzinger et al. . The resulting correlation equation for PCBs containing up to six chlorine
atoms is:
0
11
10
31
32
log F
o w
= log 5 - 0.01
x
m.p. + 0.05
(4)
The root mean square of the deviation in log P for the 20 points is 0.28, corresponding to a factor of 1.9 in / ° .
The difference between the a terms (eqn. 3) of the P C B and the P A H correlations is 0.5. Following the interpretation of Mackay et al. , it must be concluded
that the mean y of PCBs (up to hexachlorobiphenyl) is about 14, three times as high
as y for P A H s .
As shown by Fig. I, the correlation between P and S failed for PCBs with
calculated log P higher than 7.5 or solubilities below 0.5 //g/1 (ca. 10" mol/1). A p parently, the hydrophobicity that can be measured by aqueous solubility has reached
its maximum here. Similarly, coronene (log P = 7.7, S = 0.14 ^g/1 or 10" mol/1,
m.p. = 438 ' C ) was not included by Yalkowsky and V a l v a n i in their P A H correlation.
0 w
11
B
a
9
9 3
3 3
10
RP-TLC
Whatman KC-18 T L C plates, compared with other makes, have the advantage
of larger surface areas and faster elution , which is especially important in ascending
tank elution systems. The reproducibility of the ascending tank elution was moderate,
presumably due to insufficient acclimatization of the elution chamber, coupled with
formation of a solvent gradient on the plate. A better equilibration and reproducibility was obtained by horizontal elution in the sandwich system. Comparison of the
behaviour of PAHs in both systems (Table lib) shows that the retention of relatively
fast eluting compounds, at the top of the table, was stronger in the tank system. This
might be the result of a decreasing percentage of methanol at the solvent front during
34
79
T A B L E II
RP-TLC RETENTION, R , C O M P A R E D WITH HYDROPHOBIC PARAMETERS O F PCBs (a), PAHs (b)
A N D SUBSTITUTED BENZENES (c)
M
m.p. = Melting point (°C); S = aqueous solubility (mol/1); P „ = octanol-water partition coefficient; R
(l/flf - 1); /V = n-alkylbenzene retention index. Whatman KC-18 RP-TLC, methanol-water (95:5).
= log
Compound
N
M
ab
(a) PCBs
Biphenyl
2-Chloro342,2'-Dichloro2,42,53,3'4,4'2,2',5-Trichloro2,4,4'2,4,52,4',53,4.4'2,2',5,5-Tetrachloro2,2',6.6'2,3,4,52,3',4',53,3',4,4'3,3',5,5'2,2',3,4,5'-Pentachloro2,2',4,5,5'2,3,4,5,62,2'.3.3',4.4'-Hexachloro2,2',4,4',5,5'2,2',4,4',6,6'2,2',3,4,5,5',6-Heptachloro2,2',3,3',4,4',5,5-Octachloro2,2',3,3',5,5',6,6'2,2',3,3',4,4',5,5',6-NonachloroDecachlorobiphenyl
b) PAHs
Naphthalene
Fluorene
Anthracene
Phenanthrene
Fluoranthene
Pyrene
Chrysene
Benzo[a]pyrene
Benzo[g,/i,/]perylene
•) Substituted benzenes
n-Butyl«-Hexyln-Octyln-Decyl1,2,3,4-TetrachloroPentachloro-
m.p.
-logS
- log S +
-0.01 x m.p.
log P „
R
71
34
17
78
61
24
23
4.28
4.66
5.16
5.32
5.45
5.55
5.58
3.57
4.32
4.91
4.54
4.84
5.31
5.35
149
44
57
78
6.60
5.60
5.99
6.44
5.11
5.16
5.42
5.66
-0.21
-0.24
-0.10
-0.13
-0.32
-0.12
-0.05
0.02
-0.10
-0.11
2.56
2.28
3.57
3.29
1.55
3.39
4.03
4.67
3.57
3.48
-0.01
4.40
88
7.23
6.35
4.10
4.56
4.72
4.69
5.02
5.15
5.18
5.34
5.28
5.64
5.74
5.77
5.77
5.90
6.26
5.94
6.39
6.39
6.52
6.58
6.85
6.85
6.85
7.44
7.44
7.12
7.93
8.68
8.42
9.14
9.60
0.00
-0.22
4.49
2.47
0.15
5.87
0.34
7.62
0.24
6.70
0.38
0.58
7.99
9.83
0.67
0.90
10.65
12.77
92
7.18
6.26
180
8.59
6.79
112
77
124
150
103
114
149
159
162
206
305
7.86
7.89
7.68
8.91
8.47
8.60
8.92
9.19
9.37
9.61
10.49
6.74
7.12
6.44
7.41
7.44
7.46
7.43
7.60
7.75
7.55
7.44
M
Tank
80
116
216
101
111
156
255
175
277
3.61
4.93
6.38
5.15
5.90
6.18
8.06
7.82
9.02
2.81
3.77
4.22
4.14
4.79
4.62
5.51
6.07
6.25
3.35
4.18
4.63
4.63
5.22
5.22
5.91
6.50
7.10
-0.08
0.00
0.07
0.03
0.10
0.16
0.22
0.40
0.54
4.18
5.24
6.30
7.35
4.94
5.69
-0.05
0.16
0.38
0.60
0.05
0.23
ab
Sandwich.
-0.25
-0.10
-0.03
-0.06
0.05
0.12
0.16
0.38
0.54
2.19
3.57
4.21
3.94
4.95
5.59
5.96
7.99
9.46
4.03
5.96
7.99
10.01
4.95
6.61
10
i
I
I
i
i
i
1
g
8
• y
»'
• •
• y• / ' ' '
7
—
a
I
6
PCB
/ * '
o
?
5
PA H
co
f
4
A*
i
3
S /
oo
/
7
2
1
3
4
1
5
1
6
1
7
1
8
1
9
1
10
log Pow
Fig. 1. Relationship between octanol-water partition coefficient, />„„, and aqueous solubility, S (mol/1),
when corrected for melting point (m.p.). • — • PCBs; O
O , PAHs; V . coronene.
tank elution. Consequently, the correlation of R with log P obtained by linear
regression analysis was somewhat better for the retention data from the sandwich
systems (eqns. 5 and 6):
M
ov
sandwich
l o g / » = 4.52 R + 4.81
n = 9, r = 0.98, s = 0.20
ow
(5)
M
tank
log P = 5.31 R + 4.39
n = 9, r m 0.96, s = 0.26
o w
(6)
M
a non-linear relationship would give a better fit to the tank data. Therefore, the
sandwich system was preferred for further experiments.
The relationship between calculated log P and R for 20 PCBs (Table Ila) is
given by:
o w
M
log P
= 4.52 R + 5.79
n = 20, r = 0.95, s = 0.51
ow
(7)
M
This correlation is far inferior to that of the P A H s (eqn. 5), as demonstrated by the
root mean square deviation in log P. Two compounds with equal R value have
completely different log P values, viz., 4.1 and 5.9 for biphenyl and 2,2',6,6'-tetrachlorobiphenyl respectively. In addition, eqns. 5 and 7 show a difference of one log P unit.
Important improvements are obtained when the PCBs are distinguished acM
81
cording to their number of chlorine atoms in ortho positions (numbers 2 and 6 of both
phenyl rings). As indicated by the four compounds at the top of Table Ha and the
four tetrachlorobiphenyls, substitution of hydrogen by chlorine in one of the ortho
positions gives no contribution, or even a small negative one, to R , in contrast to
meta- or para-chlorine substitution (positions 3, 4 and 5: AR
= 0.13 + 0.06). The
new lines in Fig. 2 correspond to the following equations:
M
MC]
no o-C\
one o-Cl
two, three or four o-Cl
: log P
: log P
ow
M
ow
= 4.5 R
= 4.5 R
: log P„ = 4.2 R
M
+ 5.2 (n = 6)
+ 5.6 (n = 5)
M
+ 6.3 (n = 9)
(8)
(9)
(10)
Deviations between calculated and observed values were now generally below 0.25
log P units (factor 1.75 in P); exceptions were 4,4'-di-, 2,2',6,6'-tetra- and decachlorobiphenyl, with log P deviations of ca. 0.5 (factor of 3 in P). This accuracy is
comparable to that obtained by the P A H correlation (eqn. 5).
The four «-alkylbenzenes (Table II) fitted the equation:
log P
= 4.85 R
ow
M
(11)
+ AAA
Tetra- and pentachlorobenzene were shown to be in intermediate positions between
alkylbenzenes and P A H s (Fig. 2).
Direct comparison of aqueous solubility and R P - T L C retention was only
1.0 |
-0.5
I
3
1
I
4
1
1
I
5
r
i—
6
I
7
I
8
I
9
10
log P w
0
Fig. 2. Relationship between octanol-water partition coefficient, P„, and reversed-phase retention, R .
• . n-Alkylbenzenes;
polychlorinated benzenes; O , polycyclic aromatic hydrocarbons; A , polychlorinated biphenyl (PCB) w ithout ortho-C\; V , PCB with one o-Cl; • PCB with two or more o-Cl. Whatman
KC-18 RP-TLC; methanol-water (95:5).
M
82
possible for a restricted number of compounds from Table II (Fig. 3). The correlation
between R and log 5 + 0.01 m.p. is evident for the P A H s :
M
- log S - 0.01
x
m.p. = 4.3 + 4.7 R
M
(n = 9)
(12)
mean deviation: 0.3 log S units
max. deviation: 0.5 log S units
For the higher chlorinated biphenyls, (log S + 0.01 m.p.) had reached a maximum
value, whereas too few data were available for the lower chlorinated biphenyls to
allow reasonable correlations according to the different degrees of ori/io-chlorine
substitution.
Changes in the eluent composition resulted in a linear increase of the R values
of PCBs with the water content of the eluent, in the range of 0-20 % (v/v) (Fig. 4)
M
2,2',3,4,5,5',6-heptachlorobiphenyl:
2,2',5,5'-tetrachlorobiphenyl:
2-chlorobiphenyl:
biphenyl:
R
R
R
R
M
M
M
M
=
0.06
= -0.24
= -0.34
= -0.41
+
+
+
+
7.6
6.4
4.5
4.5
x
x
x
x
*
<P
<*>
d>
(13)
(14)
(15)
(16)
where <P = the volume fraction of water in the eluent.
i
Fig. 3. Relationship between aqueous solubility, S (mol/1), corrected for melting point (m.p.), and reversed-phase retention, R . O , Polycyclic aromatic hydrocarbons; polychlorinated biphenyls: A , no ortho-Cl; V , one o-Cl; • , two or more o-Cl. Whatman KC-18 RP-TLC; methanol-water (95:5).
u
Fig. 4. Retention of polychlorinated biphenyls at different percentages of water in the eluent (Whatman
KC-18 RP-TLC, methanol-water). • , Biphenyl; V , 2-chloro-; • . 2,2',5,5'-tetrachloro-; O ,
2,2',3,4,5,5',6-heptachlorobiphenyl.
83
possible for a restricted number of compounds from Table II (Fig. 3). The correlation
between R and log S + 0.01 m.p. is evident for the PAHs:
M
- log S - 0.01
x
m.p. = 4.3 + 4.7 R
M
(n = 9)
(12)
mean deviation: 0.3 log S units
max. deviation: 0.5 log S units
For the higher chlorinated biphenyls, (log S + 0.01 m.p.) had reached a maximum
value, whereas too few data were available for the lower chlorinated biphenyls to
allow reasonable correlations according to the different degrees of or/Ao-chlorine
substitution.
Changes in the eluent composition resulted in a linear increase of the R values
of PCBs with the water content of the eluent, in the range of 0-20% (v/v) (Fig. 4)
M
2,2',3,4,5,5',6-heptachlorobiphenyl:
2,2',5,5'-tetrachlorobiphenyl:
2-chlorobiphenyl:
biphenyl:
R
R
R
R
M
M
M
M
=
0.06 + 7.6
= -0.24 + 6.4
= -0.34 + 4.5
= -0.41 + 4.5
x *
(13)
x <P
(14)
x <t> (15)
x tf> (16)
where <P = the volume fraction of water in the eluent.
Fig. 3. Relationship between aqueous solubility, 5 (mol/1), corrected for melting point (m.p ), and reversed-phase retention, R . O , Polycyclic aromatic hydrocarbons; polychlorinated biphenyls: A , no ortho-C\; V , one o-Cl; • two or more o-Cl. Whatman KC-18 RP-TLC; methanol-water (95:5).
u
Fig. 4. Retention of polychlorinated biphenyls at different percentages of water in the eluent (Whatman
KC-18 RP-TLC, methanol-water). • , Biphenyl; V , 2-chloro-; • . 2,2',5,5-tetrachloro-; O .
2,2',3,4,5,5',6-heptachlorobiphenyl.
84
liquid chromatography. Besides the homologous series on which the retention index
is based, only the type of reversed-phase material and the eluent composition should
be specified. It is expected that the retention index will show only a minor change with
variation in the percentage of methanol in the eluent. The exact determination of this
dependence will be simplified by linearity of the relationship between R and the
methanol percentage over a certain range (see Fig. 3).
The reversed-phase retention index may replace the unreliable log P determinations of highly lipophilic chemicals (log P > 5), for prediction of biological activity
and environmental distribution coefficients, such as bioaccumulation factors and
sorption coefficients. For this purpose, more knowledge of different group contributions to R P - L C retention and their influence on environmental and biological
parameters is desirable.
Thin-layer chromatography is a rapid, simple and inexpensive method. However, high-performance liquid column chromatography may be preferred for reasons
of accuracy, sensitivity and reproducibility. In addition, H P L C offers the possibility
of gradient elution, which may be very useful for mixtures of unknown chemicals, as
found in chemical wastes, to estimate environmental mobility and accumulation potential .
M
ov
28
REFERENCES
1 W. A. Bruggeman, in O. Hutzinger (Editor), The Handbook of Environmental Chemistry, Vol. 2, Part B,
Springer, Heidelberg, 1982, p. 83.
2 A. Leo, C. Hansch and D. Elkins, Chem. Rev., 71 (1971) 525.
3 R. F. Rekker, The Hydrophobic Fragmental Constant, Elsevier, Amsterdam, 1977.
4 C. Hansch and A. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, Wiley.
New York, 1979.
5 C. Hansch, J. E. Quinlan and G . L. Lawrence, J. Org. Chem., 33 (1968) 347.
6 C. T. Chiou, V. H. Freed, D. W. Schmedding and R. L. Kohnert, Environ. Sci. Technol., 11 (1977) 475.
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10 S. H . Yalkowsky and S. C. Valvani, J. Chem. Eng. Data, 24 (1979) 127.
11 D. Mackay, A. Bobra, W. Y. Shiu and S. H . Yalkowsky, Chemosphere, 9 (1980) 701.
12 D. Mackay, R. Mascarenhas, W. Y. Shiu, S. C. Valvani and S. H . Yalkowsky, Chemosphere, 9 (1980)
257.
13 D. C. Locke, J. Chromatogr. Sci., 12 (1974) 433.
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18 U . R. Tjaden, Ph. D. Thesis, University of Amsterdam, Amsterdam, 1976.
19 S. R. Bakalyar, R. Mcllwrick and E. Roggendorf, J. Chromatogr., 142 (1977) 353.
20 F. Takano and T. Tanimura, Chem. Pharm. Bull, 25 (1977) 1157.
21 N. Tanaka and E. R. Thornton, J. Amer. Chem. Soc, 99 (1977) 7300.
22 E. Tomlinson, Proc. Anal. Div. Chem. Soc. 14 (1977) 294.
23 E. Tomlinson. H. Poppe and J. C. Kraak, in E. Reid (Editor), Blood Drugs and Other Analytical
Challenges, Methodological Surveys in Biochemistry, Vol. 7, Ellis Horwood, Chichester, 1978, p. 207.
24 H. Konemann, R. Zelle, F. Busser and W. E. Hammers, J. Chromatogr.. 178 (1979) 559.
25 L. Renberg and G. Sundstrom, Chemosphere, 8 (1979) 449.
26 L. Renberg, G. Sundstrom and K. Sundh-Nygard, Chemosphere, 9 (1980) 683.
27 G. D. Veith, N. M . Austin and R. T. Morris, Water Res., 13 (1979) 43.
28 G . D. Veith, D. L. Defoe and B. V. Bergstedt, J. Fish. Res. Bd. Can., 36 (1979) 1040.
85
29
30
31
32
33
34
35
36
86
G . Sundstrom, Acta Chem. Scant!., 27 (1973) 600.
O. Hutzinger, S. Safe and V, Zitko, Int. J. Environ. Anal. Chem., 2 (1972) 95.
L. Weil, G . Dure and K. E. Quentin, Z. Wasser Abwasser Forsch., 7 (1974) 169.
O. Hutzinger, S. Safe and V. Zitko, The Chemistry of PCBs, C R C Press, Cleveland, O H , 1974.
D. Mackay and W. Y. Shiu, J. Chem. Eng. Data, 22 (1977) 399.
U. A. Th. Brinkman and G. de Vries, J. Chromatogr., 192 (1980) 331.
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W. R. Melander, B.-K. Chen and Cs. Horvath, J. Chromatogr., 185 (1979) 99.
CHAPTER
4
BIOACCUMULATION
OF
SUPER-LIPOPHILIC
CHEMICALS
IN
FISH
87
BIOACCUMULATION OF S U P E R - L I P O P H I L I C CHEMICALS
W.A.
Bruggeman*, A. Opperhuizen, A. Wijbenga
Laboratory
of
Environmental
University
of
Amsterdam, Nieuwe A c h t e r g r a c h t
Amsterdam
(The
IN
FISH
and 0.
and T o x i c o l o g i c a l
Hutzinger.**
Chemistry,
166,
1 0 1 8 WV
Netherlands)
ABSTRACT
The i n f l u e n c e
of
bioaccumulation
investigated
increasing molecular
kinetics
for
a series
comparison with other
Extremely
of
biphenyls
uptake
uptake
from contaminated
fish.
from water
were
in
a linear
contrast
appeared to
food.
direct
structure
of
biphenyls
to
food-chain
is
and p h y s i c o - c h e m i c a l
was
(PCBs)
of
the
more s o l u b l e
be l e s s
efficient
accumulation
than
octachlorodibenzoby
living
(biomagnification)
discussed in
of
in
chemicals,
were not accumulated
properties
in
higher
accumulation
Hexabromobenzene,
bioconcentration
on
compounds.
characteristic
in
andtetradeca-chloroterphenyl
The i m p o r t a n c e
versus
(Poecilia reticulata)
aromatic
and r e s u l t e d
direct
and h y d r o p h o b i c i t y
polychlorinated
halogenated
F o r these compounds,
p-dioxin
guppies
low c l e a r a n c e r a t e s
chlorinated
fish.
in
size
relation
organic
to
molecular
chemicals.
