Dynamics of isopropylphenyl diphenyl phosphate in fathead minnows

Chemosphere,Vol.12,No.6,pp P r i n t e d in G r e a t B r i t a i n

799-807,1983

0 0 4 5 - 6 5 3 5 / 8 3 $ 3 . 0 0 + .OO © 1 9 8 3 P e r g a m o n Press Ltd.

DYNAMICS OF ISOPROPYLPHENYL DIPHENYL PHOSPHATE IN FATHEAD MINNOWS James N. Huckins* and Jimmie D. Petty U.S. Fish and Wildlife Service Columbia National Fisheries Research Laboratory Columbia, Missouri 65201 ABSTRACT The fate of a triaryl phosphate (ring-UL-14C-2-isopropylphenyl diphenyl phosphate) was examined in fathead minnows. Dipheuyl phosphate was identified as a major metabolite in whole body fish samples. A significant portion of the radioactive residues appeared to be associated with biogenic compounds, indicating "in vivo" reactions of biochemicals with 14C_metabolite s or 14C-ring cleavage of 2-isopropylphenyl diphenyl phosphate and subsquent incorporation into biochemical pathways. INTRODUCTION Triaryl phosphate esters have replaced polychlorinated biphenyls for a number of applications.

Large quantities of triaryl phosphates are being used as hydraulic fluids,

plasticizers, and gasoline additives (i). The Midwest Research Institute (I) estimated potential losses to the environment and modes of entry for a number of triaryl phosphate esters.

Several

of these compounds have been detected in the aquatic environment (1-4) and their toxicity to selected aquatic organisms has been reported by a number of investigators (I, 2).

Although some

information exists on the fate of triaryl phosphate esters in the aquatic environment (I-3), little is known about the metabolism of these compounds by aquatic organisms. The 2-isopropylphenyl diphenyl phosphate ~-IFDP) ester is representative of alkyl substituted triaryl phosphates and is a major component of three commercial phosphate ester mixtures (Phosflex 31P", Kronitex I00", and Kronitex 200").

As part of an integrated research

effort ~, Buckler, D. R. and Sanders, H. 0., Columbia National Fisheries Research Laboratory, 1982) to assess the impact of triaryl phosphates on aquatic organisms, we report the fate of 2-IPDP in fathead minnows (Pimevhales vromelas).

EXPERIMENTAL Apparatus (a)

L i q u i d c h r o m a t o g r a p h y . - - A n Autoprep a u t o m a t e d g e l p e r m e a t i o n c h r o m a t o g r a p h (GPC) from

Analytlcal

B i o c h e m i s t r y C o r p o r a t i o n , Columbia, MO, was used f o r t h e s e p a r a t i o n

extracts.

The GPC column ~ . 2

Biobeads S-X3 and e l u t e d w i t h m e t h y l e n e c h l o r i d e / c y c l o h e x a n e

(HPLC).

The HPLC columns used were s e m i - p r e p a r a t i v e i 0 ~ C18 A d s o r b o s i l

~.36

cm i d X 20 om).

and A d s o r b o s i l from M i l t o n Roy Company.

799

A DuPont 870

w i t h an LDC UV I I I M o n i t o r from

by h i g h p e r f o r m a n c e l i q u i d

chromatography

Zorbax BP 7 - 8 p C18 (0.9 om i d X 25 om) and Zorbax BP was o b t a i n e d from DuPont Company

We p e r f o r m e d a l l

m i x t u r e s of m e t h a n o l and w a t e r .

was packed w i t h

( 5 0 / 5 0 , v / v , 2mL/miu).

pump module from DuPont Company, W i l m i n g t o n , DE, was i n t e r f a c e d M i l t o n Roy Company, R i v i e r a Beach, FL, f o r s e p a r a t i o n s preparative

of n o n p o l a r sample

cm i d X 30 cm, Ace g l a s s w i t h on-column i n j e c t o r )

HPLC s e p a r a t i o n s

isocratlcally

using

8OO

(b)

Gas c h r o m a t o g r a p h y . - - A V a r i a n 3700 gas c h r o m a t o g r a p h (GC) from V a r i a n I n s t r m n e n t s ,

Walnut Creek, CA, was f i t t e d

w i t h V a r i a n f l a m e p h o t o m e t r i c ( p h o s p h o r u s mode) and t h e r m i o n i c

( n i t r o g e n / p h o s p h o r u s mode) d e t e c t o r s . on-101 or SE-30 on Chromosorb W liP.

