5 Metabolism of Phthalate Esters in Aquatic Species
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M A R K J. M E L A N C O N Department of Pharmacology, Medical College of Wisconsin, Milwaukee, W I 53226
Phthalate esters, particularly, di-2-ethylhexyl phthalate (DEHP), are widely used as plasticizers. The total annual U.S. production of phthalate esters was over 400,000 tons in 1970 (1). Many of the applications of phthalate esters such as in construction, home furnishings, the automobile industry, etc. make it likely that they reach the aquatic environment. Since the init i a l report of the presence of phthalate esters in natural waters and fish in the United States by Mayer and coworkers (2), there have been reports of these chemicals in fish in Canada (3,4) and in Japan (5). The presence of phthalate esters in the ocean waters off the United States and in Gulf of Mexico biota has also been noted (6). Concern over the fate of phthalate esters in the aquatic environment has resulted in studies of the fate of phthalate esters in a number of aquatic species. Among the aquatic organisms which have been shown to metabolize phthalate esters, are microorganisms present in water, hydrosoil and sewage sludge, several invertebrates and several species of fish. Metabolism of Phthalate Esters by Microorganisms The various studies with aquatic microorganisms have shown that a variety of phthalate esters are degraded by river water, hydrosoil and sewage sludge. The data in Table I is derived from work at several different laboratories (7,8,9). While most of these phthalate esters are degraded in the systems studied, those esters containing the longer chain alcohols are broken down more slowly than those containing short chain alcohols. Monobutyl phthalate and phthalic acid were also found to be degraded by activated sludge by Saeger and Tucker (9). Johnson and Lulves (8) found that di-butyl phthalate was degraded much more rapidly than DEHP and that anaerobic degradation of DEHP and dibutyl phthalate by hydrosoil was much slower 0-8412-0489-6/79/47-099-077$05.00/0 © 1979 American Chemical Society Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 99
Butyl
d
c
d a t a f r o m Graham ( 7 ) b d a t a from Saeger and Tucker ( 9 ) d a t a f r o m J o h n s o n and L u l v e s ( 8 ) ^mixture o f d i h e p t y l , d i n o n y l and d i u n d e c y l
a
Dibutyl
Santicizer 711
phthalates
51
37
Diundecyl
96
74
A c t i v a t e d Sludge^ continuous % Degraded
99
a
E s t e r s by A q u a t i c
Butylglycol butyl
benzyl
91
Ester
Di-2-ethylhexyl
Phthalate
Activated Sludge 48 H r
Degradation o f Phthalate
TABLE I
0
10
95
80
10
R i v e r Water* 1 week
3
Microorganisms
92
Hydrosoil 2 weeks
0
5.
MELANCON
Phthalate
79
Esters
t h a n a e r o b i c ( T a b l e I I ) . DEHP, i n f a c t , was c o m p l e t e l y u n a f f e c t e d b y h y d r o s o i l o r g a n i s m s u n d e r a n a e r o b i c c o n d i t i o n s . A comp a r i s o n o f the amounts o f d i b u t y l p h t h a l a t e and m e t a b o l i t e s p r e s e n t u n d e r a e r o b i c a n d a n a e r o b i c c o n d i t i o n s i s shown i n T a b l e I I I . The most s t r i k i n g d i f f e r e n c e i n m e t a b o l i t e s i s t h e much l a r g e r amount o f m o n o e s t e r p r e s e n t u n d e r a n a e r o b i c c o n d i t i o n s . The amount o f p h t h a l i c a c i d i s a l s o h i g h e r , b u t t h i s r e p r e s e n t s a much s m a l l e r p r o p o r t i o n o f t o t a l ^ C . These r e s u l t s s u g g e s t t h a t h y d r o l y s i s p r o d u c t s o f d i b u t y l p h t h a l a t e are a c c u m u l a t i n g i n t h e a b s e n c e o f o x y g e n . T h i s c o u l d