INTRODUCTION
Bioaccumulation of
been the
mental
fish
subject
of
extensive
systems, high
or
other
by d i r e c t
(QSARs)
*present
aquatic
uptake
and u n i v e r s a l
via
present
of
chemicals in
In
most
Rijkswaterstaat
-
has
and
experi-
compounds
in
explained
1-4
alone.
structure-activity
partition
fish
natural
hydrophobic
c o u l d be
from ambient water
Relatively
simple
relationships
coefficients
(P) were
derived
R I Z A , P . O . B o x 1 7 , 8 2 0 0 AA
Netherlands
address: Chair
University
research.
organisms
quantitative
address:
lipophilic
concentrations
octanol-water
L e l y s t a d , The
**
persistent,
of
Bayreuth,
of
E c o l o g i c a l C h e m i s t r y and G e o c h e m i s t r y ,
P . O . Box 3 0 0 8 , D - 8 5 8 0 B a y r e u t h ,
Germany
89
for
bioconcentration
Q
constants
as w e l l
kinetic
and e s t i m a t i o n
simple experimental
for
the
Still,
of
u p t a k e and c l e a r a n c e
d e s c r i p t i o n of
conception of
the
bioconcentration
systems. This
food-chain transfer)
aquatic
, and f o r
. F o r many o r g a n i c c h e m i c a l s t h i s
allowed a suitable
process
factors
approach
bioaccumulation
factors
in
relatively
has been an i m p o r t a n t
equilibrium
partitioning
as the main cause o f
rate
(in
stimulus
contrast
bioaccumulation
to
in
organisms.
the q u e s t i o n
remains
with a fat
droplet
precludes
reliable
chemicals
from t h e i r
to what e x t e n t
in water.
Is
prediction
there
of
partition
the
In
constants
a previous
coefficients
paper, the
and t r a n s p o r t
is
comparable
factor
bioaccumulation of
In o u r o p i n i o n , b i o a c c u m u l a t i o n k i n e t i c s
problem.
a fish
any b i o l o g i c a l
which
certain
only?
c a n be a k e y t o
relationship
between
and exchange p r o c e s s e s i n
the
this
rate
fish
was
g
discussed
(kj)
.
It
was s h o w n t h a t
a maximum u p t a k e
is
approached f o r
PCBs w i t h
whereas
clearance rate
constants
increasing
lipophilicity.
o b t a i n e d by o t h e r
Simple f i r s t
by eqn
authors
c
k
k
l^ 2
=
k
l
C
w -
k
2
in
ratio
in
measurement o f
in
in
until
a plateau
clearance
agreement w i t h
Cl-atoms),
the
results
experiments.^'^'^
kinetics
c a n be d e s c r i b e d
C
fish,
is
(1)
C = concentration
w
in water
and
factor.
of
K
c
as t h e e q u i l i b r i u m
flow
observed, or
the
in
experiments,
fish-water
it
is
has been e s t a b l i s h e d . T h i s
concentrations
concentration
experiment.
C
continuous
a separate experiment:
equilibrium
90
K
know when t h e e q u i l i b r i u m
vals
is
similar
f = M c w- f)
determination
concentration
to
C
bioconcentration
For a d i r e c t
either
P between 5 and 6 ( 2 - 4
decrease with
order bioconcentration
w h e r e C^= c o n c e n t r a t i o n
=
in
constant
1,
^ f -
K
This
log
rate
in
fish
fish
regular
determination
time needed to
the
at
of
requires
time
half
inter-
clearance
r e a c h 50% o f
equals the
necessary
life
the
in
a
rates
Extremely
low c l e a r a n c e r a t e s
chemicals.
then
L i n e a r uptake w i t h o u t s i g n i f i c a n t
proceed for
rapidly
real
a long
retained.
"food-chain
the
time;
achieved s i n c e the
completely
of
proper
Several
In
equilibrium
way,
accumulation"
the
is
primary
fulfilled
will
is
not
almost
condition
for
a
as a c o n s e q u e n c e
theory.
have s p e c u l a t e d about
classes
certain
elimination
with water
the
relationships
and p h y s i c o - c h e m i c a l p r o p e r t i e s
accumulation of
super-lipophilic
c h e m i c a l , once a b s o r b e d , i s
this
partitioning
authors
molecular
are expected f o r
of
between
and r e s t r i c t e d
lipophilic
bio-
chemicals.
13
Tulp
and H u t z i n g e r
size
and extreme
the
relationship
possible
role
between
of
parameters
to
quantify
and t o
be a b l e
of
to
of
constants
mg/l)
is
determine
highly
hydrophobicity
(n-values),
or
a combination
more
insight
of
of
in
the
Therefore,
("size"
find
and p h y s i c o and
chemicals
xenobiotic
study
size
a
and
solubility
. The
direct
coefficients
estimations
of
constants
reverse-phase l i q u i d
these m e t h o d s ^
of
factors
partition
reliable
of
independently.
(aqueous
g
publication
octanol-water
i m p o s s i b l e , but
a series of
"lipophilicity")
vary
c a n be made u s i n g s u b s t i t u t i o n
influence
suitable
bioconcentration
an e a r l i e r
attendant
the experimental
bioaccumulation of
food.
the
bioaccumulation potential
aqueous s o l u b i l i t i e s ,
The p u r p o s e o f
of
the
reducing
these parameters
hydrophobic
described in
virtually
is
the
which
determination
of
necessary to
desired structural
(log
6)
is
compounds
the
P>
factor
the
measurement of
the
molecular
linearity
and s t r e s s e d
the
chemicals for
The e x p e r i m e n t a l
< 0.1
c
the
potential.
properties
series
increasing
P and l o g K ,
these hypotheses, i t
chemical
rate
log
both
could affect
m e t a b o l i s m as a n o t h e r
bioaccumulation
To t e s t
suggested that
lipophilicity
chromatography
^ .
d e s c r i b e d h e r e was t o
obtain
and h y d r o p h o b i c i t y
the
chemicals in
fish,
polychlorinated
on
via water
biphenyls
and
was
91
compared w i t h
some o t h e r
They were e x p e c t e d t o
lipophilicity
factors.
were
halogenated aromatic
fall
does not
in
the
critical
studied
r e g i o n , where
necessarily result
For these c h e m i c a l s , the
separately.
compounds.
in
higher
bioaccumulation
aqueous and d i e t a r y
The i n t e r p r e t a t i o n
of
the
increasing
route
results
was
9
b a s e d on t h e
kinetic
approach d e s c r i b e d p r e v i o u s l y
.
M A T E R I A L S AND METHODS.
The e x p e r i m e n t a l
system f o r
was e s s e n t i a l l y
the
bioaccumulation of
are described
Chemicals.
same a s u s e d i n
lower
for
described in
structure
(tetra-,
see f i g .
.
study
from a commercial
from syntheses
Tetradecachloro-p-terphenyl
99%, as c o n f i r m e d
final
mixtures
(toluene,
of
Pb-Na a l l o y
to
as u s e d i n
remove w a t e r
1,2,3,4-
f r o m A l d r i c h . The c h e m i c a l s w e r e
Final
purity
by G C - E C D ; o n l y
n-hexane)
was
P e n t a c h l o r o - and
o f most
the
c o m p o u n d s was
10%, p r e s u m a b l y
fish
exposure
were d i s t i l l e d
and t r a c e s
of
in
the
another
i n the gas chromatogram
experiments.
presence of
a
electron-capturing
substances.
Fish.
Instead
reticulata)
variation
0.1
g).
In
of
goldfish,
were chosen i n
(age,
the
12+3
food
laboratory
order
months;
experiment,
age w e r e u s e d a s w e l l
(length,
A q u a r i u m w a t e r was a m i x t u r e
tap water
of
92
(1:1).
of
to
bred male guppies
reduce e f f e c t s
length,
of
(Poecilia
individual
1 4 - 1 8 mm; a v e r a g e
female guppies
of
the
2 3 - 2 5 mm; a v e r a g e w e i g h t ,
demineralized water
Water temperature
1 2 / 1 2 h r s was c r e a t e d b y
higher
2,2* ,3,3',4,4* , 5 , 5 * - o c t a c h l o r o b i p h e n y l
ca.
c h l o r i n a t e d b i p h e n y l , which, h o w e v e r , d i d not i n t e r f e r e
the
(octachlorodioxin)
Hexabromo-benzene
preparation.
by r e c r y s t a l l i z a t i o n .
Solvents
the
Modifications
and o c t a c h l o r o - d i b e n z o - p - d i o x i n
by p e r c h l o n " n a t i o n .
was a c c o m p a n i e d b y a n i m p u r i t y
of
on
fish
o c t a - and d e c a c h l o r o -
originated
.
t e t r a c h l o r o b e n z e n e were o b t a i n e d
than
biphenyls
hexa-,
1)
publications
(perchloroterphenyl)
purified
our previous
g
chlorinated
PCBs
earlier
were p r e p a r e d
purified
exposure of
below.
Individual
biphenyl;
aqueous and d i e t a r y
weight,
same
0.35
and Amsterdam
was 2 2 - 2 3 ° C ; a d a y - n i g h t
"daylight"
fluorescent
g).
lamps.
cycle
Exposure
Water. Chemicals were administered i n water by a f l u i d i z e d bed
column containing the t e s t mixture (each component ca. 25 mg)
coated on 5 g of a porous, i n e r t support (Chromosorb W,
45-60 mesh). This column was part of a water r e c i r c u l a t i o n
system f u r t h e r including a 3 1 sedimentation tank and an oxygen
control unit ( 0
2
concentration i n water: 7.5 + 1.0 m g / l ) , feeding
the 40 1 exposure aquarium at a flow of ca. 40 1/h.
Food. F o r t i f i e d dry f o o d , containing 50 mg/kg of each t e s t compound
and a standardized mixture of polydimethylsiloxanes ( s i l i c o n e s :
PDMS; f o r d e t a i l s see r e f . 18), was fed guppies placed i n a 40 1
aquarium (clearance system), i n d a i l y amounts of ca. 20 mg/g f i s h .
The aquarium f u r t h e r contained s n a i l s (Planorbis s p . ) , feeding
on s p i l t food r e s t s , and green plants ( S e l a g i n e l l a sp.)on a layer
of f i n e g r a v e l .
Clearance system. Hydrophobic chemicals eventually excreted by
the f i s h a f t e r the exposure were continuously removed from the
aquarium water by a combination of aeration and activated carbon
fi ltration.
Chemical analysis
E x t r a c t i o n . Samples of f i s h and food were extracted with ca. 100 ml
r e f l u x i n g toluene as described previously.
C l a r i f i e d extracts were divided i n equal portions f o r determination
of extracted l i p i d weight (by evaporation of the solvent) and
f o r quantitative chemical analysis by gas chromatography with
electron-capture detection (GC-ECD) a f t e r clean-up.
Water samples were extracted with toluene at room temperature,
d i l u t e d or concentrated by evaporation i f necessary and used
d i r e c t l y f o r GC-ECD a n a l y s i s .
Clean-up. Crude extracts were concentrated to a small volume
(0.5 - 2 ml) by evaporation and passed through two microcolumns
(bed volume ca. 0.5 m l ) , successively containing s i l i c a (100 120 mesh) impregnated with s u l f u r i c acid (ca. 40 % by weight) and
s i l i c a with 1 N NaOH (ca. 33 % by weight).
In t h i s
way, most polar, a c i d i c and b a s i c , as well as hydrolyzable com-
93
pounds
The
( e . g . , body l i p i d s )
recovery of
extraction
extracts
Gas
the
halo-aromatic
chromatography.
in
to
(1 m x 2 mm i . d . ) ,
20 m l / m i n ;
detector,
360°C.
of
packed with
detector
10°C/min;
limit
0 . 0 5 mg/kg f i s h
t h a n 80%.
the
halogenated
a
GC-ECD.
test
a
2% ( w / w )
Dexsil
and o u t l e t
standard
300 GC on
(95:5);
and c a . 0.01 yjg/1
program:
280°C;
comparison
The
(per
glass
chromosorb
Temperature
injection.
practical
compound)
ca.
water.
RESULTS
Aqueous e x p o s u r e . The c o n c e n t r a t i o n s
in water without
fish
the
recirculation
of
start
of
the
the most v o l a t i l e
2,5-dichlorobiphenyl
this
experiment)
is
compounds, i . e . ,
saturation
of
their
the
level,
other
during
Plateau concentrations
octa-
reported
pentachlorobenzene
solubility.
'
10 d a y s .
. The
c o m p o u n d s was m a i n t a i n e d
the extremely
over the
and
in
The
mg/l,
water
at
stock
hydrophobic
and d e c a c h l o r o b i p h e n y l , o c t a c h l o r o d i o x i n
t e r p h e n y l , were h i g h e r
and
included
b o t h c o m p o u n d s was 0 . 2
9 19
recirculation
of
after
concentrations
( t e t r a c h l o r o b e n z e n e was n o t
observed of
compounds
20 d a y s
s y s t e m . The w a t e r
slowly declined already after
c l o s e to
concentration
most t e s t
reached a plateau w i t h i n
maximum c o n c e n t r a t i o n
which
of
column.
chemicals,
perchloro-
than 0 . 8 u g / 1 , exceeding the
expected
20
solubilities
Filtration
gation
at
of
o v e r a 0 . 2 ji
of
c e l l u l o s e membrane f i l t e r
c a . 1000 x g d i d
concentrations:
94
t h e s e c o m p o u n d s b y some o r d e r s
not
apparently,
significantly
stable
solutions
on
flow
temperature,
p r o c e d u r e was
1 ^il
Spectra-
GC-conditions: column,
m a k e - u p , 80 m l / m i n .
this
of
linearized
C o n c e n t r a t i o n s w e r e c a l c u l a t e d by
through
Clean
and a n a l y z e d by
g a s , argon-methane
inlet
peak a r e a s and e x t e r n a l
detection
1 ml
and c o n n e c t e d w i t h
integrator.
750 ( 1 0 0 - 1 2 0 m e s h ) ; c a r r i e r
rate
-
this
w e r e d e t e r m i n e d by i n j e c t i o n
detector
4100 c o m p u t i n g
80-320°C;
0.5
extract.
compounds a f t e r
chromatograph, equipped with
electron-capture
rate,
test
Concentrations of
clean extracts
a T r a c o r 550 g a s
63
Physics
removed from tne
a n d c l e a n - u p p r o c e d u r e was b e t t e r
were c o n c e n t r a t e d
compounds
Ni
are e a s i l y
magnitude.
or
centrifu-
reduce the
were
observed
formed.
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95
Six days a f t e r placing 102 male guppies i n the aquarium, the
stock column was removed from the r e c i r c u l a t i o n system i n order
to accelerate e q u i l i b r a t i o n of the concentrations i n water
and f i s h . A f t e r s t a b i l i z a t i o n of the aqueous concentrations
of the n o n - v o l a t i l e compounds, the f i s h were transferred to the
clearance system f o r depuration of accumulated chemicals.
During the whole experiment, f i s h (3-4 i n d i v i d u a l s ) and water
were sampled at i n t e r v a l s to determine concentrations of the
test compounds. Hexabromobenzene, octachlorodioxin and perchloroterphenyl have not been detected i n samples of l i v i n g
f i s h , whereas aqueous concentrations amounted to values between
1 and 10 ^ig/1. In contrast, samples of dead f i s h from the uptake
experiment contained considerable quantities of these compounds:
3
apparent concentration factors i n dead f i s h were ca. 10
for
octachlorodioxin and perchloroterphenyl and ca. 10^ f o r hexabromobenzene. Decachlorobiphenyl also showed s i g n i f i c a n t l y higher
3
concentrations i n dead f i s h (apparent K ^ I O ) .
For the other test compounds there was no s i g n i f i c a n t d i f f e r e n c e
between the concentrations i n l i v e and dead f i s h sampled at the
same date.
The course of the concentrations of PCBs and pentachlorobenzene
in f i s h and water i s shown i n f i g . 1. The high v a r i a t i o n i n the
f i s h concentrations i s mainly a t t r i b u t e d to the bad condition
of the guppies: the aqueous exposure was l e t h a l to ca. 50% of the
f i s h ; surviving guppies showed a very low a c t i v i t y and loss of
color and appetite. Gradual recovery was observed i n the f i r s t
two weeks of the clearance experiment. Concurrently, the concentrations of pentachlorobenzene and dichlorobiphenyl f e l l to
ca. 20% of the i n i t i a l value, whereas only a minor decrease of the
other compounds was observed. Therefore, the t o x i c i t y of the
mixture was not a t t r i b u t e d to the higher chlorinated biphenyls.
The s l i g h t decrease of the concentrations of hexa-, octa- and
decachlorobiphenyl can be f u l l y explained as the r e s u l t of d i l u t i o n
by gain of f i s h weight, since the absolute amounts per individual
remained v i r t u a l l y constant (table 1).
96
Uptake r a t e
(k )
constants
(kj)
and apparent
were c a l c u l a t e d from c o n c e n t r a t i o n s
2
sampled i n
days
the
first
six
days o f
clearance experiment
the
observed concentration
Apparently,
biphenyl
this
the
the
factors
ratios
or
by a h i g h
(microparticles
Indications
of
or
the
low s o l u b i l i t i e s
proportion
colloids
solubility.
changes
of
6
'
2
latter
which
1
.
the
suggesting
a fast
these
compounds i n
(K =
2 x 10
c
the
dilution
by f i s h
The r e l a t i o n s h i p
chlorinated
past
the
factor
the
2,
in
between the
1 1
for
higher
these
partition
of
than
in
the
(log
mem-
compound i n
water
in
of
the
extremely
relationships
P) and aqueous
that
no
significant
o c t a - and d e c a c h l o r o b i p h e n y l
(without
resaturation),
available fraction
attained
by
This
of
hexachlorobiphenyl
factor
will
be c l o s e t o
complete absorption
followed
of
by c o m p l e t e
the
a chemical
retention
results
fish.
these extremely
bioconcentration
the
that
biphenyls
(table
from
hydrophobic
,
low s o l u b i l i t y
their
as p r e s e n t e d
22
Mackay
corresponds better
partitioning
of
9) a n d
accumulation
compounds a r e a l m o s t
The v e r y
1 6
suggestions of
a linear
factor
2 and r e f .
coefficient
an e q u i l i b r i u m
"super-lipophilic"
retained
principle,
biological
first
concentration
lipid
agreement w i t h
chlorinated
situation
found
is
and b i p h e n y l s
s h o u l d be e m p h a s i s e d h o w e v e r ,
actual
In
growth.
benzenes
is
gills,
steady-state
estimated octanol-water
figure
o c t a - and d e c a c h l o r o -
water.
b a s e d on w e t w e i g h t ) .
fat:
the
equilibration.
dissolved molecules).
10 d a y s e q u i l i b r a t i o n
only
60
compared w i t h
through
s h o u l d be n o t e d
exhaustion of
flowing
fish
of
the
1).
10 d a y s
unavailable test
free
and i n
fig.
are
2
factors
concentrations
a b s o l u t e maximum, c o r r e s p o n d i n g t o
from water
w
lipophilicity
bioconcentration
5
1
after
permeation
of
weight)
c a l c u l a t e d from e s t a b l i s h e d
Secondly, i t
were observed d u r i n g
The h i g h e s t
f
2,
k /k )
c
phenomenon a r e
are
aqueous and f i s h
test
(!< =
(C /C )
instead of
between m o l e c u l a r s t r u c t u r e ,
1
(wet
from a reduced uptake e f f i c i e n c y .
m i g h t be c a u s e d by h i n d e r e d
branes,
fish
(table
lower bioconcentration
result
in
constants
uptake experiment
respectively
Calculated bioconcentration
in
clearance rate
to
.
in
It
model
the
model,
since
completely
or
chemicals in water
availability
now f o c u s s e s
97
™o
•- "o
"o
o
99
Fig.