The GC columns used were g l a s s 2 mm i d X 1.1 m, w i t h 3% The c a r r i e r

gas was N2 and t h e f l o w was s e t a t 30 mL/min.

The minimum d e t e c t i o n l i m i t s o f 2-IPDP, u s i n g t h e flame p h o t o m e t r i c and t h e r m i o n i c d e t e c t o r s , were 0.5 ng and 0.05 ng, r e s p e c t i v e l y . (c)

Mass s p e c t r o m e t r y . - - A F i n n i g a n Model 4023 automated GC-MS s y s t e m from F i n n i g a n

I n s t r u m e n t s , S u n n y v a l e , CA, e q u i p p e d w%th a q u a d r a p o l e mass f i l t e r

and a J & W DB5 c a p i l l a r y

column ( 0 . 2 5 mm i d X 30 m), was u s e d f o r c o n f i r m a t i o n o f d i p h e n y l p h o s p h a t e . p e r f o r m e d i n t h e e l e c t r o n impact mode; e l e c t r o n e n e r g y was s e t a t 55 eV.

The a n a l y s e s were

Samples were i n j e c t e d

a t 60 ° C, h e l d i s o t h e r m a l l y f o r 1 min and programmed t o 210 ° C a t 20 ° C/min.

The program r a t e

was t h e n changed t o 5 ° C/min up t o 290 ° C and h e l d a t 290 ° C f o r 5 min. (d)

Liquid scintillation

and b i o l o g i c a l m a t e r i a l s

was done w i t h a Beckman L i q u i d S c i n t i l l a t i o n Fullerton,

CA.

Scintillation

oxidizer. --All

scintillation

f l u i d r o u t i n e l y u s e d c o n t a i n e d Beckman F l u o r a l l o y dry mix d i s s o l v e d

i n t o l u e n e and T r i t o n X-100.

R a d i o a c t i v e 1 4 C - r e s i d u e s i n f i s h t i s s u e s were l i b e r a t e d w i t h a

B i o l o g i c a l M a t e r i a l s O x i d i z e r (BMO) from R. J . Harvey I n s t r u m e n t C o r p . , H i l l s d a l e , r e c o v e r y o f 14C-CO 2 e v o l v e d from c o m b u s t i o n o f 14C-samples was 80 t o 85%. was u s e d f o r t r a p p i n g e v o l v e d 14C-C02 and s u b s e q u e n t LS c o u n t i n g . efficiencies

counting

System (LS-3133T) from Beckman I n s t r u m e n t s I n c . ,

of Beckman and Harvey c o c k t a i l s

NJ.

The

Harvey 14C c o c k t a i l

D i f f e r e n c e s in the counting

r e q u i r e d t h e e s t a b l i s h m e n t o f a s e p a r a t e LS quench

c u r v e f o r each c o c k t a i l . (e)

D i l u t o r s y s t e m s . --Two d u a l - c e l l

Micromedic a u t o m a t i c p i p e t t e r s

S y s t e m s , Horsham, PA, were used f o r i n t e r m i t t e n t glass aquaria.

Each p i p e t t e r

from Micromedic

f l o w - t h r o u g h e x p o s u r e s of f i s h i n f o u r 40 L

d e l i v e r e d two 100-AtL a l i q u o t s o f t h e a c e t o n e s t o c k s o l u t i o n s .

ReaEents Ca)

Solvents. --All solvents were nanograde and were purchased from Burdick and Jackson,

Muskegon, MI. (b~

Eadiochemicals. --Two lots of ring UL-14C-2-1PDP were purchased from Pathfinder

Laboratories, Saint Louis, MO.