be due t o a s h i f t t o a n o t h e r r o u t e o f m e t a b o l i s m due t o t h e l a c k o f oxygen o r t o g r e a t l y s l o w e d o x i d a t i o n o f the monoester. Because the C was i n t h e c a r b o n y l , i t was n o t p o s s i b l e f r o m t h e s e s t u d i e s t o d e t e r m i n e w h e t h e r t h e b e n zene r i n g r e m a i n e d i n t a c t . S t u d i e s b y S a e g e r and T u c k e r , h o w e v e r , showed t h a t CO2 e v o l u t i o n f r o m d e g r a d a t i o n o f d i b u t y l p h t h a l a t e , DEHP and S a n t i c i z e r 711 was 85-95% o f t h e t h e o r e t i c a l ( 9 ) . J o h n s o n a n d c o w o r k e r s ( 1 0 ) l a t e r showed t h e e v o l u t i o n o f C 0 f r o m r i n g l a b e l e d DEHP. I t seems c l e a r , t h e n , t h a t a q u a t i c m i c r o o r g a n i s m s a r e a b l e t o c o m p l e t e l y degrade p h t h a l a t e e s t e r s . 1 4
Metabolism
2
o f Phthalate E s t e r s by F i s h i n v i v o
A l t h o u g h t h e m e t a b o l i s m o f s e v e r a l p h t h a l a t e e s t e r s has b e e n s t u d i e d in_ v i t r o , e s s e n t i a l l y a l l o f the in_ v i v o s t u d i e s have i n v o l v e d DEHP. A summary o f t h e s e e x p e r i m e n t s w h i c h i n v o l v e d e x p o s u r e o f f i s h t o aqueous C-DEHP i s p r e s e n t e d i n T a b l e I V ( 1 1 , 1 2 ) . Tissue C was i s o l a t e d and s e p a r a t e d i n t o p a r e n t a n d t h e v a r i o u s m e t a b o l i t e s b y p r e p a r a t i v e t h i n l a y e r c h r o m a t o g r a p h y on s i l i c a gel. M e t a b o l i t e s were h y d r o l y z e d where a p p r o p r i a t e a n d i d e n t i f i e d b y gas chromatography-mass s p e c t r o s c o p y . I n whole c a t f i s h , w h o l e f a t h e a d minnow a n d t r o u t m u s c l e , t h e m a j o r m e t a b o l i t e was the m o n o e s t e r w h i l e i n t r o u t b i l e t h e m a j o r m e t a b o l i t e was t h e m o n o e s t e r g l u c u r o n i d e . The f a c t t h a t i n a l l c a s e s t h e major^metabo l i t e was m o n o e s t e r o r m o n o e s t e r g l u c u r o n i d e d e s p i t e t h e d i f ferences i n s p e c i e s , exposure l e v e l and d u r a t i o n , e t c . represented by t h e s e d a t a , s u g g e s t s t h a t h y d r o l y s i s o f DEHP t o m o n o e s t e r i s i m p o r t a n t i n t h e b i o t r a n s f o r m a t i o n o f DEHP b y f i s h . li+
Metabolism
o f Phthalate E s t e r s by F i s h i n v i t r o
S t a l l i n g a n d c o w o r k e r s ( 1 1 ) s t u d i e d t h e m e t a b o l i s m o f CDEHP a n d d i b u t y l p h t h a l a t e b y c h a n n e l c a t f i s h l i v e r m i c r o s o m e s . They f o u n d t h a t t h e r e s p e c t i v e m o n o e s t e r s and more p o l a r metabol i t e s were p r o d u c e d , b u t t h a t DEHP was m e t a b o l i z e d t o a much l e s s e r e x t e n t than d i b u t y l p h t h a l a t e . Additional studies ( T a b l e V) showed t h a t t h e p r o d u c t i o n o f t h e r e s p e c t i v e m o n o e s t e r s was u n a f f e c t e d b y t h e p r e s e n c e o f NADPH, b u t t h a t t h e p r o d u c t i o n o f more p o l a r m e t a b o l i t e s i s v e r y d e p e n d e n t upon NADPH. The r e a sons f o r t h e a p p a r e n t l a c k o f a r e q u i r e m e n t f o r oxygen are u n c l e a r ±H
Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
a
3 5 8 3
5
7
14
30 2
39
59
69
100
recovery of
d a t a from Johnson and L u l v e s ( 8 ) ^ r e c o v e r y from h e a t k i l l e d controls=100%
95
%
di-n-butyl phthalate aerobic anaerobic
1
Incubation D u r a t i on (days)
1 4
Λ
41
53
100
C from h y d r o s o i l
U.