2
R e l a t i o n between b i o c o n c e n t r a t i o n
water
partition
(partially
the
O
chlorinated
benzenes
•
chlorinated
biphenyls
attention
fish
via
Dietary
for
in
on t h e
and
c
octanol/
)
11)
(PCBs)
possible role
of
chemicals entering
the
e x p o s u r e . T h e 110 m a l e a n d 20 f e m a l e g u p p i e s , e x p o s e d
food,
a concentration
s h o w e d no s i g n s
as m e a s u r e d by t h e
The c o u r s e o f
the
number o f
of
of
consisted of
Roughly,
according
six
three
to
new-born
concentrations
in
f i s h was
fish
is
each chemical
Their
reproduction
normal.
sampled during
given in
fig.
of
compounds can be
distinguished
u p t a k e and a c c u m u l a t i o n
behavior:
dietary
3. Samples
individuals.
types
their
50 y g / g o f
intoxication.
e x p o s u r e and s u b s e q u e n t d e p u r a t i o n
100
(P
9 and
(K )
food.
10 w e e k s t o
the
coefficient
b a s e d on r e f s .
factor
1. Compounds which could not be q u a n t i f i e d since they were at
or below detection l i m i t i n most f i s h samples:
hexabromoben-
zene, octachlorodioxin and perchloroterphenyl. Apparently,
uptake of these chemicals i n f i s h does not occur or i s very
inefficient.
2. Compounds which could be q u a n t i f i e d i n most f i s h samples
during the period of dietary a d m i n i s t r a t i o n , but had
disappeared a f t e r ' 1 4 days clearance: dichlorobiphenyl and
pentachlorobenzene.
The concentrations of these compounds
i n f i s h during exposure were f l u c t u a t i n g : as a r u l e , they
did not exceed the amount d a i l y consumed (ca. 1-2 | i g / g f i s h ) .
It i s concluded that a f a s t e l i m i n a t i o n precludes real
accumulation of these compounds, which were the
least
l i p o p h i l i c of the t e s t s e r i e s .
3. Compounds which showed a gradual increase i n f i s h during
exposure, and a gradual decrease a f t e r dosing had stopped:
tetra-,
hexa-, octa- and decachlorobiphenyl. In p r i n c i p l e , clearance rate
constants,
h a l f l i v e s , absorption e f f i c i e n c i e s and steady state
biomagnification f a c t o r s (K„) can be c a l c u l a t e d f o r these chemicals
(table 3). The clearance of the most l i p o p h i l i c compounds (octa- and
decachlorobiphenyl) was n e g l i g i b l e : t h e i r h a l f l i f e exceeded
the duration of the experiment (84 days). Therefore, only t h e i r
absorption e f f i c i e n c y ( e ) could be determined with
reasonable
accuracy. "Clearance" rates and biomagnification factors w i l l
then
be highly dependent on c o n d i t i o n , growth and reproduction
of the f i s h , as shown by the s i g n i f i c a n t l y higher
"clearance"
rate constants ( k ) of octa- and decachlorobiphenyl i n female
2
guppies (table 3). The e l i m i n a t i o n of t e t r a c h l o r o b i p h e n y l ,
in contrast, was lower i n females, which i s i n agreement with
t h e i r higher f a t content:
the r e l a t i v e l y f a s t e l i m i n a t i o n
of the higher chlorinated biphenyls presumably results from
t r a n s f e r to the progeny ( f i g . 4.table
3).
101
102
KBIcklysl
100 [daysl
Fig. 3
Bioaccumulation of PCBs by guppies
Dietary exposure: uptake and clearance
103
104
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DISCUSSION
The e x a m p l e o f
increasing
the
PCB s e r i e s
hydrophobicity
in
these experiments
ultimately
retention
of
bi phenyl)
o n c e a b s o r b e d by a q u a t i c
On t h e
non-degradable
other
hand, i t
"super-lipophilic"
and w i l l
the
n e v e r become s i g n i f i c a n t
p r e c l u d e d by t h e
bility
(log
is
very
high
in
the water
is
ug/1),
or
simple centrifugation
B o t h 1 / S and K ( t h e a d s o r p t i o n
the
of
to
lipophilicity
(P).
trations
found a s s o c i a t e d w i t h
reduced
aqueous
fraction
solu-
will
"dissolved"
are
as
is
organic
filtration
coefficient)
proportional
these
in water
colloidal
or
of
conditions,
Their
and a major
phase: even d e t e r m i n a t i o n
after
uptake
considerably
under n a t u r a l
be a d s o r b e d by s u s p e n d e d p a r t i c l e s
trations
direct
P>6)
hydrophobicity.
(S<10
complete
o c t a - and d e c a c h l o r o -
dissolved concentrations
extreme
low
(e.g.,
that
organisms.
was s h o w n t h a t
chemicals
occurrence of
may l e a d t o
compounds
shows
matter
concen-
is
dubious.
roughly
R e l a t i v e l y high
concen23
which
are
form the
and o t h e r
It
of
unaffected
warrant
rather
the
the
0.5).
conclusion that
quantitative
implies
that
proportional
in
a steady-state
to
the
efficiency
combination,
a linear
of
fish.
the
In
that
energetics
b a s e d model
on t h e
results
higher
authors
nature
of
the
in
of
model
pure
concentration
organism
lake
fish
organisms.
observations
transfer
is
higher
s u c h a model
fish
or
model
appropriate
form,
in
after
(e)
bioaccumulation of
its
is
inversely
gross
(g g r o w t h a t t a i n e d
assimi/g
food
25
, after
for
Norstrom
the
trout:
, d e s c r i b e d such a
accumulation of
his
assumptions and c o n c l u s i o n s
described above; but
biphenyls
paid attention
to
the
bio-
a PCB m i x t u r e
b i o a c c u m u l a t i o n p r o c e s s a r e s u p p o r t e d by
experiments
chlorinated
,
relatively
these
food-chain
24
weininger
1254)
is
biomass conversion f a c t o r
of
consumed),
(Aroclor
efficiency
biphenyls
partitioning
description
biphenyls
leading to
deposit-feeding
absorption
In
and m i c r o - o r g a n i s m s
chains
or
chlorinated
than an e q u i l i b r i u m
chlorinated
lition
that
-
food
filtering
higher
(E = 0 . 2
the
aquatic
via
was s h o w n f u r t h e r
ingestion
for
basis of
predators
detritus
(Cl^-Cl^).
only
Recently,
development of
for
the
the
several
bioaccumulation
107
models
26-29 accounting
DDT
in
aquatic
and
bioconcentration
for
(direct
s h o u l d be d e p e n d e n t f i r s t
organisms
in
Bioconcentration,
small
aquatic
(mainly
moderate
atoms
show i n t e r m e d i a t e
In
in
since it
consumption,
the
PCBs
and
biomagnification
chemicals
r a p i d exchange
model
and
behavior.
The r e l a t i v e
versus food-chain
d e p e n d e n t on s i z e ,
growth,
up t o
reproduction
-
fat
for
chemicals
P between 3 and 5 ) ,
a complete b i o e n e r g e t i c s
is
between
i m p o r t a n t mechanism
o r g a n i s m s , and f o r
(log
e.g.,
six
chlorine
importance
transfer
of
can
only
based accumulation
content,
and r e s p i r a t i o n
food
rate
of
organism concerned.
contrast
to
the
polychlorinated
hexabromobenzene, o c t a c h l o r o d i o x i n
were
not
likely
accumulated in
to
aromatic
be
of
partitioning)
of
b e n z e n e s . PCBs c o n t a i n i n g
exchange w i t h water
determined
of
planktonic)
chlorinated
study,
type
be t h e m o s t
lipophilicity
lower
be
fish-water
on t h e
as a r e s u l t
and o r g a n i s m , w i l l
direct
transfer
question.
water
with
food-chain
e c o s y s t e m s . The c h o i c e b e t w e e n t h e
be r a p i d
biodegradable.
by p a r t i c u l a r
structural
w i t h membrane
In
principle,
of
accumulation of
mw= 7 1 3 , b u t
or
is
is
not
expected
to
hindered
inter-
might e x p l a i n the
(estimated
log
lack
P>10;
P = 7.9;
mw = 5 5 2 )
P = 8 . 5 ; mw = 4 6 0 ) , w h i c h
s h o u l d be
higher
factor:
and n o t
uptake
hexabromobenzene ( l o g
comparable w i t h
the
this
halogenated
physico-chemical properties
hydrophobicity
(log
1 2
hydrophobic
Probably, their
or octachlorodioxin
Hutzinger
The r e a s o n f o r
since these f u l l y
perchloroterphenyl
of
perchloroterphenyl
transport.
extreme
not
and
fish.
elimination,
compounds a r e e x t r e m e l y
readily
fering
living
benzenes and b i p h e n y l s ,
PCBs
(Clg -
C1
1 Q
have s u g g e s t e d m o l e c u l a r s i z e
a molecular weight
of
).
to
Zitko
and
be t h e
c a . 600 w o u l d be t h e
limiting
upper
limit
for
absorption.
From a m e c h a n i s t i c p o i n t
volume o r
diameter)
membrane p a s s a g e .
of
rather
view,
t h a n mass w o u l d
dimensions
interfere
Indeed, decachlorobiphenyl
have a somewhat s m a l l e r e f f e c t i v e
108
spatial
diameter
(molecular
with
(mw = 4 9 9 )
than
will
hexabromobenzene
(since
chlorine
p-dioxin
(as
Apparently,
restricted
is
smaller
a result
the
bioaccumulation
bromine)
different
relationship
uptake
lipophilic
of
than
octa-chlorodibenzo-
positioning
of
between m o l e c u l a r
by o r g a n i s m s
correlation
or
the
size
phenyl
d e s e r v e s more a t t e n t i o n
studies,
especially
rings).
and
for
in
highly
chemicals.
REFERENCES
1.
J . L . Hamelink,
Soc,
2.
F.
100,
207-214
Moriarty,
organic
1975,
R . C . Waybrant
in:
F.
D.M. Rosenberg, Quest.
4.
J.L.
5.
Hamelink
6.
1113-1115
C T .
and A .
Entomol.,
Spacie,
Sci. Technol.,
8.
W.B. N e e l y , E n v i r o n .
D.L.
9.
W.A.
1040-1048
Chemosphere,
Ann.
(1975)
Rev. Pharmacol. T o x i c o l . ,
Environ.
Sci.
Schmedding and R . L .
1 1 , 475-478
17,
Technol.,
Kohnert,
(1977).
Bergstedt,
S c i . Technol.,
Martron,
10, 811-832
K.
S u g i u r a , N.
Tsukakoshi
J . Fish.Res.Board Can.,
Ito,
13,
D.
1506-1510
H.
Kbnemann a n d K.
12.
V.
Zitko
and 0 .
(1979).
Kooiman and 0 .
Hutzinger,
(1981).
N. Matsumoto,
Y. Mihara,
and M. G o t o , C h e m o s p h e r e , 7 ,
11.
K.
Bull.
Environ.
Murata,
731-736
van Leeuwen, Chemosphere, 9 ,
Hutzinger,
(1978).
3-19
(1980).
Contam. T o x i c o l . ,
16,
(1976).
13.
M . T h . M . T u l p and 0 .
14.
H.
15.
B. M c D u f f i e ,
16.
W.A. Bruggeman, J . van der
K o n e m a n n , R.
238,
London,
(1979).
Y.
178,
1 1 , 97-110
DeFoe and B . V .
Bruggeman, L . B . J . M .
665-673
Academic P r e s s ,
B r a n s o n and G . E . B l a u ,
G.D. V e i t h ,
10.
(Ed.),
persistent
(1974).
7.
36,
insecticides:
Moriarty
C h i o u , V . H . F r e e d , D.W.
Environ.
Fish.
(1977).
W.B. N e e l y , D.R.
8,
Amer.
29-71.
3.
167-177
Trans.
(1971)
Organochlorine
pollutants,
pp.
and R . C . B a l l ,
Hutzinger,
Zelle,
F.
Chemosphere, 7,
Buser
849-860
and W . E . Hammers, J .
(1978).
Chromatogr.,
559-(1979).
335-346
Chemosphere, 10,
73-83
(1981).
S t e e n and 0 .
Hutzinger,
J.
Chromatogr.,
(1982).
109
17.
0.
Hutzinger,
18.
W.A. Bruggeman,
CRC P r e s s ,
D.
Fung, A.
Hutzinger,
(silicones)
(submitted
Zitko,
for
Absorption
in
fish:
Kbnemann, T o x i c o l o g y ,
L.
W e i l , G . Dure and K . E . Q u e n t i n ,
19, 209-221
Yalkowsky, Chemosphere, 9 ,
D. M a c k a y , E n v i r o n .
23.
W.C. S t e e n and S.W. K a r i c k h o f f ,
24.
D. W e i n i n g e r ,
25.
R . J . Norstrom,
Board C a n . , 3 3 , 248-267
M.P. Brown, J . J . A .
28.
A.L. Jensen,
Modelling,
R.
39,
727-735
H a q u e , D.W.
139-142
110
Wisconsin-Madison,
de F r e i t a s ,
700-709
(1981).
1978.
J.
Fish.
(1976).
Aquat.
S c i . , 3 8 , 280-296
(1981).
Wyman,
(1982).
S.A. Spigarelli
S c i . , 39,
(1982).
M c L a u g h l i n , J . M . O ' C o n n o r a n d K.
15, 29-47
and
a n d M . M . Thommes, C a n . J .
(1974).
Fish.
(1982).
S . G r i e s b a c h , R . H . P e t e r s and S . Y o u a k i m , C a n . J . F i s h .
Sci.,
7,
(1980).
16, 274-278
A . E . M c K i n n o n and A . S . W .
27.
30.
S.C. Valvani
Chemosphere, 10, 27-32
Ph.D. Thesis, Univ.
R . V . Thomann, C a n . J . F i s h .
Aquat.
polydimethyl-
experiments
(1981).
257-264
Sci. Technol.,
26.
Ecol.
der
(1974).
22.
Res.
J . van
Z . Wasser Abwasser F o r s c h . ,
D. M a c k a y , R. M a s c a r e n h a s , W . Y . S h i u ,
S.H.
PCBs,
publication).
H.
29.
and r e t e n t i o n o f
preliminary
19.
21.
of
Opperhuizen, A. Wijbenga,
20.
169-175
The C h e m i s t r y
C l e v e l a n d , OH, 1974.
S t e e n and 0 .
siloxanes
S . S a f e and V .
Aquat.
(1982).
Schmedding and V . H . F r e e d , E n v i r o n .
S c i . Technol.,
8,
CHAPTER
5
ABSORPTION
(SILICONES)
AND R E T E N T I O N
IN
FISH:
OF
POLYDIMETHYLSILOXANES
PRELIMINARY
EXPERIMENTS
ABSORPTION AND RETENTION OF POLYDIMETHYLSILOXANES
(SILICONES) IN FISH
W.A.
J.
: PRELIMINARY EXPERIMENTS.*
Bruggeman**, D. Weber-Fung, A. Opperhuizen,
van der Steen,
A . Wijbenga and 0.
Hutzinger.
Laboratory of Environmental and T o x i c o l o g i c a l Chemistry,
U n i v e r s i t y of Amsterdam, Nieuwe Achtergracht 166,
1018 WV Amsterdam, The Netherlands.
ABSTRACT
Hydrophobicity and bioaccumulation p o t e n t i a l
and c y c l i c p o l y d i m e t h y l s i l o x a n e
estimated
by reversed-phase
of
linear
(PDMS) oligomers were
l i q u i d chromatography and
feeding experiments
with guppies
(Poecilia
reticulata).
PDMS concentrations
i n f i s h were determined by c a p i l l a r y
column gas chromatography and gas chromatography - mass
spectrometry.
In c o n t r a s t
to p o l y c h l o r i n a t e d
biphenyls
(PCBs), only very small amounts of PDMS were r e t a i n e d by
the f i s h a f t e r six weeks f e e d i n g .
INTRODUCTION
Among the o r g a n o s i l i c o n compounds,
(PDMS) represent
the most important c l a s s as to
q u a n t i t y produced (ca.
*
This a r t i c l e
of
**
polydimethylsiloxanes
their
1
45.000 tons i n 1978 ") . These
i s dedicated to P r o f . Korte f o r the
occasion
h i s 60th b i r t h d a y .
Present address
P.O.B.
17,
: Governra. I n s t . Waste Water Treatm.
(RIZA),
8200 AA L e l y s t a d , The Netherlands.
113
" s i l i c o n e o i l s " have found widespread use as thermally
stable,
hydrophobic f l u i d s of low flammability and
r e l a t i v e l y highoxidative resistance
inertness.
One of t h e i r p o t e n t i a l
and b i o l o g i c a l
applications
is
the
replacement of PCB formulations i n transformer o i l s .
Commercial preparations are d e f i n e d by v i s c o s i t y
and
u s u a l l y c o n s i s t of mixtures of l i n e a r polymers of v a r y i n g
chain lengths.
be present
special
Low molecular weight c y c l i c s t r u c t u r e s may
i n small q u a n t i t i e s
as byproducts, o r ,
in
f o r m u l a t i o n s , as main components.
Considerable information on environmental f a t e and
e f f e c t s of PDMS compounds has been obtained from
laboratory t e s t s .
oils
The b i o l o g i c a l inertness
of
silicone
i s demonstrated by t h e i r low t o x i c i t y and lack of
2
biodegradation by m i c r o b i a l populations
. L i g h t induced
and c l a y c a t a l y z e d h y d r o l y s i s appear to be the most
important environmental t r a n s f o r m a t i o n r e a c t i o n s .
A n a l y t i c a l data on t h e i r a c t u a l environmental
d i s t r i b u t i o n is scarce.
Pellenbarg
3
reported the
occurrence of PDMS i n aquatic sediments,
using atomic
absorption spectrometry i n combination with
extraction for total
organosilicon
determinations.
Traces of octamethylcyclo t e t r a s i l o x a n e
i n Rhine River water
solvent
(D ),
4
detected
(1979) by r e s i n absorption and
combined gas chromatography - mass spectrometry
(GC-MS)
4
have been reported i n a Dutch p a p e r . The presence
of
PDMS i n aquatic organisms in the environment has not been
demonstrated,
and bioaccumulation of 1 4 „ - l a b e l e d PDMS
2
d i d not appear to take place i n an experimental study
The bioaccumulation p o t e n t i a l
of x e n o b i o t i c
combination with d e g r a d a b i l i t y and t o x i c i t y ,
interest
in
i s of
i n environmental hazard assessment.
To a large extent,
114
chemicals,
.
it
is possible
to p r e d i c t approximate
bioaccumulation f a c t o r s of many organic chemicals based on
t h e i r chemical s t r u c t u r e and physico-chemical
characteristics.