One lot was ring labeled on the diphenyl moiety (D) and the other

lot only on the isopropylphenyl moiety tiP) o dpm/pg and t h e p u r i t y was >95% baaed o n t h i n

The specific activity of both lots was 6.4 x 10 4 l a y e r chromatography ( s i l i c a

g e l 60 F-254 p l a t e s

w i t h h e x a n e / a c e t o n e , 4 / 1 ) , GC, and s e m i - p r e p a r a t i v e C18 HPLC d e t e r m i n a t i o n s . Freshwater Institute, specific activity

(c)

Winnipeg, M a n i t o b a , k i n d l y p r o v i d e d r i n g UL-14C-diphenyl p h o s p h a t e .

was 1 . 1 X

methylated material

Derek Muir o f t h e

104

The

dpm/~g and t h e p u r i t y was >75% b a s e d on GC a n a l y s i s o f t h e

( s e e "Residue A n a l y s i s " ) .

C h e m i c a l s . - - P h o s f l e x 31P was from S t a u f f e r Chemical Company, W e s t p o r t , CT, and

p u r i f i e d ~ - G l u c u r o n i d a s e (Type VII) was from Sigma Chemical Company, S a i n t L o u i s , MO. F a t h e a d Minnow Exnosur~s F a t h e a d minnows ( P i m e v h a l e s v r o m e l a s ) ware r e a r e d a t our l a b o r a t o r y t o a w e i g h t o f 1.0 to 1.5 g .

F o r t y f i s h were p l a c e d i n each of t h e f o u r d i l u t o r

a q u a r i a and h e l d i n f l o w i n g w e l l w a t e r

801

at 25 ° C for 3 days before we began the exposure.

Well water was characterized by a pH of 7.4,

alkalinity of 237 mg/L as CaCO3,and hardness of 272 mg/L as CaCO 3.

The stock solutions used in

these exposures were mixtures of ring UL-14C-2-1PDP (2.5 pg/L, two label positions) and Phosflex 31P (497.5 #g/L).

The actual amount of 2-1PDP in stocks was 120.0 pg/L, based on multiple GC

analysis (2-1FDP represented 24 Z of the area under Phosflex 31P GC trace). specific activity of 2-1PDP in these exposures was 1.30 X 103 dpm/~g.

exposed continuously and those in the other two aquaria served as co,trols. libitum throughout the study.

Therefore the

Fish in two aquaria were All fish were fed ad

After the exposure period of 28 days, the fish remaining in

treated aquaria were transferred to the parallel control aquaria for a 14-day elimination phase (control fish were removed at 28 days and frozen at -25 o C).

Fish were collected and weighed on

days 3.5, 7, 14, 21, 28 of the exposure period and days 3.5, 7 and 14 of the elimination period. Water samples were collected every 3.5 days of the study.

Residue Analysis At least three whole body fish samples were analyzed from each sample period of the IP-labeled and D-labeled exposures and elimination phases.

Individual fish were ground with four

times their weight of dry Na2SO 4 in an Osterizer blender and placed in glass columns I cm id with teflon stopcocks for sequential solvent extraction (5).

Blended samples were extracted with 150

mL of toluene/isopropanol/methylene

5/25/70, v/v/v) and then 150 mL of

methanol (solvent II).

chloride (solvent I,

The flow rate was maintained at <2 mL/min and a number of samples were

further extracted or hydrolyzed with 100 mL of hot water, 5 % glacial acetlc acid in water, and/or 0.1 N NaOH in an attempt to improve the recovery of 14C-residues from fish tissues. Tissues remaining after multiple extraction of 3 fish from the D-exposure were hydrolyzed for I hour with high purity 6N HCI (100 ° C under N 2) and examined for water solubilized radioactivity. The concentration of 14C-residues in tissues after extraction with solvent I and II was determined by analysis of 3 to 4 subsamples with the BMO. Solvent I tissue extracts were fractionated by using GPC. lipid eluates (GPC) from a number of samples

We concentrated the 0 to 40 mL

under N 2 and further fractionated them by

semi-preparative HPLC (Zorbax, C18) using a mixture of methanol and water (60/40, v/v).

The 50

to 120 mL GPC fractions, which contained 2-1PDP and Phosflex 31P components, were analyzed radiometrically and by GC.

Selected solvent II tissue extracts were cleaned up by using

preparative or semi-preparative HPLC.