3
Κ
100
100
100
di-2-ethylhexyl phthalate aerobic anaerobic
Biodégradation o f C - d i - n - b u t y l P h t h a l a t e a n d 'C-di-2-ethylhexyl Phthalate i n Freshwater H y d r o s o i l
TABLE I I
Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 3
1 4
d a t a from Johnson and L u l v e s ( 8 ) j for % of i n i t i a l C r e c o v e r e d see Table I I
29.0 63.0 1.3 4.6 trace
29.6 45.3 4.3 16.3 4.6
68.3 30.7 trace 0.5 trace
di-n-butyl phthalate mono-n-butyl p h t h a l a t e Unknown I Unknown I I Other
Anaerobic incubation
a
70.0 26.0 0 0 3.0 1.0
85.3 7.0 trace 0 2.3 3.3
7
5.0 85.6 1.0 6.3 trace
74.0 22.0 0 0 2.3 1.0
14
duration (days)
46.3 46.3 2.3 0.1 0.9 2.3
5
1
Incubation
di-n-butyl phthalate mono-n-butyl p h t h a l a t e Unknown I Unknown I I Phthalic acid Other
1 I f
Aerobic incubation
H
C h a r a c t e r i z a t i o n o f C Recovered from H y d r o s o i l a f t e r Incubation with C-di-n-butyl phthalate
TABLE I I I
37.6 16.9 0 35.0 9 .0
76.0 18.6 0 0 3.6 1.0
30
oo
ST
w δ g ^
ÇA
Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979. a
a
c
d a t a f r o m S t a l l i n g , e_t a l . ( 1 1 ) ^ d a t a f r o m M e l a n c o n and L e c h (12) and exposure duration
Melancon , e t a l . (15) .
1.3
4.4
25 1.4
60
2
37 0.7
50
4.9 3.7
Other
a
as d i - 2 - e t h y l h e x y l p h t h a l a t e
5.2 3.0
14
C
4 0.3
1 4
Phthalic acid free conjugated
%
c
Fathead Minnow Whole f i s h 29d 56d £
21
0 2
0.5 72
1
39
46
metabolites
Rainbow T r o u t 24 h r Bile Muscle
C - d i - 2 - e t h y l h e x y l P h t h a l a t e by F i s h In V i v o
66 14
phthalate
l l +
Catfish Whole f i s h 24 h r
of
Mono-2-ethylhexyl phthalate free conjugated
Di-2-ethylhexyl
Biotransformation
TABLE I V
ζ
i
η
I
>
t—I
S
ΗΗ
w ο r
Ο
Η
w ο
Ο
§
Ο
%
M
9
8
oo to
Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979. (DPM)
3
data
from S t a l l i n g , e t a l . (11)
478 864 2,768 6,130
5 ,49 8 4,776 4,259 5 ,676
0 + 0 +
0 0 + +
Di-2-ethylhexyl phthalate
a
2,955 1,992 26 ,9 80 21,125
27,662 31,376 34,569 36 ,234
0 + 0 +
0 0 + +
Polar Metabolites 0 , 1 8 2
formed
Microsomes
Di-n-butyl phthalate
2
Monoester
Metabolites
E s t e r s by C a t f i s h H e p a t i c
0
Additions
C-Phthalate
NADPH
Metabolism o f
TABLE V
84
PESTICIDE A N D XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS
Sanborn, e t a l . (13) r e p o r t e d t h a t mosquito f i s h l i v e r microsomes gave r i s e t o t h e m o n o e s t e r as a m e t a b o l i t e f r o m d i o c t y l p h t h a l a t e a n d t h a t t h i s m e t a b o l i s m was b l o c k e d b y p a r a o x o n . The m e t a b o l i s m o f C - D E H P b y r a i n b o w t r o u t l i v e r s u b c e l l ular f r a c t i o n s a n d serum was s t u d i e d b y M e l a n c o n a n d Lech ( 1 4 ) . The d a t a i n T a b l e VI show t h a t w i t h o u t added NADPH, t h e m a j o r m e t a b o l i t e p r o d u c e d was m o n o - 2 - e t h y l h e x y l p h t h a l a t e . When NADPH was added t o l i v e r homogenates o r t h e m i t o c h o n d r i a l o r m i c r o s o m a l f r a c t i o n s , two u n i d e n t i f i e d m e t a b o l i t e s more p o l a r t h a n t h e m o n o e s t e r were p r o d u c e d . A d