For t h i s purpose,
information is
needed
on :
1.
Hydrophobicity. Hydrophobic chemicals tend to
accumulate i n f a t t y t i s s u e s .
coefficients
(P)
Octanol-water p a r t i t i o n
c o r r e l a t e well with b i o c o n c e n t r a t i o n
f a c t o r s (K ) i n a q u a t i c organisms f o r many organic
s t r u c t u r e s . Estimations of the hydrophobicity can a l s o
c
be made by a combination of aqueous s o l u b i l i t y
(S)
and
5-9
melting point d a t a .
2. Molecular s i z e .
D i f f u s i v e t r a n s p o r t through
biomembranes w i l l be hindered f o r molecules
certain size.
1
exceeding
a
0
Zitko and Hutzinger " have suggested a
molecular weight upper l i m i t of c a . 600 f o r
xenobiotic
chemicals.
3.
Presence of "biodegradable groups". The o r i g i n a l
s t r u c t u r e may be changed by metabolism.
For i n s t a n c e ,
transformation of apolar s u b s t i t u e n t s by o x i d a t i o n
p o l a r groups and formation of h y d r o p h i l i c
will
facilitate
general,
the e x c r e t i o n of the chemicals.
a reasonable
stability
For PDMS compounds, t h i s
considerations
for
organisms.
leads to the
following
:
The bulk of commercial formulations c o n s i s t s of
of
In
(or even p e r s i s t e n c e )
i n the aquatic environment i s a p r e r e q u i s i t e
bioaccumulation i n a q u a t i c
into
conjugates
mixtures
long chain polymers with molecular weights above
mw = 7 50 and up to s e v e r a l
thousands.
with water and only s l i g h t l y
soluble
They are immiscible
i n lower
alcohols.
115
Because of t h e i r molecular weight ranges,
bioaccumulation
of
the higher v i s c o s i t y
polymers i s not expected,
in spite
of
t h e i r apparent h y d r o p h o b i c i t y . On the other hand, low
molecular weight oligomers w i l l be l e s s hydrophobic, but
may be small enough f o r membrane passage. As discussed
5
a previous paper, bioaccumulation v i a food
(biomagnification)
i s probably the most important route
for extremely hydrophobic chemicals
(log P > 5.5).
For a
r e l i a b l e estimation of the bioaccumulation p o t e n t i a l
PDMS compounds, i t seemed advisable, f i r s t ,
insight
i n t o the r e l a t i o n s h i p s
h y d r o p h o b i c i t y , and second,
to s e l e c t a s e r i e s of
i.e.
mw < 1000 and
f o r p r e l i m i n a r y bioaccumulation
experiments
with f i s h . For t h i s procedure, s e n s i t i v e methods
separation,
of
to obtain
between chain length and
compounds i n the c r i t i c a l range,
l o g P > 3,
in
for
i d e n t i f i c a t i o n and q u a n t i f i c a t i o n of
i n d i v i d u a l PDMS compounds were a l s o devised or adapted.
MATERIALS AND METHODS
Chemicals. Linear and c y c l i c PDMS oligomers and polymeric
PDMS of v a r i o u s v i s c o s i t i e s were provided by Dow Corning
corporation.
1).
(For s t r u c t u r e s and abbreviations see
Solvents were d i s t i l l e d before
Hydrophobicity determinations.
table
use.
The apparent
hydrophobicity of PDMS oligomers was measured by c o r r e c t e d
r e t e n t i o n times ( t ,
= t-t
) in reversed-phase
high
performance l i q u i d chromatography (RP-HPLC) on an
o c t a d e c y l s i l i c a ( C ) column with methanol-water
II 12
(90: 10) as eluent
'
. Apparent log P values were
1Q
determined from r e t e n t i o n
116
i n d i c e s using a homologous
s e r i e s of n-alkylbenzenes
13).
A dual detector
absorption)
and
as reference compounds (see
ref.
system ( r e f r a c t i v e index and UV
was used f o r simultaneous
d e t e c t i o n of PDMS
alkylbenzenes.
Bioaccumulation t e s t s .
The experimental
bioaccumulation
t e s t procedure has been described i n previous
papers
1 4 , 1 5
.
For d i e t a r y exposure,
a standardized
mixture containing l i n e a r and c y c l i c PDMS oligomers
added to the food of 110 male guppies
reticulata),
was
(Poecilia
together with c h l o r i n a t e d benzenes and
biphenyls to allow a d i r e c t comparison of
bioaccumulation of these chemicals.
the
The t e s t food was
prepared from commercial dry food by adding 3 ml of a
toluene
s o l u t i o n c o n t a i n i n g 250 ^Ug/ml of the
individual
compounds (MM-MD^M, D^-D^, D , c h l o r i n a t e d
g
benzenes and biphenyls)
and 2.5 mg/ml of DC 200
(5cs);
Toluene was removed by e v a p o r a t i o n . A n a l y s i s of the
food showed the presence of compounds of
volatility
(i.e.,
t e t r a - and
g
and D^) i n
of 2 0-5 0 jug per gram of dry f o o d . The more
v o l a t i l e materials
Dj.)
intermediate
pentachlorobenzene,
c h l o r i n a t e d b i p h e n y l s , MD^M - MD^M, D
concentrations
(e.g.,
MM, MDM, MD^M, D , D^,
3
apparently evaporated together with toluene
during
the p r e p a r a t i o n and t h e r e f o r e were not s t u d i e d .
of
dry food was fed per gram of f i s h
Samples of six
test
Ca. 20 mg
(wet weight) per day.
f i s h were taken each week a f t e r a p e r i o d of
two days without f e e d i n g .
A n a l y s i s of f i s h e x t r a c t s
chromatography with e l e c t r o n capture d e t e c t i o n
by gas
showed
most c h l o r i n a t e d aromatics reached t h e i r maximum l e v e l
f i s h a f t e r seven weeks feeding
For aqueous exposure,
5
that
in
.
water was continuously
r e c i r c u l a t e d over a f l u i d i z e d bed c o n t a i n i n g the
test
mixture coated on an i n o r g a n i c support. However, chemical
a n a l y s i s of the water phase showed that the
aqueous
117
concentrations
of the PDMS oligomers,
in contrast
to
c h l o r i n a t e d benzenes and PCBs, remained below our
detection
l i m i t of 0.1 y.g/1,
presumably as a r e s u l t of
high v o l a t i l i t y and low s o l u b i l i t y of the PDMS compounds.
T h e r e f o r e , the system was considered unsuited f o r d i r e c t
b i o c o n c e n t r a t i o n experiments
Chemical a n a l y s i s .
oligomers
For q u a n t i t a t i v e a n a l y s i s
i n water,
consisting
with PDMS.
food and f i s h , a method was developed
of batch e x t r a c t i o n with b o i l i n g
c l e a n up by F l o r i s i l
o f PDMS
n-hexane,
column chromatography with 5 pc
dichloromethane i n n-hexane,
and i d e n t i f i c a t i o n and
q u a n t i f i c a t i o n by temperature programmed gas
chromatography (GC), using high r e s o l u t i o n
with low s p e c i f i c i t y d e t e c t i o n
( c a p i l l a r y ) GC
(FID) f o r primary
q u a n t i f i c a t i o n (combination 1) and low r e s o l u t i o n
column) GC with high s p e c i f i c i t y d e t e c t i o n
c o n f i r m a t i o n (combination
(packed
(MS) f o r
2).
Combination 1 : Packard-Becker 428 GC; 15 m. CP S i l 5
WCOT column, 0.25 mm i . d . , s p l i t
injection;
temperature
programme, 5 0 - 3 0 0 ° C ; flame i o n i z a t i o n detector
Combination 2 : GC - mass spectrometer
(FID).
- data system
Hewlett Packard 5982 A; GC, g l a s s column, 2 m, 2 mm. i . d . ,
packed with "ultrabond" (0.2% Carbowax 20 M); temperature
programme 4 0 - 2 5 0 ° C ; MS, e l e c t r o n - i m p a c t at 70 eV, s e l e c t e d
ion monitoring (SIM; f o r mass spectrometry of PDMS,
ref
15).
D e t a i l s of these procedures are described
16
elsewhere .
118
see
RESULTS
A n a l y t i c a l procedure
Good s e n s i t i v i t y
and r e p r o d u c i b i l i t y was obtained both by
c a p i l l a r y GC-FID and by GC-MS-SIM : the d e t e c t i o n
limit
was c a . 0.3 Ug/g i n samples of c a . 0.2
g of f i s h
weight).
GC-MS was p r e f e r r e d
for
In case of i n t e r f e r i n g peaks,
identity confirmation.
For the gas chromatographic determination,
of
(wet
the upper l i m i t
PDMS chain length i s determined by the v o l a t i l i t y of
the higher PDMS oligomers
(MD M,
13
D)
in relation
g
the maximum operating temperature of the
chromatographic column (2 50 or 3 0 0 ° C ) .
low-boiling solvents
(e.g.,
to
gas
The use of
n-pentane),
is desirable
prevent evaporation of the more v o l a t i l e
to
compounds d u r i n g
e x t r a c t i o n and clean up.
Apparent Hydrophobicity
L i n e a r r e l a t i o n s h i p s between chain length and log t
n-alkylbenzenes
and p o l y d i m e t h y l s i l o x a n e s
of
s i m p l i f i e d the
determination of "apparent log P" v a l u e s ,
listed
in
table
1. From these v a l u e s , an "apparent hydrophobic
17
fragmental constant" (f)
of the d i m e t h y l s i l o x y group
could be c a l c u l a t e d ,
representing the
c o n t r i b u t i o n of the - O S i ( C * ^ ^
l o g P: f= 0.59.
The r e s u l t s
-
u n : L t
in table
oligomers MDM-MD^M and D^-D^ have
constant
t
o
t
n
e
apparent
1 show that PDMS
lipophilicities
s i m i l a r to p o l y c h l o r i n a t e d benzenes and b i p h e n y l s , and
t h e r e f o r e must be c o n s i d e r e d ,
bioaccumulating
i n p r i n c i p l e , as
potential
chemicals.
119
F i s h Feeding of PDMS
During the whole 10 weeks feeding p e r i o d , no adverse
e f f e c t s on the f i s h were observed.
Detectable
quantities
of the l i n e a r PDMS oligomers
MD M-MD_M have been found only i n f i s h sampled a f t e r
b
o
6-8 weeks f e e d i n g . The other l i n e a r and c y c l i c PDMS
compounds that were present
MDgM, M D ^ q M ,
i n food
Dg, Dg) remained below
(MD^M, MD^M,
detection
limit in a l l f i s h
samples.
The concentrations
of PDMS oligomers detected i n f i s h d i d
not exceed 2 | i g / g whereas the average d a i l y intake was c a .
1 L(g/g of f i s h . Apparently, l i t t l e
absorption and
r e t e n t i o n of PDMS oligomers does occur; accumulation of
these compounds v i a food i s not s i g n i f i c a n t .
concentrations
of these PDMS oligomers
The
i n f i s h were
comparable to those of t e t r a - and pentachlorobenzene
and
2 , 5 - d i c h l o r o b i p h e n y l , whereas the higher c h l o r i n a t e d
biphenyls
(tetra-,
reached 10-20
hexa-,
o c t a - and decachlorobiphenyl)
times higher concentrations
i n the same f i s h
samples.
A comparison of the t e s t compounds i s presented
in f i g . 1
In t h i s f i g u r e , the r a t i o between concentrations
found i n
f i s h (C )
f
and i n t h e i r food ( C
extractable
lipids,
"biomagnification f a c t o r "
against
),
adjusted
for
(i.e.
: C-/C-.)
and p l o t t e d
the hydrophobicity (apparent log P) of
compounds.
the
Both f o r PCEs and l i n e a r PDMS, the maximum
f i s h / f o o d concentration r a t i o
was found at log P =s 7.5.
120
f d
i s expressed as an apparent
( a f t e r six weeks feeding)
Figure 1.
M a g n i f i c a t i o n of l i p o p h i l i c chemicals by
guppies
a f t e r s i x weeks f e e d i n g .
P = octanol-water
partition
coefficient
•
l i n e a r polyd-imethylsiloxanes
O
c y c l i c PDMS
•
p o l y c h l o r i n a t e d biphenyls
O
c h l o r i n a t e d benzenes :
T = tetra-,
Q =
(PDMS)
(PCBs)
pentachlorobenzene
121
TABLE 1
Polydimethylsiloxanes
weight,
: structures,
abbreviations,
l i p o p h i l i c i t y and m a g n i f i c a t i o n by guppies.
CH.
L i n e a r PDMS s t r u c t u r e :
CH.
CH.
CH - S i - 0 - S i - O - S i - C H - .
I
a b b r e v i a t i o n : MD M
n
CH.
CH-
CH'n
CM
m •f
o o o
o
o
V
s/s
o
CO
<H
o
CO
CO
1*1
o
o
o
o
o
o
en
o
o
CN CN
o
o
o
o
\M \& V
ON
\f
LTI
o
+
o
c N C O ^ o v o c N l ^ c ^ c n L O ' H r ^ r o c T i L O
IN
• s r ^ L O c o c o r s - t ^ c o c o c T . o o ^ i H c N
X
CN
CO
o
<*i
rH
CN
ro
co
en
00
CN
cn
tr
en
cn
co
o
CO
o
CO
CO
"cr
in
r»
oo
CN
CO
CN
o
cn
rcn
oo
o
in
o
rH
s s s s S s
s Q Q1*1 Qcn Q Qr* s
s S
(N
CO
T
s
S
2
a
a
£
co
S
a
0)
CD
f—
X
0
r3
X
0
rH
•H
rH
•rH 0 1
CO •H
SH
•H
T3 •P
rH rH
>1
xj
4->
CU
5
io
X
<D
ji
E
r
- -
p
ft)
rH
nj
P
C
OJ
id
+J
3
4-1
01
-M
(JJ
id
X
<D
•p a •C
pO H
OJ
4-1
td*
>1
si
u
B
=
X
-r-l
e
a
-H
•H
0
rH
m
to
0
CD
id
o
0J
o
OJ
-a
rd T )
-p
c
CD
o
o
id
Ul
CN
0
fa
to rd CU M
•U
u
-a
p
U
OJ o 0 )
o T J -a + 1
X
OJ
si
0)
M
DH
y
a
T
CN
oo
oi
+
rH
rH
TT
f-H
122
molecular
CN
s s s ss
o
i-
CN
en
rH
rH
T
rH
Q Q Q
S S S
s
c
Q
s
CH,
I
C y c l i c
PDMS
s t r u c t u r e
a b b r e v i a t i o n
:
-Si-O-
:
CH.
D
ro
o
I
—
•H
XI
HH C r<
•H O O
C-H
P
B.P
o
ro
o
o
o
V
V
id id id
ro
in
tr
CN
LD
CO
LO
in
o
o
tn
O
LD
3"
CM
CN
CM
tr
tr
tr
LO O
cr.
r-
C N ro
O
rH
r-l
CO
tr O N
iH
o o o
O
rH
S u m
CM
uo
CO
tr
cn
co
ON
LD
rH
CN
tr
in
LO
r-
ro
CM
ON
CM
LD
co
o
LD
LD
o
ro
tr
tr
P
O X!
0) tn
LO
LD
LD
tr
rH *H
o OJ
rH
r»
rH
o
m
CN
CM
CM
rH
ro
ON
ON
>l
c
OJ
ro tr in L O cn
Q Q Q Q
Q
>1
a •8
>1
G
0)
X!
CD
X
TJ
PH
•H
fa
•8
u
o
•rH
gg
id
U
•H
P
p
o
P
u
OJ
ro
P
C
rd
X
oj
oj
IX A
n)
C
>1
o
OJ
C
rH
O
rH
>1
x.
P
1
f8
X
<D
A
c
a
•H
XI
TJ
>1
o
=
:
Id
o
id
o
0)
TJ
id
O
a>
TJ
oj <d
TJ
P
o
TJ
o
O
x:
a
-H
OJ
p
id
c
-H
M
ui
OJ
C
OJ
O N
rH C
OJ
O JH
rH
>1
as
0J
0)
0J
a
N
c
N
c
C
0J
XI
OH
TH
OJ 0J
XI XI
o
0
XI
o
rH
rH
x;
u
XI
o
ro
u
P
cu
p
u
0
rH
XI
o
0
0
•H
id
TJ
I
in
OJ
*
a
OH
id
SH
P
0)
P
1
SH
o
P
o
rH
si
o
CM
a>
X!
D H
•H
Xt
o
H
o
X!
o
H
o
rH
XI
o
id
P
I
-
id in
X
te
OJ in
rH
X!
>1
Cl
1
-in— tr-
0)
XI
»
m
tr
te te
r-
—
—
in
tr
ro
tr
rote
te
in
—
CM
-C M -C M
CM
CN
(X
•H
X!
O
SH
0
rH
XI
O
ra
o
te C Nte
OJ
TJ
123
Apparent log P were measured by C18-RP-HPLC with
methanol/water (90:10)
as reference
as eluent and
n-alkylbenzenes
compounds
b)
concentration i n f i s h
(lipids)
concentration i n food
(lipids)
Magnification factor =
at day 42
The concentration of the PDMS oligomers
20-50 / J - g / g
i n food was
dry weight.
E x t r a c t a b l e l i p i d s i n food : 10% of
(dry)
weight
E x t r a c t a b l e l i p i d s i n f i s h : 1.7% of wet weight
c
'
C a l c u l a t e d log P from Bruggeman e t a l .
(1982)
13
DISCUSSION
These experiments
give some b a s i c information on the
bioaccumulation behaviour of PDMS compounds. The
hydrophobicity of most PDMS oligomers
RP-HPLC i s
as estimated
s u f f i c i e n t l y high (log P > 5) to
bioaccumulation i n f i s h l i p i d s by d i r e c t
typical
anticipate
bioconcentration
or by food-chain m a g n i f i c a t i o n . Nevertheless,
concentrations
by
the
observed
i n f i s h were much lower than expected f o r
l i p o p h i l i c chemicals
such as PCBs. The same
phenomenon has been observed with p e r c h l o r o t e r p h e n y l ,
o c t a c h l o r o d i b e n z o - p - d i o x i n and penta- and hexa5 18
bromobenzene '
. Presumably, the molecular s i z e i s an
important l i m i t i n g f a c t o r ,
124
at l e a s t f o r the higher
linear
polymers (MDgM and higher)
and f o r the c y c l i c compounds.
The c o u p l i n g of the bulky - 0 S i ( C H . j ) 2
especially
i n c y c l i c s molecules,
c o n s i d e r a b l e e f f e c t i v e diameters,
will
-
groups,
result in
i n comparison with
c h l o r i n a t e d aromatic hydrocarbon s t r u c t u r e s of the same
molecular weight.
Principally,
t h i s would i n f l u e n c e the
a b s o r p t i o n e f f i c i e n c y . The present r e s u l t s
suggest that
the absorption of PDMS from the g a s t r o - i n t e s t i n a l
tract
is
very low, or h i g h l y i n e f f e c t i v e by e f f i c i e n t clearance
mechanisms. Several compounds that were present i n the
food were not detected
at a l l i n the f i s h samples.