Purification of relatively large quantities (> 1 #g) of

14C-diphenyl phosphate (solvent II extract) for GC analysis was accomplished with preparative HPLC.

Semi-preparative HPLC of solvent II extracts was used to separate smaller quantities (< 1

~g) of 14C-resldues.

These separations provided higher resolution (26,000 plates/m) and reduced

the possibility of coeluting impurities. A 70/30, methanol/water phase was used for preparative separations of solvent II extracts, whereas a number of methanol/water mixtures were used for semi-preparative separations. The 14C-diphenyl phosphate eluted in the 45 to 50 mL fraction of preparative separations.

Polar 14C-fractions from HPLC separations of solvent II extracts were

concentrated in acetonitrile and methylated with diazomethane (6) for GC analysis. Control fish and fish samples spiked with 14C-2-IPDP were processed with each set of 3 to 12 samples.

Losses of 14C-residues during isolation procedures were monitored by LS counting.

802

The 1 4 C - 2 - I P D P i n f i s h

was recovered

Only 1 . 5 5 Z o f 1 4 C - 2 - I P D P s p i k e s i n t h e 0 t o 40 mL GPC l i p i d concentrations (14C-residues averaged

by s o l v e n t

(Nil6)

eluted

fraction.

I and eluted

These values

not resolved values

from lfpids in tables

as polar

by GPC a n d HPLC).

were corrected

II

tissue

were subtracted

in Tables 1 and 2, which are reported

89Z; a l l

i n t h e 50 t o 90 mL GPC f r a c t i o n .

in the solvent

extract

and 1.60Z eluted

from the residue

metabolites

and lipid

associated

R e c o v e r y o f 1 4 C - 2 - I P D P t h r o u g h GPC

for procedural

losses

and counting

efficiencies. RESULTS AND DISCUSSION Concentrations (Table

I,

half-life

3.5-day

o f t h e 2-1PDP i n f i s h 14C-IP)

and 51.1 ~g/g

o f 2-1PDP i n f i s h

s a m p l e d (N=6)

increased

was l e s s

phase.

Little

peaked at 64.2 ~g/g

2 , 7 - d a y 14C-D) a n d d e c l i n e d

than 7 days in both exposures.

f r o m 1 . 5 0 g a t day 3 . 5

end of the 14-day elimination control

samples from the t~o exposures

(Table

thereafter.

The a v e r a g e w e i g h t

t o 1 . 8 5 g a t d a y 21 a n d d e c l i n e d

difference

in mortalities

The of fish

to 1.16 g at

was o b s e r v e d

between

and exposed fish.

Table 1.

14 a Whole b o ~ C - r e s i d u e s (~ug/g) i n f a t h e a d minnows c o n t i n u o u s l y exposed to 2.5 ~ s / L of r i n g UL- C-XP-labeled 2-IPDP and 497.5 ~g/L of P h o s f l e x 31P (120 # g / L t o t a l 2-IPDP)

Type o f 14C-Residue

DaTe of Exposure 14 21

7

2-IPDP

64.2 _.+16.4

59.7 --+15.2

39.7 -+ 8 . 0

41.8 +22.2

24.9 _+12.0

15.6 -+11.0

4.5 _+0.9

0.9 _+0.2

Lipid Associated b

5.4 + 0.5

4.8 + 1.9

5.6 + 1.4

3.3 _+ l.O

0.2 + 0.1

3.3 + 2.9

1.2 _+0.2

1.1 _+0.3

P o l a r Compoundsc

9.0 +_. 4.2

8.6 _+ 4 . 3

7.1 + 4.6

10.5 _+ 2.0

14.1 _+ 5.9

3.5 + 1.1

1.6 _+0.3

1.8 _+0.3

Non_Extracted d

5.7 + 1.6

7.2 _+ 3 . 0

8.0 + 2.2

8.0 +_ 1.4

8.5 _+ 5.3

18.9 +_ 9.8

9.8 +_4.0

84.3

80.3

60.4

63.6

47.7

41.3

17.1

T o t a l 14C-Residues

28

Days of El~Jninacion 3.5 7 14

3.5

a b

Mean and s t a n d a r d d e v i a t i o n of t h r e e or more samples.

c

These r e s i d u e s were p r e s e n t i n s o l v e n t I I and r e p r e s e n t a t l e a s t two compounds.

d e

These r e s i d u e s r e p r e s e n t a t l e a s t two compounds t h a t were not r e s o l v e d from l i p t d s . Values r e p o r t e d a r e based on BMO d e t e r m i n a t i o n s of e x t r a c t e d t i s s u e s . Values

represent

moan o f t ~

samples.