d i t i o n a l s t u d i e s showed t h a t t h e m e t a b o l i s m o f DEHP b y t h e m i t o c h o n d r i a l a n d t h e m i c r o s o m a l f r a c t i o n s were v e r y s i m i l a r ( F i g u r e 1 ) . B o t h f r a c t i o n s show an i n c r e a s e d p r o d u c t i o n o f m e t a b o l i t e s o f DEHP r e s u l t i n g f r o m a d d i t i o n o f NADPH a n d t h e s h i f t f r o m p r o d u c t i o n o f m o n o e s t e r t o t h a t o f more p o l a r m e t a b o l i t e s . The r e d u c e d a c c u m u l a t i o n o f monoe s t e r w h i c h a c c o m p a n i e d t h i s NADPH m e d i a t e d p r o d u c t i o n o f more p o l a r m e t a b o l i t e s may h e l p i n i n t e r p r e t i n g t h e pathway o f DEHP metabolism i n t r o u t l i v e r . T h i s d e c r e a s e d a c c u m u l a t i o n o f monoe s t e r c o u l d be e x p l a i n e d e i t h e r by metabolism o f t h e monoester t o more p o l a r m e t a b o l i t e s o r t h e s h i f t o f DEHP f r o m t h e h y d r o l y t i c r o u t e t o a d i f f e r e n t , o x i d a t i v e p a t h w a y . The l a t t e r e x p l a n a t i o n i s u n l i k e l y because i n these e x p e r i m e n t s l e s s than 20% o f t h e DEHP was m e t a b o l i z e d . B e c a u s e t h e m e t a b o l i s m o f DEHP was c a t a l y z e d b y s o many f r a c t i o n s o f t h e t r o u t l i v e r h o m o g e n a t e , t h e s e f r a c t i o n s were c h a r a c t e r i z e d b y measurement o f m a r k e r enzymes t o d e t e r m i n e w h i c h o r g a n e l l e s a c t u a l l y were r e s p o n s i b l e f o r t h e o b s e r v e d DEHP metabolism. S u c c i n i c d e h y d r o g e n a s e a c t i v i t y was u s e d as a m a r k e r f o r m i t o c h o n d r i a , w h e r e a s g l u c o s e - 6 - p h o s p h a t a s e was u s e d as a marker f o r microsomes. The d i s t r i b u t i o n o f DEHP o x i d a s e a c t i v i t y ( p r o d u c t i o n o f p o l a r m e t a b o l i t e s 1 a n d 2 w i t h added NADPH) a n d o f DEHP e s t e r a s e a c t i v i t y ( p r o d u c t i o n o f m o n o e s t e r w i t h o u t added NADPH) were a l s o d e t e r m i n e d . I t was f o u n d ( F i g u r e 2) t h a t t h e d i s t r i b u t i o n o f DEHP o x i d a s e a c t i v i t y p a r a l l e l s t h e d i s t r i b u t i o n o f m i c r o s o m a l a c t i v i t y a n d t h e d i s t r i b u t i o n o f DEHP e s t e r a s e a c t i v i t y p a r a l l e l s the d i s t r i b u t i o n o f microsomal a c t i v i t y , b u t i s also present i n the cytosol f r a c t i o n . I n an e f f o r t t o c h a r a c t e r i z e f u r t h e r t h e m e t a b o l i s m o f DEHP by t r o u t , t h e e f f e c t o f t h e m i x e d f u n c t i o n o x i d a s e i n h i b i t o r , p i p e r o n y l b u t o x i d e , upon t h e m e t a b o l i s m o f DEHP b y t h e s e t r o u t l i v e r f r a c t i o n s a n d s e r u m was e x a m i n e d . B e c a u s e o f t h e use o f p i p e r o n y l b u t o x i d e as an i n s e c t i c i d e s y n e r g i s t , i t i s p o s s i b l e t h a t f i s h m i g h t be e x p o s e d t o t h i s c h e m i c a l i n t h e e n v