T h e r e f o r e , i t must be excluded that undigested food r e s t s
i n the g a s t r o - i n t e s t i n a l
t r a c t were r e s p o n s i b l e f o r the
presence of MD M, MD M and MD M i n f i s h .
C
As discussed i n a previous paper
, absorption a f t e r
i n g e s t i o n of p a r t i c u l a t e m a t e r i a l ( e . g . ,
food)
is
thought
to be the main uptake route f o r s u p e r - l i p o p h i l i c chemicals
(log P > 7).
However, the r e s u l t s presented here i n d i c a t e
that t h i s process does not r e s u l t i n a s i g n i f i c a n t
accumulation of PDMS polymers (M > 850)
in f i s h .
The present study does not provide s u f f i c i e n t i n f o r m a t i o n
t o draw d e f i n i t i v e conclusions about the p o t e n t i a l
bioaccumulation of PDMS. In f u t u r e s t u d i e s ,
more a t t e n t i o n
should be paid to the uptake r a t e during aqueous
exposure,
f o r the smaller l i n e a r and c y c l i c PDMS
especially
o l i g o m e r s , and to determination of clearance r a t e
constants
i n r e l a t i o n to chain length and aqueous
solubility.
Acknowledgement
T h i s work was supported by a grant from Dow Corning
C o r p o r a t i o n , which i s g r a t e f u l l y acknowledged. We thank
Dr.
C L . Frye f o r u s e f u l comments d u r i n g p r e p a r a t i o n of
the manuscript.
125
REFERENCES
1.
R.R. Buch and D . N . Ingebrightson,
13,
2.
Environ. S c i . Technol.,
676-679 (1979).
E . J . Hobbs, M . L . Keplinger and J . C . Calandra,
E n v i r o n . R e s . , 10, 397-406
(1975)
3.
R. P e l l e n b a r g ,
E n v i r o n . S c i . T e c h n o l . , 13, 565-569 (1979)
4.
R. de Groot, H_ 0, 12_, 333-336
5.
W.A. Bruggeman, A. Opperhuizen, A. Wijbenga and
(1979)
2
O. Hutzinger,
Bioaccumulation of
chemicals i n f i s h
(in Dutch)
super-lipophilic
(This thesis, chapter 4 )
6.
W.B. Neely, D.R. Branson, G . E . Blau, E n v i r . S c i . T e c h n o l . ,
7.
C T . Chiou, V . H . Freed, D.W. Schmedding and R . L . Kohnert,
8, 1113-1115
Envir.
8.
(1974)
S c i . Technol.,
11, 475-478
(1977)
C D . V e i t h , D . L . De Foe and B . V . Bergstedt,
J.
F i s h . Res. Board C a n . , 36, 1040-1048
(1979)
9.
D. Mackay, E n v i r o n . S c i . T e c h n o l . , 16, 274-278
10.
V. Zitko and 0. Hutzinger,
16,
11.
B u l l . E n v i r o n . Contam. T o x i c o l . ,
665-673 (1976)
H. Konemann, R. Z e l l e ,
J.
F . Busser and W.E. Hammers,
Chromatogr., 178, 559
12.
B. Mc D u f f i e , Chemosphere,
13.
W.A. Bruggeman, J . vander Steen and 0.
14.
W.A. Bruggeman, L . B . J . M .
J.
10, 73-83
Chromatogr. , 238, 335-346
(1981)
Hutzinger,
(1982) (This thesis, chapter 3)
Martron, D. Kooiman and
0. Hutzinger, Chemosphere,
15.
(1982)
10, 811-832
J . E . Coutant and R . J . Robinson,
(1981) (chapter 2)
i n : A . L . Smith
A n a l y s i s of S i l i c o n e s (Chemical A n a l y s i s ,
(Ed.),
V o l . 41), Wiley,
New York (1974), pp. 325-348.
16.
17.
D. Fung, Ph. D. T h e s i s ,
U n i v e r s i t y of Guelph, Can. (1982).
R . F . Rekker, The Hydrophobic Fragmental Constant,
Elsevier,
Amsterdam, 1977
18.
W.A. Bruggeman, C J . Peeters, J . van der Steen and
O. Hutzinger,
Uptake from contaminated
food and e l i m i n a t i o n
of brominated benzenes, pentabromotoluene,
benzene and decachlorobiphenyl
126
pentachloro-
by f i s h , (chapter 6)
CHAPTER
6
ABSORPTION
AND
BROMOTOLUENE,
BY
ELIMINATION
OF
BROMINATED
P E N T A C H L O R O B E N Z E N E AND
BENZENES,
PENTA-
DECACHLOROBIPHENYL
FISH
127
ABSORPTION AND
E L I M I N A T I O N OF BROMINATED BENZENES, PENTABROMOTOLUENE,
PENTACHLOROBENZENE AND DECACHLOROBIPHENYL
W.A.
B r u g g e m a n * , G..J. P e e t e r s , J . v a n d e r S t e e n
Laboratory
o f Environmental
University
BY
a n d O.
and T o x i c o l o g i c a l
WV
AMSTERDAM, T h e
Hutzinger
Chemistry,
o f A m s t e r d a m , Nieuwe A c h t e r g r a c h t
1018
FISH
166,
Netherlands
ABSTRACT
Dietary
exposure
of guppies
(Poecilia
robenzene ,
1, 3 , 5 - t r i b r o m o b e n z e n e ,
motoluene,
hexabromobenzene
cant
accumulation
depuration
lifes
were
study
between
was
in fish.
negligible,
2.5 a n d 5 d a y s .
the
h y d r o p h o b i c i t y o f these
the
lack of efficient
during
and tetrabromobenzene,
was n o t d e t e c t e d
recovered
to a test
1,2,4,5-tetrabromobenzene,
and d e c a c h l o r o b i p h e n y l
of the t r i -
b e n z e n e . Hexabromobenzene
bromotoluene
reticulata)
and o n l y
The e l i m i n a t i o n
whereas
78 d a y s
resulted
pentabro-
in a
and
of decachlorobiphenyl
test
factors
a b s o r p t i o n and accumulation
pentachlo-
signifi-
pentachloro-
t r a c e s o f pentabromobenzene and
compounds
i n accumulation
compounds. S t e r i c
containing
pentabromobenzene,
decachlorobiphenyl
the other
The d i f f e r e n c e s
mixture
showed
kinetics
a r e suggested
in a
penta-
subsequent
clearance
half
were n o t r e l a t e d
to
t o be r e s p o n s i b l e f o r
o f h i g h l y brominated
benzenes.
INTRODUCTION
Bioaccumulat ion
direct
The
of xenobiot i c chemicals
b i o c o n c e n t r a t i o n from
relative
version
importance
of the organism,
Bioaccumulat ion
ambient
o f these
in fish
water
processes
a n d on w a t e r
c a n be d e s c r i b e d
i s dependent
solubility
as a
can proceed
balance
between
chemicals
absorption
and e x t r e m e l y
fer
to a clean
* Present
The
from
water
and f o o d
on f e e d i n g
or l i p o p h i l i c i t y
Character i s t i c of s t r o n g l y bioaccumulat ing
efficiencies
via different
routes, i . e . ,
and b i o m a g n i f i c a t i o n v i a c o n t a m i n a t e d
uptake
(e . g . ,
food.
h a b i t s and e n e r g y
of the o r g a n i c
and e l i n . i n a t i o n
hexachlorobiphenyl)
low c l e a r a n c e
con-
compound.
processes.
are high
rates after
trans-
environment.
address:
G o v e r n m . I n s t . Waste Water
Treatm.
(RIZA),
P.O.B.
17, 8200 AA
Lelystad,
Netherlands
129
In
a
recent
found
able
for
study,
the e l i m i n a t i o n o f s e v e r a l
t o be n e g l i g i b l e ' .
Combined
absorpt ion e f f i c i e n c y
a classical
after
"food-chain"
In g e n e r a l , b i o a c c u m u l a t i o n
accumulation
factors)
parameters
as the octanol-water
at log P
food.
Examples
phenyls3,6,7,
Several
suggested
accumulat ion
of
ting
In
the present
study
in fish.
an
Since
i t was f e l t
hexabromobenzene
Dietary
parameters,
compounds,
potent i a l
to
1 0
as
fish
was
reason-
are favourable
related
1
or sigmoid
to explain
.
and h i g h l y
However,
relationships
the reduced
these
models
hexabromobenzene
lipophilic
have
made
to
and
between
l o g Kc
bioaccumulat ion of
cannot
explain
the lack
octachlorodibenzo-p-dioxin.
and a c c u m u l a t e s
suggested
to high
molecular
approximate
experiments
with
levels i n
size
the molecular
give
aqueous
solubility.
efficiencies
a series
could
to direct
defective
a reduced
brominated b i -
as a
limi-
5
preferred
between
Several
water o r
biomembranes .
t o low a q u e o u s
mechanisms c a u s i n g
5
to contaminated
8
pentabromotoluene,
i . e . , absorption
distinguish
exposed
o f d i - , t r i - and t e t r a b r o m o b e n z e n e s
and
was
1
and p o l y d i m e t h y l s i l o x a n e s .
through
was
and b i o -
chemicals,
close to 1 ug/l. ~3.
hexabromobenzene ,
i s more
attempt
rate constants
( P ) . F o r b i o c o n c e n t r a t i o n , an o p t i m u m
by f i s h
Z i t k o and H u t z i n g e r
that accumulation
exposure
complications
9
such
uptake
including
kinetic
3
f o r comparison,
reported^,
prevent
coefficient
accumulated
, , ,
f o r uptake and passage
absorption
question.
2
compounds
For hexabromobenzene,
factor
from
and a
of environmental
t o an a q u e o u s s o l u b i l i t y
and l o g P,
compounds
Decachlorobiphenyl,
uptake and c l e a r a n c e
parabolic, bilinear,
factor)
some h i g h l y l i p o p h i l i c
1
partition
octachlorodibenzo-p-dioxin1
authors
chemicals
s o l u b i l i ty
the condi t ions
the l i p o p h i l i c i t y
are chlorinated paraffins*,
c
fish .
with
a r e not r e a d i l y
(K =bioconcentrat ion
of
(i.e.,
well
7, c o r r e s p o n d i n g
chemicals
lipophilic
low aqueous
ingest ion of the chemical,
correlate
expressed
lipophilic
highly
an e x t r e m e l y
accumulation.
was
found
with
limits
in fish
o f brominated
for
has
been
benzenes,
more
insight
exposure
i n this
in
this
case,
to
E m p h a s i s was on d e t e r m i n a t i o n o f
and c l e a r a n c e
absorption
and
rate
constants
of the
enhanced
elimination
as
catalyzed
brominat ion
of
accumulat ion i n f i s h .
MATERIALS AND METHODS
Chemicals.
Penta-
and
hexabromobenzene
were
synthesized
by
b e n z e n e a t 370* C.
Pentachlorobenzene
was o b t a i n e d
1,2,4,5-tetrabromobenzene
Decachlorobiphenyl
was
1260). The c h e m i c a l s
higher
than
130
prepared
Aldrich,
1,2,4- a n d 1 , 3 , 5 - t r i b r o m o b e n z e n e
and p e n t a b r o m o t o l u e n e
by p e r c h l o r i n a t i o n o f a
were p u r i f i e d
determination.
P, was e s t i m a t e d
cm, 3 mm
from
Eastman
by r e c r y s t a l l i z a t i o n
from
commercial
until
White
from
Fluka,
Chemical
Corp.
PCB-mixture
(Aroclor
a gas chromatographic
purity
9 9 % was o b t a i n e d .
Hydrophobicity
matography
from
(C
1 8
from
The h y d r o p h o b i c i t y
r e t e n t i o n times
_RP_HPLC)
i . d . , stainless
using
(tr'
i
n
n-alkylbenzenes
steel,filled
with
of the test
octadecylsilica
as reference
Z o r b a x ODS
compounds,
expressed
reversed-phase
liquid
compounds. The column
(5 u ) ; m e t h a n o l - w a t e r
as l o g
chrowas
(80:20} was
15
used
as e l u e n t .
Hold-up
n-alkylbenzenes
(1980)
1 1
.
Log
constants
carbon
were
(f)
1
2
atoms
brominated
P
values
.
From
benzenes
of
50 <>
j
female
a n d 500*^
78 d a y s .
guppies
analysis.
extraction
columns
and
capture
detection
The
operation
toluene
test
kept
test
1 4
i n water
between
The c h e m i c a l
with
log t
rate
for dietary
experiment,
20.7 mg
food
was
at
and
c
Berendsen
fragmental
t h e number
3
of
of the
) .
exposure
100 g u p p i e s
has been
(Poecilia
desreti-
fed a diet
con-
(for pentachlorobenzene:
o f f o o d p e r gram
fed during
regular
by
log P values
c h e m i c a l s and were
of dry food
was
of a series of
0
hydrophobic
and a p p a r e n t
of test
of
series
the test
of
hexachlorobenzene
compounds
test
166
f i s h (wet
240 d a y s .
intervals
the (linearized)
test
compounds i n s p i k e d
76%
f o r tribromobenzene
by
1
was
The
i n samples
were
aromatic
concenof
male
gas
Dexsil
chromatography
300 GC
determined
by
isothermal
quantified
temperature
runs
at
capture
by an i n t e r n a l
3
detector
by t h i s
(*> Ni,
extraction
electron-
210"C.
as
liquid
programmed
pentabromoThe
detection
The c o n c e n t r a t i o n s o f t h e
s t a n d a r d method
a S p e c t r a - P h y s i c s 4100 c o m p u t i n g
samples
wi t h
(2%) was u s e d
by
(wet w e i g h t ) .
hydrocarbons
and N a O H - s i l i c a m i c r o
4
( < 0 . 1 ug/g) o f p e n t a b r o m o b e n z e n e ,
was c a . 1 ng/g
using
electron
the
2
compounds
measured
samples
f o r halogenated
up by H S 0 - s i l i c a
previously .
compounds
were
in fish
in injected
procedure
toluene, clean
(GC-ECD) a s d e s c r i b e d
complete
compounds
free
determined
analysis
refluxing
quant i f i c a t i o n
f o r these
r
Bruggeman e t a l . , 1982
uncontarninated
were
c
described
from
l o g P,
indices
p e r gram
feeding
(90-300"C a t 1 0 * C / m i n ) . T r a c e s
and
calculated
. For t h i s
compound
chemicals
2,2',5,5*-tetrachlorobiphenyl)
with
„
(t =t -t )
1
(see a l s o
1
time
procedure
(3 i n d i v i d u a l s ) .
included
limit
were
The a v e r a g e
the t e s t
Chemical
phase.
were
The e x p e r i m e n t a l p r o c e d u r e
per day. Subsequently,
of
linearization
relationships
i n previous papers
c a . 100 pg o f e a c h
trations
the
were d e t e r m i n e d
taining
and
by
the l i n e a r
culata,
weight)
retention
0
n-alkylbenzenes
experiment.
in detail
Mg/g) d u r i n g
( t ) and c o r r e c t e d
of the n-alkylbenzenes, r e t e n t i o n
Bioaccumulation
cribed
time
determined
integrator
( i .s.=
connected
Tracor).
The s t a n d a r d
recovery of
and c l e a n
up p r o c e d u r e
varied
from
t o 9 5 % f o r d e c a c h l o r o b i p h e n y l (mean, 8 2 % ) .
RESULTS
Hydrophobicity.
compared
with
Retention indices
l o g P values
calculated
(TT - o r f - v a l u e s ) f o r c h l o r i n e
culated
than
This
those determined
phenomenon
before
is
l o g P values,
1 3
.
practically
significance
clearly
uncommon
impossible,
in correlation
Bioaccumulation.
are
by r e v e r s e d - p h a s e
Typical
demonstrated
both
and
liquid
has
from
substitution
i n aromatic
o f the h i g h l y
measurement
l o g P values are presented
directly
and bromine
especially
i s not
Since direct
and apparent
systems.
halogenated
I t i s seen
that
1 and
the c a l -
a r o m a t i c s , a r e somewhat
higher
chromatography.
been
reported
for polychlorinated
of octanol-water p a r t i t i o n
types
i n table
or fragmental c o n s t a n t s
o f "apparent
l o g P"
coefficients
values
c a n have
biphenyls
above
l o g P-:-.5
only
relative
studies.
differences
i n the accumulation
by t h e g a s chromatograms o f f i s h
behaviour
sampled
of the test
after
50 d a y s
compounds
u p t a k e and
131
after
28
days
benzene
(i.e.,
Only
and
depuration
amounts h i g h e r
traces
benzene
2.
than
the
not
detected
had
at
substituted
decachlorobiphenyl
This
is a
trations
Clearance
rance
can
the
absorption
from
be
linear
28
days
in
(k )
M
(£)
C,(t)=
x
were
gram
amounts
of
measured,
depuration,
fish
in
wet
whereas
a l l test
been
plotted
for
male
fish.
in
male
male
mainly
guppies,
fish
the
was
fish
weight).
hexabromo-
chemicals,
from
by
against
The
slower
of
the
time
clearance
whereas
even
decrease
to d i l u t i o n
were c a l c u l a t e d
2
per
have
found
the
1,3,5-tribromo-
significant
except
the. f i s h .
guppies
regression using
the
than
in
of
the
decline
of
the
in
the
females.
decachlorobiphenyl
growth
concentrations
and
figure
rates
concen-
r e p r o d u c t i o n 1.
in fish
during
the
clea-
eqn.1,
.
and
concentrations
K
2 ug
pentabromotoluene
attributed
efficiency
the
intake: ca
higher
f
The
daily
p o s t u l a t i o n that
C,(t) = C (o) x e ~
derived
to
were
somewhat
concentrations
rate constants
p e r i o d by
curves
were
c o n f i r m a t i o n of
in guppies
accumulated
a l l . After
i n female
accumulation
benzenes
and
pentachlorobenzene,
were
been e l i m i n a t e d from
c o n c e n t r a t i o n s measured
Comparable
average
pentabromobenzene
of
was
decachlorobiphenyl,
The
(fig.1) . Decachlorobiphenyl,
1,2,4,5-tetrabromobenzene
(D
the
i n the
equilibrium
uptake
biomagnification factor
p e r i o d by
e q n s . 2 and
(K )
m
were
x d - e - M ,
C ( d
then
3,
( 2 )
2
and
_
K
C((o°)
C
where
of
f=feeding
the
test
_
f^f
k
f d
rate
(g o f
compound
i n the
food
bioaccumulation
parameters
The
most
differences
tion
(t
efficiencies.
^
(£. =
The
0.7),
rance
by
rate
a
of
only
consumed
per
gram
of
fish
per
day)
and
C
f d
=concentration
the
very
thus
obtained
between
the
are
presented
brominated
tetrabromobenzene
small
fract ions
of
is
in table
benzenes
taken
up
by
are
2.
found
the
pentabromobenzene
in their
fish
and
after
absorp-
ingestion
pentabromotoluene
absorbed.
c o n c e n t r a t i o n s of
followed
132
whereas
0.01-0.03) a r e
Most
(3)
food.