9.1 e

12.9

the

803

Table 2.

Whole b o ~ 14C-res£duesa (,ug/8) in fathead minnovs continuously exposed to 2.5 ~8/L of r i n g UL-" C-D-labeled 2-IPDP and 497.5 ~g/L of Phosflex 31P(120 ~g/L t o t a l 2-IPDP)

Type of 14C-Residues

Days of v'rposure 14 21

Days of Elimination 3.$ 7 14

3.5

7

29.3 +13.9

51.1 +26.6

49.0 +17.I

45.4 +17.5

36.4 +11.9

6.0 + 1.9

0.8 + 0.2

0.2 b

2-IPDP

1.6 +0.9

2.9 +2.0

4.7 +0.6

4.5 +2.0

3.8 +1.3

3.2 +2.8

0.6 +0.2

1.6 b

Lipid ~ s ° c t a t e d e

C~o~ds d

1.0 +0.7

+1.6

~2.2

+2.2

4.3

12.3 +0.7

+0.8

+0.2

0.3

0.2 ~.3

2.8 + 1.2

+ 4.4

5.8

24.2 + 7.1

25.5 +22.1

64.9 +.34.3

40.0 +--44.8

5.6 + 3.4

7.1 +--5.8

34.7

64.1

80.3

79.7

49.6

7.3

9.1

Polar

Non_£xtracted e

Total 14C-Residues

a

4.3

2.4

28

0.4

117.4

Nean and standard deviation of three or more samples.

b

Values represent mean of two samples.

c

These r e s i d u e s represent at l e a s t two compounds t h a t were not resolved from l i p i d s .

d

These r e s i d u e s were present in solvent II and represent a t l e a s t two compounds.

•

Values reported are based on BHO determinations of extracted t i s s u e s .

A bioconcentration

factor

o f 970 ( c a l c u l a t e d

from t h e o c t a n o l / v a t e r

partition

f o r a 2-IPDP c o m m e r c i a l m i x t u r e ( K r o n i t e x 100) h a s b e e n r e p o r t e d i n t h e l i t e r a t u r e minnows i n t h i s

s t u d y c o n c e n t r a t e d 2-IPDP 550 t i m e s i n t h e I P - l a b e l e d

the D-labeled exposure.

The d i f f e r e n c e

in the reported calculated

v a l u e s may be due t o t h e p r e s e n c e o f o t h e r p h o s p h a t e e s t e r

coefficient) (3).

Fathead

e x p o s u r e and 440 t i m e s i n

v a l u e and o u r e x p e r i m e n t a l

c o m p o n e n t s i n t h e K r o n i t e x m i x t u r e or

t o t h e o b s e r v e d m e t a b o l i s m o f 2-IPDP by £ a t h e a d minnows. The GC a n a l y s i s differential eluting

o f 50-120 mL GPC e l u a t e s

(solvent

I) c o n t a i n i n g P h o s f l e x 31P r e v e a l e d

u p t a k e of P h o s f l e x components by f a t b e a d minnows ( F i g u r e 1 ) .

In general early

(GC) P h o s £ 1 e x 31P c o m p o n e n t s were r a p i d l y a c c u m u l a t e d , and r a p i d l y e l i m i n a t e d d u r i n g t h e

el~nination procedure,

phase (Figure 1).