i r o n m e n t . The d a t a i n T a b l e V I I show t h a t p i p e r o n y l b u t o x i d e i n h i b i t e d o v e r a l l m e t a b o l i s m o f DEHP b y l i v e r homogenates a n d microsomes w h e t h e r NADPH was added o r n o t . The h y d r o l y s i s o f DEHP b y s e r u m was a l s o b l o c k e d b y p i p e r o n y l b u t o x i d e a n d a l t h o u g h n o t shown, t h i s was a l s o t h e case w i t h l i v e r c y t o s o l . These l a t t e r r e s u l t s w e r e s u r p r i s i n g b e c a u s e p i p e r o n y l b u t o x i d e h a s b e e n known as a m i x e d f u n c t i o n o x i d a s e i n h i b i t o r o n l y , a n d w o u l d n o t be e x p e c t e d 14
Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
l 4
data
f r o m M e l a n c o n and Lech ( 1 4 )
0
Serum
a
0
100,000xg supernatnat
0.08
0.20 2.93
0.07 0.73
0.53 3.38
0.61
0.01
serum
nmol/hr/g l i v e r
n m o l / h r / 0 ,,08ml
4.13
4.51 2.23
0 +
Microsomes (100,000xg
pellet)
1.99 2.19
0 +
Mitochondria (10 000xg p e l l e t )
5
3.91 3.11
0 +
2000xg supernatant
NADPH
a
0.04
0.10
0.13 1.41
0.07 0.33
0.20 . . . 1.66
M e t a b o l i t e s Formed Mono-2-ethylhexyl Polar Metabolites phthalate 1 2
M e t a b o l i s m o f C - d i - 2 - e t h y l h e x y l P h t h a l a t e by Rainbow T r o u t L i v e r P r e p a r a t i o n s and B l o o d S e r u m
TABLE VI
0>
Co
ft
*
If
^
86
PESTICIDE A N D XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS
Mitochondia CONTROL
+ NADPH
Microsomes CONTROL
+ NADPH
6h
4 h
JH
iff 2h
0.25 0.5 1 2
0.2505 1 2
O250.5 1 2
0.25 a s 1 2
incubation time (hours) Figure 1. Influence of time on metabolism of C-DEHP by trout liver mitochondrial and microsomal fractions. Incubation contained 0.010 pmol of C-DEPH in a total volume of 2 mL. Mitochondria equivalent to 0.254 g of liver or microsomes equivalent to 0.361 g of liver were used in each incubation. Open bars represent monoester, striped bars Polar Metabolite I and solid bars Polar Metabolite 2. Each column represents an individual incubation (14). 14
14
Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
5.
MELANCON
Phthalate Esters
87
Succinic Dehydrogenase A B C D
0
20
40
60
80
100
Glucose-6- Phosphatase
0 3
1
20
40
60
80
100
80
100
DEHP Esterase
20
40
60
Percentage of total protein Figure 2. Distribution of marker enzymes and DEHP-metabolizing enzymes in trout liver homogenate fractions. DEHP esterase and DEHP oxidase were each measured by 1-hr incubations of 0.010 μmol of C-DEHP in a total volume of 2 mL. Fraction (A), 2,000 g pellet; (B), 10,000 g pellet; (C), 100,000 g pellet; and (D), 100,000 g supernatant. Relative Specific Activity = percent of total activity/ percent of total protein (14). 14
Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
b
a
serum
0 90yM
0 0
+ +
15 .96 + 0 .96 1.08 + 0 . 1 2
b
b
b
0 .62 0 .08 0 .47 0 .01
+ ± + ±
3.93 0.20 3.10 0.12
0 2mM 0 2mM
0 0
+ +
0 .82 0 0 .54 0 .37
+ ± + +
7.51 1.05 1.14 1.39
0 90yM 0 90μΜ
0 0
± ± ± ±
± ± ± ± 0.05 0.02 0.64 0.08
0.13 0.18 0.93 0.33
liver
Polar 1
formed
a
b
b
0.11 0.03 1.03 0.04
0.14 0.06 1.41 0.08
0.01 0.02 0.20 0.02
b
+ + + ±
b
0.02 0.01 0.46 0.01
+ ± + +
Metabolites 2
a d d e d ) , P