The
remarkable
'
2
s e v e r a l compounds seem
slow d e c l i n e .
constants,
which
T h i s might
i s not
be
an
accounted
to a t t a i n
indication
for
a maximum a f t e r
of
in this
a change
first
s i x weeks
i n the
order
uptake
exposure,
or
accumulation
cleamodel.
Fig.
1: Gas chromatograms
of fish
a f t e r d i e t a r y exposure and
extracts
clearance:
b r o m i n a t e d benzenes
133
Table
1.
Hydrophobicity
of
brominated
benzenes
determined
by
reversed
phase
chromatography.
P
log
compound
t (min)
N*)
r
b
c
RP-LC )
calc )
ethylbenzene
0.53
2.00
3.13
n-butylbenzene
0.83
4.00
4.19
n-hexylbenzene
1.33
6.00
5.25
n-octylbenzene
2.37
8.00
6.31
n-decylbenzene
4.45
10.00
1.2.4- t r i b r o m o b e n z e n e
0.90
4.5
4.4
4.9
1.3.5- t r i b r o m o b e n z e n e
1.20
5.6
5.0
4.9
7.37
1,2,4,5-tetrabromobenzene
1.40
6.2
5.3
5.9
pentabromobenzene
2.15
7.7
6.1
6.9
pentabromotoluene
3.35
9.1
6.9
7.4
hexabromobenzene
3.20
8.9
6.8
7.9
pentachlorobenzene
decachlorobiphenyl
N=
retention
c)
P - 2.60 +
from
(N-1) x
Log P c a l c u l a t e d
d) L o g P d e t e r m i n e d
134
c
9.6
0.27
i n d e x , o r number
b) Log P c a l c u l a t e d
Log
5.6
8.8 )
t
a)
d
d
5.6 )
o f C-atoms
hydrophobic
i n the a l k y l
fragmental constants
chain of
n-alkylbenzenes
for n-alkylbenzenes
0.53
directly
by RP-TLC
from
1
3
C l a n d Br f r a g m e n t a l c o n s t a n t s
1 2
,13
1
2
:
liquid
Table
2.
Bioaccumulation
decachlorobiphenyl
parameters
i n male and f e m a l e
compound
of
brominated
guppies
benzenes,
{Poecilia
1
m o l e c u l a r weight
pentachlorobenzene
reticulata,
k2(d"" )
£
dietary
Km
ty
2
(d)
1, 3 , 5 - t r i b r o m o b e n z e n e
315
0 26
0 3
0 08
2 7
1
394
0 16
0 7
0 3
4 3
pentabromobenzene
473
0 23
0 03
0 01
3 0
pentabromotoluene
487
0 16
0 01
0 005
hexabromobenzene
552
#2,4,5-tetrabromobenzene
>°
a
0005 )
<o
0003
pentachlorobenzene
250
0 15
0 3
0 15
decachlorobiphenyl
494
0 010
0 26
1 8
1,3,5-tr ibromobenzene
>°
4
0 22
1,2,4,5-tetrabromobenzene
pentabromobenzene
>
pentabromotoluene
>0.3
0 3
> o
2
0 8
>0
02
>0.02
h e x a b r omobe n ze ne
0 03
0 2
0 4
0 1
0 35
4 8
biomagnification
concentration
•
from
food
factor
in fish
(lipids)
concent r a t i o n i n food
(lipids)
clearance
half
2. 6
140
constant
= absorption efficiency
•
3. 2
<3
0 27
rate
2
01
0 005
= clearance
<
0 005
pentachlorobenzene
2
4 6
70
< 3
decachlorobiphenyl
k
4 3
<150
0 004
<o
and
exposure)
life
at steady state
= ln2
*2
a
)
minimum d i l u t i o n
r a t e by w e i g h t
increase
135
136
DISCUSSION
Comparison
of
the
properties
decachlorobiphenyl
pentabromotoluene
explanation
therefore
in
(tables
cannot
terms
w o u l d be
the diameter
of
configurations
is
of
indeed
than
pentabromobenzene,
this
principle,
of
the
proved
be
The
which
the
a
bromine
which
is
alternative
or
atom
(Van
required.
effective
reduced
this
the
type
of a molecule
can
pass.
diameter
Waals
radius
with
their
a
can
From
be
weight.
passage
be
1.80
As
vs.
be
would
that
as
this
chlorine
ft)
1.95
,
the
s m a l l e r than
be
An
structural
the
absorption efficiences
effiency
of
and
defined
the
derived
(fig.3).
and
absorption
t o membrane
pentabromobenzene can
der
low
or m o l e c u l a r
decachlorobiphenyl w i l l
that
(£~0.3).
expected
for
the
experiment.
rapid
transformation (e.g.,
absorption of
of
"effective
a b s o r p t i o n o f compounds s u c h
related
diameter
absorption
after
extremely
the m o l e c u l e
i n a comparative
directly
by
However,
and
i n agreement
a
be
smaller effective
hypothesis^ of
before or
disproved
and
the
lipophilicity
may
effective
through
have
size
1,2,4,5-tetrabromobenzene
with
that
to high
molecular
1,2,3,5-tetrabromobenzene
covalent binding
would
must
interpretation,
In
be
clear
directly
effective
hole
makes
1,2,4,5-tetrabromobenzene
or
not
2)
diameters of pentachlorobenzene
Following
isomeric
and
more s a t i s f y i n g .
of
smaller
effective
pentabromobenzene
related
the s m a l l e s t
tetrabromobenzene
atom
1
be
of
of
highly
experimental
diameter"
procedure;
approach
debromination)
brominated
might
benzenes
radiotracer
also
can-
studies
explain
the
lack
1
as o c t a c h l o r o d i b e n z o - p - d i o x i n a n d p o l y d i m e t h y l -
siloxanes**.
REFERENCES
1.
W.A.
Bruggeman,
super-lipophilic
chapter
H.
Konemann and
3.
K.
S u g i u r a , N.
Chemosphere,
4.
V.
Zitko,
5.
V.
Zitko
6.
V.
Zitko,
V.
Zitko,
8.
9.
chemicals
K.
van
I t o , N.
Bull.
and
Bull.
and
0.
Hutzinger,
E n v i r o n . Chem.
L e e u w e n , C h e m o s p h e r e , 9,
3-19
Murata,
Bioaccumulation
submitted
(this
of
thesis,
i n : Pesticide
and
(Eds),
J . J . Menn
Bruggeman,
D.
Absorption
preliminary
experiments,
M.Th.M. T u l p and
A.
S p a c i e and
Berendsen
12.
R.F.
Rekker,
13.
W.A.
and
12,
406-412
Toxicol.
Y.
T s u k a k o s h i , and
van
der
thesis, chapter
Bruggeman,
L.B.J.M.
(this
H .
Goto,
(1974).
Opperhuizen,
of
16,
van
der
T_, 849-860
Chromatogr.,
Chem.,
3,
O.
Khan,
Steen,
A.
Wijbenga
(this
thesis,
and
in
chapter
0.
fish:
5).
(1978).
1,
309-320
1669-1686
fragmental constant, E l s e v i e r ,
and
M.A.Q.
177-182.
polydimethylsiloxanes (silicones)
Environ. Toxicol.
Steen
(1976).
i n a q u a t i c organisms,
(1979), pp.
J.
665-673
(1977).
E n v i r o n . Chem., s u b m i t t e d
Hutzinger,
J.
(1982).
(1980).
Amsterdam,
1979.
Chromatogr.,
238,
335-346
3).
Martron,
thesis,
248-292
metabolism
H u t z i n g e r , Chemosphere,
hydrophobic
J.
17,
Symp. S e r . , 99
A.
et a l . , J . L i q u i d
The
(1981)
ACS
retention
J . L . Hamelink,
Bruggeman,
(this
O.
xenobiotic
Weber-Fung,
Hutzinger,
G.E.
(1980).
( 1978).
and
10.
M i h a r a , K.
E n v i r o n . Contam. T o x i c o l . ,
W.A.'
W.A.
wijbenga
Toxicol.
H u t z i n g e r , B u l l . E n v i r o n . Contain. T o x i c o l . ,
Lech
811-832
fish,
E n v i r o n . Contain. T o x i c o l . ,
0.
J.J.
(1982)
in
A.
M a t s u m o t o , Y.
T_, 731-736
11.
14.
Opperhuizen,
4).
2.
7.
A.
chapter
0.
Kooiman
and
O.
Hutzinger,
Chemosphere,
10,
2).
137
CHAPTER
7
INFLUENCE
OF
THE CHLORINE
CHLOROBIPHENYLS
DIETARY
ON T H E I R
SUBSTITUTION
PATTERN
ACCUMULATION IN
FISH
IN
TETRA-
AFTER
EXPOSURE
139
I N F L U E N C E OF
ON
THE
CHLORINE SUBSTITUTION
THEIR ACCUMULATION
W.A.
Laboratory
of
IN
Bruggeman*, G.J.
Environmental
and
Nieuwe A c h t e r g r a c h t
PATTERN
Peeters
Toxicological
166,
IN
TETRACHLOROBIPHENYLS
F I S H A F T E R DIETARY
1018
WV
and
O.
EXPOSURE
Hutzinger
Chemistry,
University
AMSTERDAM, The
of
Amsterdam,
Netherlands
ABSTRACT
Accumulation
lata)
lumn
simultaneously
gaschromatography.
s e v e r a l weeks,
latively
the
kinetics
small
partly
reduced
and
be
was
related
not
a b s o r p t i o n or
and
separated
and
3,3'-
quantified
and
combined
with
of
any
significant
excretion of
steric
these
isomers
extent.
factors
by
ad-
capillary
co-
isomers
were
The
might
reticuwere
clearance
most
2,3,4,5-tetrachlorobiphenyl
to
(Poecilia
Eight
individually
bioaccumulation
h y d r o p h o b i c i t y , but
active
experiment.
efficiencies,
accumulated
the
clearance
considerable
2,2',
to
t e t r a c h l o r o b i p h e n y l s i n guppies
absorption
in a
amounts o f
of
exposure
were
High
resulted
2,2',6,6'-isomer
only
a
elimination
were s t u d i e d i n a d i e t a r y
ministered
of
and
half
lifes
in fish.
found,
differences
be
Re-
whereas
could
responsible for
isomers.
INTRODUCTION
Polychlorinated
biphenyls
bioaccumulation
i n the
vious
High
studies
the
coefficient
substitution
chlorinated
therefore
in
an
Bioaccumulation,
however
organisms.
tion
i n the
cells,
processes
ral
factors
* present
The
{P)
like
may
be
address:
or
in
fish
is
by
from
passage
the
not
active
through
i n v o l v e d which
of
proceed
the
water
a
in
both
appears
lower
persistence
water
and
in
via
resulted
and
pre-
food.
from
ef-
2
to
be
measured
increasing
the
via
systems
1
be
their
been d e m o n s t r a t e d
narrowly
by
3
5
(S) ,*, .
the
related
Increasing
lipophilicity;
chlorinated
to
octanol-water
chlorine
thus,
ones,
higher
and
would
lipids.
simple
factors
can
than
i n body
for
chemicals. ,
solubility
results
levels
known
I t has
in experimental
which
lipophilic
Complicating
can
xenobiotic chemicals
system
more
are
organisms.
factors
elimination
or
to higher
living
environment
living
hydrophobicity,
biphenylsare
with
In
slow
aromatic
accumulate
of
magnificat ion
very
or
the
tissues
bioaccumulat ion of
lipophilicity
partition
in
accumulation
and
a b s o r p t i o n and
general,
their
fatty
their
bioconcentrat ion
ficient
In
that
(PCB's)
may
phase
arise
distribution
from
process,
biological
but
barriers
has
to
to
do
absorp-
e x c r e t i o n mechanisms.
biomembranes or
cannot
be
biotransformation
accounted
f o r by
G o v e r n m . I n s t . W a s t e Water T r e a t m .
(RIZA),
(metabolism),
lipophilicity
P.O.B. 17,
structu-
alone.
8200 A A
Lelystad,
Netherlands
141
Several
polar
types
of
lipophilic
metabolites
which
reduced
accumulation
seem
prevent
to
of
the
chemicals
are
are
rapidly
specific
absorption
DDT
of
not
accumulated
excreted
was
analogs^,
some
whereas
other
by
shown
types
living
to
be
spatial
of
fish.
the
The
main
dimensions
compounds
(e.g.,
formation
reason
for
(molecular
of
the
size)
hexabromobenzene,
octachlorodibenzo-p-dioxin)*„'„8.
Accumulation
(1980)
9
isomers
of
differences
have
in estuarine
differential
chlorine
the
i n the
present
sorption
and
pretation,
may
fish
the
and
pattern
was
examined
only
symmetrical
v i a the
or
steric
thought
to
influence
the
area
the
of
of
the
presence
of
preparation.
Combined
purity
of
the
other
Sixty
free
and
were
food.
The
average
per
gram
pound
ca.
test
per
32
mixture
study
dry
was
the
nation
dure
chemicals,
gram
mg
of
food
stopped,
clearance
of
the
Concentrations
tion
and
Chemical
toluene
trated
ture
GC
142
the
analysis.
of
of
The
mainly
via
fish.
isomers
were
To
pattern
simplify
chosen
and
on
the
ab-
inter-
administered
test
impurity
guppies
fish
amount
fish
of
(wet
compounds
higher
their
than
97%.
containing ca.
food
consumed
were
per
76
an
sampled
1
day
days,
of
(the
the
regular
r e t a i n e d . Experimental
were
3 ug
additional
at
are
showed
1
reticulata)
After
structures
investigation
3,3 ,5,5'-tetrachlorobiphenyl
food
fed d u r i n g
Fish
Analabs;
i n the
(Poecilia
weight).
was
from
placed
each
feeding
dosing
p e r i o d of
of
the
106
for
this
water
com-
rate)
of
intervals
details
in
test
was
test
days
to
determi-
test
proce-
2
i n previous p u b l i c a t i o n s , .
chemicals
in fish
were
i n t e r p r e t e d i n terms of
fish
samples
first
order
accumula-
each)
with
refluxing
1
kinetics .
Extraction
of
the
c o m p o u n d s was
the
H S 0 - s i l i c a and
2
by
better
(3
NaOH-silica
4
than
capillary
98%.
individuals
was
performed
Concentrations
column
gas
as
described
in purified
chromatography
with
and
2
before .
concen-
electron-cap-
(GC-ECD).
system: Packard-Becker
column,
efficiency
- mass s p e c t r o m e t r i c
c o m p o u n d s was
chemicals.
e x t r a c t s were d e t e r m i n e d
detection
possibility
coefficient.
chlorine substitution
obtained
an
fed dry
with
test
as
male
clean-up
SE-30; c a r r i e r
C;
of
the
effect
METHODS
were
uncontaminated
the
described
elimination
and
Recovery
and
of
amounts o f
have been
the
chromatographic
Bioaccumulat ion exper iment.
of
Connel
molecule.
of
substituted
isomers
gas
a pentachlorobiphenyl
The
excluded
uptake
and
hexachlorobiphenyl
food.
Tetrachlorobiphenyl
i n f i g 1.
presented
They
steric
tetrachlorobiphenyls in
one-ring
Shaw
different
factors.
a
effects
molecules.
of
developed
MATERIALS AND
Chemicals.
isomeric
and
the
elimination kinetics
simultaneously
between
concentrations
excretion
sectional
we
exist
relative
were i n f l u e n c e d by
cross
study
even
that
metabolism
substitution
differences
In
concluded
gass,
N ,
2
428
with
pressure
i s o t h e r m a l , 240"
C;
63
N i
1.75
E
C
D
.
ato;
d e t e c t o r , 300"
column,glass
split
C.
46 m,0.2
injection;
mm
i.d.,WCOT,0.5 u
temperatures,
injector,
250"
Hexachlorobenzene
quantification
Spectra-Physics
Fig.
was
added
of the t e s t
4100
to each
compounds
computing
sample
as
an
via relative
internal
retention
standard
times
f o r i d e n t i f i c a t i o n and
and
peak
areas,
using
a
integrator.
2: C a p i l l a r y column gaschromatograms (ECD) o f f i s h
a f t e r d i e t a r y e x p o s u r e and c l e a r a n c e :
tetrachlorobiphenyls
extracts
143
RESULTS
Typical
capillary
tracts
res
from
are
min)
and
the
the
extremely
i n the
the
fish
rapid
Clearance
rate
factors
fish
samples.
(drawn
Table
rate
its
high
per
in
rate
in fish
extractable
and
the
which
was
6-10
times
2
0.2
d"');
the
and
from
as
lipid
the
based
are
lower
impression
than
effect
r
biomagnificafound
fish
might
a
the
the
in fig
of
are
relatively
most
other
by
high
clearance
attributed
enhanced
ra-
calculated
i t s apparent
are
be
was
of
by
those
in
in
plotted
pentachlorobiphenyl,
i s confirmed
higher
(t =9,25
r
time.
distinctly
first
peak
ex-
featu-
( t = 1 4 - 1 3 mill)
concentrations
vs.
fish
Remarkable
min).
r
in fish
well
The
(t =12.99
(£)
2,2',6,6'-tetrachlorobiphenyl
<k J
purified
2,3,4,5-isomer
e l i m i n a t i o n curves
(£=0.5-0.7);
2,3,4,5-isomer.
and
f i g . 2.
isomers.
mainly
a
to
reduced
(C
f i S S
)
to
the
(K
m
in table
1)
concentrations
relate
i n the
the
theoretical
contaminated
steady
food
(C
per
gram
f d
state
,eqn.1),
lipids.
2
•= f e e d i n g
rate
( in
this
experiment
f •= 0.032 gram
of
dry
food
of
fish
day).
accordance
Since
fish
accumulation
biomagnification factors
with
biomagnification
the
efficiencies
ingestion
by
efficiency.
k
f
the
of
of
were c a l c u l a t e d
actual concentrations
after
1
Cfd
where
1,
mixture
shown
2,2'6,6'-tetrachlorobiphenyl
uptake
t e t r a c h l o r o b i p h e n y l s as
2
resulting
on
the
2,2'3,3'-tetrachloroniphenyl
amounts
concentrations
based
order
with
of
standard
are
2,2',3,3'-tetrachlorobiphenyl
first
(k =0.13 d" )
clearance
the
slow
2
most
for
of
relatively
of
periods
( k ) , adsorption
absorbed
rate
small
absorption
The
that
constant
very
concentration
together
efficiency
elimination
clearance
presented' i n t a b l e
Simulated
efficiently
The
low
constants
lines)
absorption
chromatograms
and
e x t r a c t s , the
(Km),
1 shows
ther
gas
e l i m i n a t i o n of
tion
1
column
accumulation
the
daily
fluctuating
do
not
its
estimated
lipophilicity,
pentachlorobiphenyl
snowed
the
highest
factor.
intake
of
each
test
concentrations of
represent
a real
c o m p o u n d was
ca.
0.1
ug
per
2,2',6,6'-tetrachlorobiphenyl
accumulation
of
this
gram o f
fish
(wet
( 0 . 0 2 - 0 . 1 2 ng/g)
weight)
in
the
compound.
145
DISCUSSION
The
use
of
hydrophobic
octanol-water)
has
only
minor
positioning
shown
of
of
that
the
of
influence.
Actually,
in aliphatic
substitution
bond
the
in
hydrophobicity
retention
rinated
b i p h e n y l s , which
A
of
reduced
of
the
the
revealed
biphenyl
lipophilicity
accordance
with
The
for the
reason
chlorine
and
atoms
9
Connel
clearance
a
much
,
of
the
At
moment,
cal
factors
cal
from
sistent
146
may
their
can
only
log p
low
may
The
Recent
been
have
be
be
complicate
compounds such
found
are
needed
i n the p o s i t i o n
than
i n other
p
for
ortho
the
have
to
the
positions!1.
Es-
liquid
resulting
log
groups
investigations
chromatography
o r t h o - s u b s t i t u t e d and
effects
dipole
other
chlo-
in a distortion
the
the
insignificant
of
the
accumulation
biomagnification
factors
2,3,5,6-tettachlorobiphenyl
be
identified
of
this
has
for
and
a
long
may
be
adjacent
might
as
molecule.
is
shown
be
factors
to a s t e r i c a l l y
possibility
free
Biotransformation
in steric
related
information
The
2,3,4,5-tetrachlorobiphenyl
moment
underestimated
been
explain
and
either
remaining
are
of
in
an
that
ortho,
The
the
hindered
and
also
play
a
role
by
of
para
in
or
capacities
metabolites
favorable for metabolic
fast
elimination
quantitative
meta
Shawn
absorption
metabolic
polar
of
adjacent
by
relatively
accelerated
time:
even
(four
suggested
of
lower
significance
positions
epoxidation
the
in
and
excretion
of
2,2',6,6'-tetrachlorobiphenyl.
concluded
reliable
s t r u c t u r e or
as
terms
Recent
(as
substituent
values.
cannot
process^.
especially
chemical
the
reverse-phase
between
a b s o r p t i o n of
increased
reactions.
i t must
with
lipophilicity
of
1".
smaller
partly
2 , 2 ' , 3 , 3 ' - t e t r a c h l o r o b i p h e n y l can
isomers,
correction
between
to s t e r i c
3,3',5,5',
processes.
hydroxylation
of
molecules
correlation
3,3',4,4'-,
lipophilicity.
others
position
for chlorine
differences
differences
the
have
-value)
small
ring)
biphenyls
of
itself
The
estimated
bioaccumulation
rings
the
in
(TT
attributed
relatively
one
types
aromatic
substantially
via
2,2',3,3'-isomer
organisms
chlorinated
for
in
the
lower
aquatic
their
or
transformation
both
in
2,2'.5.5'-,
several
and
the
the
nucleus.
2,2',6,6'-isomer.
2,2',4,4',
calculating
that
be
dramatic
was
for
suggests
constant
PCB's m i g h t
(RP-LC)
planarity
constants
compounds
halogens
phenyl-phenyl
timation
fragmental
organic
PCB's.
that
several structural,
prediction
of
the
physico-chemical
physico-chemical
accumulation
or
of environmental
p r o p e r t i e s , even
biologichemi-
for r e l a t i v e l y
per-
REFERENCES
1.
W.A.
Bruggeman,
811-832
2.
W.A.
(1981)
L.B.J.M.
Martron,
(this thesis,
Bruggeman,
super-lipophilic
A.
Opperhuizen,
chemicals
D.
Kooiman
and
o.
Hutzinger,
Chemosphere,
10,
chapter 2).
A.
in fish,
Wijbenga
Toxicol.
and
O.
Environ.
Hutzinger,
Chem.,
Bioaccumulation
submitted
(this
of
thesis,
chapter 4 ) .
3.
W.B.
Neely,
D.R.
4.
CT.
Chiou,
V.H. F r e e d ,
475-478
5.
G.D.
Branson
a n d G.E. B l a u ,
D.W.
Schmedding
Environ. S c i . Technol.,
and R.L. K o h n e r t ,
13, 1 1 1 3-1 1 1 5
Environ.
(1974)
S c i . Technol.,
11,
(1977)
Veith,
D.L.
Defoe
and
B.v.
Bergstedt,
J . Fish.Res.Board
C a n . , 36,
1040-1 048
(1979)
6.
I.P. Kapoor,
21 , 310-31 5
7.
v.
8.
W.A.
Zitko
R.L. M e t c a l f , A . S . H i r w e , J . R . C o a t s
a n d 0. H u t z i n g e r ,
Bruggeman,
elimination
and M.S.
G.J.
of
B u l l . E n v i r o n . Contam. T o x i c o l . ,
Peeters,
brominated
J.
van
der
benzenes,
0.
pentabromotoluene,
by f i s h
G.R.
C o n n e l l , C h e m o s p h e r e , 9, 7 3 1 - 7 4 3 ( 1 9 8 0
10.
R.F. R e k k e r , The h y d r o p h o b i c
11.
W.A.
12.
W.A.
of
thesis,
and
decachlorobiphenyl
Shaw and D.W.
(this
Steen
9.
(this
fragmental
Bruggeman, J . v a n d e r S t e e n
thesis,
Khalsa,
J . Agr•
Food
Chem.,
(1973)
16, 665-673
Hutzinger,
(1976)
Absorption
pentachlorobenzene
and
and
chapter 6).
constant,
and 0 . H u t z i n g e r ,
Elsevier,
A m s t e r d a m , 1977
J . Chromatogr.,
238, 335-346
(1982)
chapter 3).
Bruggeman,
W.A.
Dijkhuizen
dichlorobiphenyls in fish
and 0.
(this
Hutzinger,
thesis,
Bioaccumulation
and t r a n s f o r m a t i o n
chapter 8 ) .
147
CHAPTER
8
BIOACCUMULATION
IN
AND
TRANSFORMATION
OF
DICHLOROBIPHENYLS
FISH
149
BIOACCUMULATION AND
W.A.
Laboratory
Bruggeman*,
of Environmental
Nieuwe
TRANSFORMATION OF DICHLOROBIPHENYLS
W.A.
D i j k h u i z e n and 0.
and T o x i c o l o g i c a l
Achtergracht
1 6 6 , 1018 WV
IN
FISH
Hutzinger
Chemistry,
University
AMSTERDAM, The
of
Amsterdam,
Netherlands
ABSTRACT
Accumulation
died
and
elimination
i n aqueous exposure
vestigation
of
fish
detection
(GC-ECD)
0.1
of
-
3%
metabolites
(methyl
the
and
ester)
concluded
that
of
2,5-dichlorobiphenyl
and c l e a r a n c e e x p e r i m e n t s
and
water
and combined
samples
their
conjugates.
found
gas
as
In
a
transformat ion
body
burden
of
significant,
but i s o f minor
Absorpton
xenobiotic chemicals
- mass
per
day
addit ion, small
transformat ion
(Poecilia
in
of
of
were
(GC-MS) showed
into
In-
capture
that
hydroxylated
2,5-dichlorobenzoic
2,5-dichlorobiphenyl.
the
stu-
reticulata).
electron
converted
amounts
i n regulating
with
spectrometry
was
product
dichlorobiphenyls
importance
4,4' - d i c h l o r o b i p h e n y l
guppies
chromatography
gas chromatography
dichlorobiphenyl
were
by
and
with
aquat i c
environment
the b i o c o n c e n t r a t i o n i n
acid
It i s
may
be
fish.
INTRODUCTION
ficient
In
elimination
fish,
from
mechanisms, w i l l
uptake can proceed
food.
In t h e s i m p l e s t
in fish
i s determined
nated
tion
of
directly
case,
by
the environment
result
from
by
living
organisms,
without
ef-
in bioaccumulation.
water
o r by
f o r many o r g a n i c
ingestion
of chemicals
chemicals,
the b i o c o n c e n t r a t i o n
factor,
the steady
K ,
and
c
with
state
contami-
concentra-
the c o n c e n t r a t i o n
in
water,
K
where
and
k
Cf
2
c
= El.
Cw
•
at equilibrium,
*2
= concentrat ion
• clearance
coefficient
and
in fish,
rate constant.
i s equal
C^
The
an e x p r e s s i o n o f the l i p o p h i l i c i t y
in
fish
lipids.
concentration
*
The r e l a t i o n s h i p
(e.g., P octanol-water)
Present
Lelystad,
o f new
compounds
address:
The
of
Governm.
the uptake
of the chemical
k-| =
has
and c l e a r a n c e
i f Cf
obvious
rate
i s given
a n d c a n be u s e d
uptake
the form
between the b i o c o n c e n t r a t i o n f a c t o r
i s then
3
i n water,
bioconcentration factor
to the r a t i o
is
ficient
• concentrat ion
as
rate
of a
constant
par t i t i o n
constants
1
2
, .
K
c
the c o n c e n t r a t i o n
and a p a r t i t i o n
for prediction
coef-
of the
bio-
4
, .
Inst.
Waste
Water
Treatm.
(RIZA),
P.O.B.
17,
8200
AA
Netherlands.
151
The
situation
is
concentrations,
mote
high
sical
food
Other
complications
hanced
in
a
chain
accumulation
of
Polychlorinated
the
demonstrated
To
biphenyls
that
depending
a
large
the
e l i m i n a t i o n of
lower
of
metabolites
PBCs
4,4'-dichlorobiphenyl.
pronounced.
fish
are
indeed
transformation
have
been
of
goldfish
In
by
study
mination
not
lower
of
hydroxylated
of
could
clear
than
in
and
and
a
porous,
and
determined
inorganic
support
by
the
PCBs
persistence
i t has
of
PCB
in
been
conge-
lipophilicity
a l . ' have
rats
of
the
did contribute
in
I
are
fish
n
of
to
chemicals
we
acid
after
the
purpose
benzoic
1 0
laboratory
study ,
is
p- 450)
inducible
2
variety
forcefed
(cytochrome
aquatic
previous
a
a
were
xenobiotic
readily
and
reported
which
activities
but
are
.
in
Polar
systems
reported
the
exposure
of
of
and
kinetics
in
metabolization
were
exposed
to
aquarium
water
45-60 mesh)
in
the
pre-
the
eli-
of
solutions
high
of
purity
aquaria.
with
a
aqueous
(2,5-DCB)
GC-MS) i n g l a s s c o v e r e d
G-AW,
of
for
fish.
2,5-dichlorobiphenyl
equilibrating
(Chromosorb
I t was
of
METHODS
reticulata)
and
scarce.
importance
bioaccumulation
GC
en-
the
chemical
fluidized
bed
coated
(see
on
a
refs.
2
5).
After
8-10
days
for
r e l e a s e and
was
followed
served
152
by
from
resulting
tetrachlorobiphenyls.
(poecilia
capillary
as
or
papers
behaviour
the
of
chlorinated
relative
s o l u t i o n s were p r e p a r e d
et
oxidase
11-13,
of
(>99%
to
urine
biphenyl
biotransformation
(4,4'-DCB)
related
mammals,
impression
guppies
,
both
stability,
in previous
transformation
on
Female
7
far biotransformation
the
an
the
6
molecules
(metabolism) ,
chemical
unknown. T u l p
in
investigators
4,4'-dichlorobiphenyl
The
be
organisms,
of
bulky
bioaccumulation
i n how
MATERIALS AND
Exposure.
clas-
2
chlorinated
of
aqueous
the
biphenyls ,8.
metabolites
part
low
favour
chlorine substitutions.
data
PCBs, as
their
mixed-function
di-,tri-
of
processes
the
the
t o get
of
extremely
rates
factor.
for
in
of
excreted
that
several
to a mixture
Quantitative
known
position
aquatic
appears
products
absorption
i n mammals i s n o t
much
found
formation
sent
It
chemicals:
neglible clearance
p r o p e r t i e s . However,
chlorinated
Biotransformation
less
are
i t was
of
hydroxylated
reduced
differences
now
and
transformation
differences exist
these
until
super-lipophilic
bioconcentration
(PCBs)
number and
extent,
compounds5, but
a
bioaccumulating
marked
on
from
biochemical
apparent
and
for
coefficients
(biomagnification)5.
arise
by
the
environment,
ners,
may
elimination
reduction
complicated
adsorption
by
t o remove
uptake
and
equilibration
re-equilibration
a
16
the
days
apolar
during
clearance
chemicals
with
10-38
water,
days.
fish
In
the
period: a c t i v e carbon
from
the
water
phase.
were
t r a n s f e r r e d to clean
2,5-DCB e x p e r i m e n t ,
filtration
and
this
strong
water
stage
aeration
To
prevent
but
excessive
a small
mg/l.
stream
The water
Chemical
with
ducts
of
Concentrate
ml
sulfuric
Stirr
volume
Adsorption
with
ca.
extract
elute
mainly
two f r a c t i o n s .
Hewlett
Packard
"Ultrabond"
8"C/min.
spectra
Methyl
determined
by gas c h r o m a t o -
5
combined
gas
fish
pro-
chromatography-mass
and water
samples,
after
h y d r o l y s i s and e x t r a c t i o n :
(1/40) a n d 2 0 - 4 0 % d i s t i l l e d
4
minutes.
Separate
layers
Methylation:
carbonate,
(to c a .
e . i . mode
stirr
by
toluene
centrifugation.
a d d 100 m l o f a c e t o n e ,
and r e f l u x
for ca.
5 hrs.
0.5 m l ) a n d a d d i t i o n o f d i s t i l l e d
ethers
column, a c t i v a t e d s i l i c a ,
i n n-hexane
of potential
fraction,
whereas
column
were p r e p a r e d
20
5
Trans-
n-hexane,
ethers)
are given
(SE-30,
8
1.8 m,
injected
2 mm
by T u l p
9
et a l . ,
i f better
a
0
i . d . , packed
compounds and s y n t h e t i c
with
100-240*C a t
with
mass
o f p o t e n t i a l PCB
. PCBs, as w e l l a s t h e i r
5
1 m packed
separation
i n the
on a
by c o m p a r i s o n
1
frac-
and c a r b o x y l i c
programme,
identified
5
PCB's a r e found
were
GC-MS c h a r a c t e r i s t i c s
b y GC-ECD, u s i n g
WCOT )
from p a r e n t
the parent
elution
to give
(phenolic
temperature
were
of synthetic standards.
stepwise
by volume)
fractions
column,
M),
a t 70 e v . M e t a b o l i t e s
were q u a n t i f i e d
(0-60%
metabolites
GC c o n d i t i o n s : g l a s s
c a . 0.2% C a r b o w a x
methyl
products,
capillary
o f acetone
i n the third
5982 A s y s t e m .
(as t h e i r
transformation
standards
2
evaporation.
evaporation
and r e t e n t i o n times
metabolites
glass
1 N H S0
GC-MS: 1-3 j i l o f t h e c o n c e n t r a t e d
(Chrompack;
MS:
o f 9-10
1ml.
o f c a . 1 ml e a c h .
first
was n o t a e r a t e d ,
concentration
chromatography.
chromatography: p a s t e u r - p i p e t t e s i z e
compounds)
water
e x t r a c t s . Transformation
wet w e i g h t ) , 1-3 g . A c i d
f o r 100
b y vacuum
increasing concentrations
tions
adsorption
to obtain ca.
reflux
by
extracts of hydrolyzed
and 3 g o f p o t a s s i u m
t o h e x a n e by r e p e a t e d
final
identified
(homogenized
acid
and
toluene
of methyliodide
fer
periods,
an oxygen
(GC-ECD) o f t o l u e n e
were
by si<lica
2.5 L ) f i s h
volume).
to maintain
o f 2,5-DCB a n d 4,4-DCB were
(GC-MS) o f m e t h y l a t e d
up and f r a c t i o n a t i o n
concentrated
(by
Concentrations
the dichlorobiphenyls
Sample: water,
add
the e q u i l i b r a t i o n
electron-capture detection
spectrometry
clean
during
g a s was a p p l i e d
t e m p e r a t u r e was 19.5 ± 1"C.
analysis.
graphy
evaporation
o f oxygen
column ,
was
o r a 46 m
required.
(methylated)
External
metabolites.
RESULTS
4,4' - D i c h l o r o b i p h e n y l .
Aquarium water,
After
i n 23 1 o f a q u a r i u m
placing
creased
at
22 f i s h
t o 6 wg/1 w i t h i n e i g h t d a y s
t h e same
time
of intoxication
clean
water
and
was f o u n d
o n wet f i s h
re-equilibration.
this
a n d was r e m o v e d . T e n d a y s
i n water. A f t e r
were
found.
38 d a y s
From
uptake
after
i n fish
figures,
i n fish
for
31 | i g / l .
one f i s h
taken
showed
o f t h e 16 r e m a i n i n g
had d r o p p e d
fish to
t o 60 Mg/g, w h e r e a s 2.2
concentrations
o f 15 u g / g f i s h
a bioconcentration factor
4,4'-DCB, d e t e r m i n e d
were c a .
i n water d e -
i n a sample o f 5 f i s h
period only
transfer
re-equilibration,
these
w e i g h t ) c a n be c a l c u l a t e d
Extractable lipids
4,4'-DCB, c o n t a i n e d
t h e 4,4'-DCB c o n c e n t r a t i o n
( f i g 1 ) . The c o n c e n t r a t i o n
(20 1 ) , t h e 4,4'-DCB c o n c e n t r a t i o n
0.9 pg/1 w a t e r
(based
water,
was 80 n g / g w e t w e i g h t . D u r i n g
signs
,ig/l
saturated with
o f c a 15x103
by e q u i l i b r a t i o n and
3.5% o f t h e wet w e i g h t .
153
Fig.
1
1: 4 , 4 - d i c h l o r o b i p h e n y l uptake i n f i s h , r e - e q u i l i b r a t i o n
and
production of metabolites
Hydroxylated
in
fish
main
metabolites
and
water
product
of
was
and
from
(3.3
pg
the
the
total
ratio
fish
and
means
the
a
total
mass
solution
(average
154
life
During
conversion
complete
patent
the
the
kinetics,
recovery
particulate
of
of
half
water).
net
end
methyl
of
the
of
balance
compound and
also
two
the
for
46
last
the
DCB
imply
0.2%
total
of
the
compound
day)
might
m a t e r i a l , micro-organisms
the
transformation product
fish.
body
the
faeces.
fish
by
The
product
of
after
of
first
of
2 pg/1,
day.
was
given.
evaporation
0.015
compound
only
per
than
Loss
from
10:1.
20 a n d
of
order
d
- 1
and
between
which
since
50%,
a
aqueous
adsorption
concentration ratio
between
ten
15%
However,
less
or
fish
The
i s ca
(k2*)
The
3,4'-di-
this
case
s a m p l e s was
both
1).
approximately
I was
be
a
pg/1).
parent
burden
cannot
explained
I i n the
In
rate constant
of
is
of
sampled
(2.4
formed;
i t s metabolites
period
and
fish
was
I was
i n c r e a s e of
DCB
plus
be
(I/II)
of
GC-MS
i n a l l samples of
this period
exchange
the
in
by
(table
amounts
compound
highest
ug
a metabolic
re-equilibration
per
36
period
small
latter
of
in
(without
28 d a y s ,
but
products
ca.
identified
were d e t e c t e d
were
end
present
days
than
parent
10%
The
but
the
transformation
would
of
less
the
at
re-equilibration,
this
of
(I),
found.
period,
i n water
parent
t y'
rate ca
organic
were
e t h e r s ) were
re-equilibration
transformation products
g r a m ) , and
10 d a y s o f
amount
biological
(as t h e i r
the
re-equilibration
I per
first
transformation
a
the
of
concentration
During
(II)
9
" N I H - s h i f t " . These
water
at
4,4'-dichloro-3-methoxybiphenyl
chloro-4-methoxybiphenyl
metabolic
days
4,4'-DCB
e x t r a c t s taken
on
between
50.
2,5-Dichlorobiphenyl.
The
The s a t u r a t i o n c o n c e n t r a t i o n o f 2,5-DCB was c a . 500 p g / 1 .
steady-state concentration
weight,
and i n water
3
15x10 .
low
i n fish,
measured
after
10 d a y s e x p o s u r e , was 690 u g / g wet
45 y g / 1 ( f i g 2 ) ; t h e c o n c e n t r a t i o n
Ca 40% o f t h e f i s h
died
during
this
exposure
factor,
based
o n wet w e i g h t , was
period, the other
fish
showed
a very
was o b s e r v e d
and a
activity.
After
transfer
o f the remaining
new d i s t r i b u t i o n
tion
fish
to clean
e q u i l i b r i u m o f the chemical
o f 2,5-DCB i n f i s h
water,
a fast
was a p p r o a c h e d .
was 430 u g / g a n d i n w a t e r
recovery
After
27 u g / 1 ; a g a i n ,
7 days,
the c o n c e n t r a -
the concentration
ration
3
was c a 1 5 x 1 0 .
The
clearance
0.08 d
-
1
rate constant
forfish
GC-MS a n a l y s i s showed
and
water
after
formed
In
VIII
tion
time
type
of
was i d e n t i c a l
compounds
biphenyls
1 4
,
1 5
.
Formation
concentrations
pounds
these
body
o f the main
burden
i n water
per day ( k ' =
2
i n fish
experiment.
The t o t a l
this
loss
experiment
was
bacter i a l
product
in f i g
after
0,01 d ~
1
) . This
i n relation
o f 2,5-DCB
loss
2,5-DCB
aqueous
into
of
fish
fairly
a
mixture
III-V
and f i s h
a s measured
stage
o f com-
t e n days o f exposure.
was c a 1% o f t h e f i s h
by t h e c o n c e n t r a t i o n s
i n fish
from
of
per
day
by
was c a 4 0 % . A b o u t
processes
these
the r e - e q u i l i b r a t i o n
27% was
i n w a t e r . T h e d i f f e r e n c e c a n be e x p r e s s e d
solut ion
of
2
before .
The c o n c e n t r a t i o n s o f
the f i r s t
compounds
in this
to
This
chlorinated
o f the c o n c e n t r a t i o n s
constant.
was c o n f i r m e d
o f 2,5-DCB.
lower
reported
( I I I ) i n water
ratio
t o 2,5-DCB
by f i s h
a s 2,5-DCB a n d i t s m e t a b o l i t e s
of
identi-
a s shown by GC r e t e n -
product
2,5-DCB) h a s b e e n
product
o f 2,5-DCB.
2 8 2 , M-15, M-43) Com-
ester)
exposure
2 a r e probably
ring
was t e n t a t i v e l y
M =
fission
increase during
o f 2,5-DCB
phenyl
transformation
. The mean
a linear
rate
(methyl
2,5-dichloro-4•-
i n table
+
fragments:
acid
o f 2,5-DCB i n f i s h
product,
V I I , which
as a r i n g
transformation
showed
and w a t e r
from
mass
containing
recovered
of
compound
c a 7:2:1 a n d r e m a i n e d
transformation
metabolites
from
products
2 ) . The main
i n the unsubstituted
(important
(also
GC-ECD, a r e p l o t t e d
metabolites
apparent
sites
o f a product,
known
of
was
depuration
(mono a n d d i m e t h o x y )
It i s considered
i n water
I I I , IV and V
transformation
t o 2,5-dichlorobenzoic
i s also
biphenyls
capillary
The
found
a n d mass s p e c t r u m .
chlorinated
The
of different
were
the subsequent
(see t a b l e
IV-VI
as a dichlorodimethoxybiphenyl
pound
by
( I I I ) and compounds
addition, traces
fied
of several
equilibration
v i ahydroxylation
from
3.5% l i p i d s .
the presence
10 d a y s
methoxybiphenyl
(R2> d e t e r m i n e d
containing
like
a s 6%
evaporation
and
adsorption.
O C H
Table
1
1: 4 , 4 - d i c h l o r o b i p h e n y l and
its
transformation
products
3
II
ci
155
156
DISCUSSION
The
aqueous
turation
al,
solubilities
method
1 6
1980
.
explained
compare
o f t h e d i c h l o r o b i p h e n y l s a s measured
well
The r e l a t i v e l y
by
i t shigher
factors
studies, lipid
2,5-DCB i n g o l d f i s h
the
values
found
1
0.008 d ~ )
Lower
was
weight
clearance
and
rate
transformation
major
9
by
methylation)
loss
study2
ted
of
favourable
of
30%
excretion
rate,
of
Still,
found
t o be
and 5.2x10
5
but twice
are
t h e two
( k ) and
2
similar, in
were
found f o r
different
i n the female
guppies
from
(k
-
2
2
as high
as i n g o l d f i s h .
responsible
f o r these
type
here
(compounds
a n d seem
higher
I and I I ) a r e i d e n t i c a l
to indicate
3,4-epoxidation
hypothetical intermediate
compound
atom.
in fish,
I I . In r a t s ,
This
would
third
produce
com-
metabolite i s
-
compound,
nor i n water
a
to
as
4 chloro-4-methoxybiphenyl
extracts after
fish
exposure,
b y GC-MS.
products
of
the 2,5-dichlorobenzoic
2,5-DCB
acid
The
has
also
been
derivative,
o f the b i o d e g r a d a b i l i t y
apparently
o f 2,5-DCB, e v e n
presence
of
an
found
without
unsubstituted
in a
formed
previous
v i a ring
specially
phenyl
adap-
ring
is
of reaction.
total
metabolism
compounds
elimination
chemical
has
a
t o the m e t a b o l i t e s
of
dichlorobiphenyl
i s the main
minor
process
influence
found
from
here
fish.
determining
on
the
accounts
for less
Apparently,
the o v e r a l l
bioaccumulation
direct
clearance
of
these
in fish.
the biotransformation
a metabolic
exposure,
o f 4.8
activity
and r a t s
chlorine
investigation
o f the parent
the
dichlorobiphenyls
and
higher
of this
was n o t d e t e c t e d
o f t h e unchanged
and
5
guppies ,
(NIH-shift),
micro-organisms.
for this
Transformation
than
shift
i s an i n d i c a t i o n
cultures
and
i n rabbits
transformation
Especially
4
i n male
o f 4,4'-DCB
o f the m i g r a t i n g
o f thorough
fission,
than
rate constants
are not s i g n i f i c a n t l y
r a t e o f 2,5-DCB
partially
of
p o i n t s ; the hydrophobicities
. Therefore, clearance
clearance
column s a -
by Mackay e t
in solubility
d i c h l o r o b i p h e n y l s a r e expected
2
Rearrangement
I and, v i a chlorine
variety
7
melting
r e s p e c t i v e l y , 5 which
content
formed
step .
(after
The
lower
products
metabolites
intermediate
spite
1
different
reviewed
t o 2,5-DCB i s a t l e a s t
The d i f f e r e n c e
biocencentration factors
The o v e r a l l
lipid
formed
in
these
c
based
exposure
by t h e c o n t i n u o u s
authors,
constants.
the
pound
to their
(K ) of
slightly
by o t h e r
concentration.
mainly
and g u p p i e s
here.
The
an
reported
a s l o g P) a r e h a r d l y d i f f e r e n t
bioconcentration
previous
those
high m o r t a l i t y during
aqueous
c o m p o u n d s c a n be a t t r i b u t e d
(expressed
with
half
life
i s not n e g l i g i b l e .
o f 50-70 d a y s ,
c a n be a s i g n i f i c a n t
factor
The p r o c e s s
as found
i s essentially
irreversible
f o r the d i c h l o r o b i p h e n y l s during
i n the environmental
fate
o f these
fish
chemicals.
157
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10,
811-832
(1975)
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W.B.Neely, D.R.Branson and
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super-li-
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A b s o r p t i o n and
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4)
elimina-
decachlorobi-
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310-315
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E.Herbst,
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K . B a l l s c h m i t e r , C h . u n g l e r t and
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D.Mackay,
264
and
and
F . K o r t e , C h e m o s p h e r e , 5,
H.J.Neu, C h e m o s p h e r e , i ,
O.Hutzinger,
R.Mascarenhas, W.Y.Shiu,
C h e m o s p h e r e , 7,
S . C . V a l v a n i and
181-188
127-130
(1976)
(1976)
51-56'(1977)
103-108
(1978)
S.H.Yalkows,ky, C h e m o s p h e r e ,
9,
257-
(1980)
17. W.A.Bruggeman, J . v a n
(this
158
W.Klein
thesis,
chapter
der
3)
Steen
and
0.Hutzinger,
J.Chromatogr.,
238,
335-346
(1982)
Dankwoord
Aan
d i t p r o e f s c h r i f t hebben v e e l mensen op een o f andere w i j z e b i j g e -
d r a g e n . Sommigen, met name de mede-auteurs van de v e r s c h i l l e n d e
kelen,
arti-
z u l l e n hun a a n d e e l g e m a k k e l i j k i n de h o o f d s t u k k e n van d i t p r o e f -
schrift
kunnen t e r u g v i n d e n , anderen hebben meer op de a c h t e r g r o n d ge-
werkt.
E n k e l e n van hen w i l i k met name noemen:
Otto H u t z i n g e r , mijn promotor,
d i e m i j met de m i l i e u c h e m i e van o r g a n i -
sche v e r b i n d i n g e n v e r t r o u w d h e e f t gemaakt, m i j t o t deze
chemische
biologisch-
s y n t h e s e h e e f t v e r l e i d en h e t g e d u l d h e e f t gehad om h e t u i t -
kristalliseren
van h e t e i n d p r o d u c t a f t e wachten, waar h i j m i s s c h i e n
meer op een r e e k s k l e i n e e x p l o s i e s had gehoopt;
I w i s h t o thank p r o f e s s o r D. Mackay f o r h i s i n t e r e s t
and s t i m u l a t i n g
remarks;
M a r t i n T u l p , d i e i n k o r t e t i j d meer i n s p i r e r e n d e , u i t d a g e n d e
den opperde
dan i n zes j a a r t i j d s
Kees O l i e , d i e me l e e r d e om n i e t a l t e v e e l v e r t r o u w e n
w i k k e l d e a p p a r a t e n en s t r u c t u r e n t e s t e l l e n
doen a l s h e t ook e e n v o u d i g
Wil
denkbeel-
b e s t r e d e n konden worden;
i n dure, inge-
(waarom zou j e m o e i l i j k
kan);
Veerkamp, d i e begon met de aanmaak
van r a d i o a c t i e f
(hexa)
broombenzeen maar d i e z i c h v e r v o l g e n s op de a f b r a a k van m i l i e u v r e e m d e
stoffen
wierp;
I
Dorcas Fung, who d e v e l o p e d an a n a l y t i c a l p r o c e d u r e
thank
f o r the
silicones;
B e r t M a r t r o n , D i r k Kooiman, B e r t R o o z e n d a a l ,
G e r t - J a n P e e t e r s en W a l t e r
D i j k h u i z e n , d i e z i c h a l s h o o f d - o f b i j v a k s t u d e n t met a d s o r p t i e , b i o a c c u m u l a t i e , b i o t r a n s f o r m a t i e o f c o m b i n a t i e s daarvan hebben
en op w i e r experimenten
beziggehouden,
d i t p r o e f s c h r i f t v o o r een b e l a n g r i j k d e e l geba-
seerd i s ;
Antoon
Opperhuizen,
d i e nu z i j n
d o c t o r a a l werk en andere bouwstenen van
d i t p r o e f s c h r i f t op hun b r u i k b a a r h e i d v o o r z i j n
e i g e n onderzoek
moet
beproeven;
Anneke Wijbenga,
bijstaat,
d i e me a l s s t u d e n t en c o l l e g a , en nu ook " i n d i t u u r "
samen met J a n v a n d e r S t e e n , d i e s y n t h e t i s e e r d e , chromatogra-
159
f e e r d e en b o v e n a l
Frans
van
a l s s t a b i l i s e r e n d e f a c t o r i n het
der W i e l e n ,
d i e de g a s c h r o m a t o g r a f e n onder z i j n hoede
meerden h o n d e r d d u i z e n d s c h o t e l s u i t een
en bemiddelde b i j h e t
Wim
S t a a l , die mij
heden" van
M a r i a van
Os,
A n i t a van
160
contacten;
s t e l d e de
"onbegrensde
der T u i n , F r i e d a H u t z i n g e r ,
leesbaar of z e l f s
Swigchem, d i e m i j
onderkomen met
Lenneke.
van moeizame
gelegenheid
hield,
glazen b u i s j e wist te halen,
(en h a a r c o l l e g a ' s ) , d i e o n d e r d e l e n
i n v e r s c h i l l e n d e fasen
en
leggen
i n de
werkte;
mogelijk-
HPLC te beproeven,-
M a r j o r i e de Leng
B e r t van
laboratorium
u i t z i c h t op
i n een k r i t i e k
zee
bood;
Anke
van
Lichtenberg,
het
manuscript
" p e r s k l a a r " hebben gemaakt;
stadium
van h e t
s c h r i j v e n een
VII
Het v e r d i e n t a a n b e v e l i n g de v e r p l i c h t e t o e v o e g i n g van h e t e l e m e n t
jodium aan b r o o d t e n behoeve vnn
de v o l k s g e z o n d h e i d t e
vervangen
door een h a l v e r i n g van de thans g e b r u i k e l i j k e t o e v o e g i n g e n
van
z o u t en v e t .
V I I I Het w e g l a t e n
lijk
van
i n chemisch
t i t e l s van
aangehaalde a r t i k e l e n ,
georienteerde wetenschappelijke
n i e t b e v o r d e r l i j k v o o r de u i t w i s s e l i n g van
informatie tussen v e r s c h i l l e n d e
IX
Gezien
tijdschriften, is
wetenschappelijke
disciplines.
h e t g e h a l t e aan h e t element a r s e e n zou R i j n s l i b
v o l g e n s de N e d e r i a n d s e
wet
a l s "chemisch
af-
sterk gestimuleerd.
Rijksinstituut
voor Zuivering
Wet Chemische
Knelpunt
van
Afvalwater,
in de rijkswateren,
Kwaliteitsonderzoek
XI
"chemisch
n a m e l i j k a l s g r o n d s t o f v o o r de b a k s t e e n i n d u s t r i e , waar-
schijnlijk
X
thans
a f v a l " aangemerkt moeten
worden. Op deze w i j z e wordt h e t n u t t i g g e b r u i k van
val",
zoals gebruike-
1982.
Afvalstoffen.
i n h e t m i l i e u c h e m i s c h en - t o x i c o l o g i s c h onderzoek i s n i e t
de o p s p o r i n g van
extreem l a g e g e h a l t e n aan t o x i s c h e s t o f f e n , maar
de i n t e r p r e t a t i e
daarvan.
Een v o l k d a t z i j n e i g e n a f v a l w a t e r d e f o s f a t e e r t en
z i j n huisdieren v r i j e l i j k
b l i j k van een
tegelijkertijd
uitwerpselen laat verspreiden, geeft
t w e e s l a c h t i g e houding
t e n o p z i c h t e van n a t u u r
en
milieu.
XII
Aan
de bodem k e n t men
h e t water.
S t e l l i n g e n b i j het p r o e f s c h r i f t : Bioaccumulation
p o l y c h l o r o b i p h e n y l s and r e l a t e d h y d r o p h o b i c
in
fish,
door W.A.
Bruggeman, 23 november
of
chemicals
1983.
STELLINGEN
I
Zowel van een s t u d e n t a l s van een w e t e n s c h a p p e l i j k o n d e r z o e k e r
worden v o o r a l de j u i s t e antwoorden op g e s t e l d e v r a g e n verwacht;
t e w e i n i g wordt e c h t e r b e s e f t d a t b i j w e t e n s c h a p p e l i j k onderzoek
h e t s t e l l e n van de j u i s t e v r a g e n v o o r o p d i e n t t e s t a a n .
II
T o e p a s s i n g van complexe mathematische m o d e l l e n v o o r b e s c h r i j v i n g
en v o o r s p e l l i n g van p r o c e s s e n i n m i l i e u en m a a t s c h a p p i j maakt h e t
v e r b a n d t u s s e n v o o r o n d e r s t e l l i n g e n en u i t k o m s t e n b e d u i d e n d minder
o v e r z i c h t e l i j k , v e r s t e r k t d a a r d o o r b i j de g e b r u i k e r e e r d e r
afhan-
k e l i j k h e i d dan b e g r i p , v e r g r o o t de kans op m o e i l i j k t e c o n t r o l e r e n f o u t e n , l o k t daarmee een i r r a t i o n e l e
stellingname
"voor" o f
" t e g e n " u i t , en d i e n t daarom v o o r t d u r e n d aan een z e e r
kritische
a n a l y s e onderworpen o f g e h e e l vermeden t e worden.
III
M a x i m a l i s a t i e van de f y t o p l a n k t o n b i o m a s s a b i j e v e n w i c h t i s o n v o l doende b a s i s v o o r m o d e l l e r i n g van a l g e n s o o r t e n s a m e n s t e l l i n g en
- p e r i o d i c i t e i t i n n a t u u r l i j k e wateren onder n u t r i e n t - g e l i m i t e e r d e
condities.
F.J.
Los, Mathematical
the model BLOOM II,
Report
IV
on investigations
simulation
of algae blooms by
Waterloopkundig
R 1310-7,
Laboratorium,
Delft,
1982.
Voor de r e l a t i e f d i e p e randmeren d i e b i j d r o o g l e g g i n g van een
Markerwaard z u l l e n o n t s t a a n , i s p e r i o d i e k e o p b l o e i van blauwalgen
van h e t g e s l a c h t
aanwezigheid van
Microcystis
Raad van Advies
Advies
V
meer t e v r e z e n dan een o v e r h e e r s e n d e
Oscillatoria-soorten.
voor de Ruimtelijke
over de ontwikkeling
1982,
Markerwaardgebied.
Z o l a n g g r o e i voorop s t a a t i n de I J s s e l m e e r p o l d e r s , z a l h e t b e g r i p
c u l t u u r daar v o o r a l b e t r e k k i n g hebben
VI
van het
Ordening,
op h e t bebouwen van grond.
Waar i n t e n s i e v e v e e h o u d e r i j z i c h l a a t kennen a l s t e g e n n a t u u r l i j k
en m i l i e u v e r v u i l e n d , i s h e t samengaan van landbouw en n a t u u r b e h e e r
v o l s t r e k t ongeloofwaardig.