A l t h o u g h no a c t u a l P h o s f l e x 31P s p i k e , c a r r i e d

i s s h o v u i n F i g u r e 1 , t h e component r a t i o s

through the cleanup

o f s p i k e s were v e r y s i m i l a r

t o t h o s e of t h e

Phosflex standard. The c h r o m a t o g r a p h i c b e h a v i o r o f 1 4 C - r e s i d u e s i n s a m p l e s f r o m b o t h e x p o s u r e s s u g g e s t e d t h a t 2-IPDP was m e t a b o l i z e d by f a t h e a d m i n n o w s . w i t h GPC i n d i c a t e d lipids

or nonpolar b i o g e n i c s .

fraction

C l e a n u p of t h e f i r s t

extracts

of I P - and D - s a = p l e s

t h a t 2 . 0 t o 9.2Z o f t h e 1 4 C - r e s i d u e s i n f a t h e a d minnows were a s s o c i a t e d v l t h These residues

and s u b s e q u e n t s e p a r a t i o n s

coeluted with lipids

i n t h e 0 t o 40 mL GPC dump

on t h e C18 s e m i - p r e p a r a t i v e HPLC column i n d i c a t e d

the

804

a. a_ m

¢1. eL m

B

A

¢1.

C

a L m

I

¢1.

a

a_ m

I

I 0

|

I 0

2'0

10 F i g u r e 1.

1'0

2'0

I 0

1'0

2'oMI N.

Fleme photometric-GC a n a l y s i s of the d i l u t o r s t o c k and p u r i f i e d i s o p r o p y l p h e n y l - l a b e l e d f a t h e a d minnow e x t r a c t s . Trace A, 21.9 ng s t o c k of P h o s f l e x 31P and 14C-2-1PDP; Bj 2 ~1 of a 3 . 5 - d a y f i s h e x t r a c t ; C, 2 ~1 of a 3 . 5 - d a y e l i m i n a t i o n f i s h e x t r a c t .

p r e s e n c e of two d i s t i n c t

bands of r a d i o a c t i v i t y

t h a t were not r e s o l v e d from b i o g e n ~ c s .

The

methanol e l u a t e s of I P - and D - f i s h samples ( s o l v e n t I f ) c o n t a i n e d 2.9 to 29.5Z of the t o t a l 14C-residues. these extracts. derivatized

The use of C18 s e m i - p r e p a r a t i v e HPLC r e s o l v e d a t l e a s t

two p o l a r 14C-compounds i n

¥1ame p h o t o m e t r i c (P mode) and t h e r m i o n i c GC a n a l y s i s (two columns) of

samples of one of the HPLC 14C-peaks i n d i c a t e d the p r e s e n c e of 14C-diphenyl phosphate

in 14C-D-exposed f i s h ( F i g u r e 2 ) .

Subsequent a n a l y s i s by GC-MS confirmed the p r e s e n c e of

d i p h e n y l phosphate and t h e e l e c t r o n impact s p e c t r a i s shown in f i g u r e 3. 14C-labeled~ d i p h e n y l phosphate was a l s o d e t e c t e d i n s i m i l i a r

fractions

Although i t was not of 14C-IP-exposed f i s h .

The o t h e r 14C-peak ( s o l v e n t I I ) r e s o l v e d by s e m i - p r e p a r a t i v e HPLC appeared to be a g l u c u r o n i d e conjugate. polarityj phenol.

Treatment of the 14C-unknown i n one sample w i t h p u r i f i e d ~ - g l u c u r o n i d a s e indicating

reduced i t s

the p o s s i b l e enzymatic c l e a v a g e of the g l u c u r o n i d e moiety from a c o n j u g a t e d

805

DP

m ul "o

A

O z

(41 rrl

B

0

Figure 2.

'

1'0

'

2'0

MiN.

Thermionic-GC a n a l y s i s of 23 ng of 14C-diphenyl phosphate (B) and a p u r i f i e d 21-day d l p h e n y l - l a b e l e d fathead minnow e x t r a c t (A). The 21-day sample (A) r e p r e s e n t s the s o l v e n t I I e x t r a c t , p u r i f i e d by HPLC.

806

lee o

~.o

A

T

1t| "

496129.

IgO O

t

50.0

800 O

77

B 5@.$-

JTO 98 65

IlI

r,,

I11

IR 0

264

54.0

~.

" ~7~

2~

~

)16 324

Figure 3.

347

334

H/E

340

44@

3N

E l e c t r o n impact GC-MS s p e c t r a o f a d i p h e n y l p h o s p h a t e s t a n d a r d (A) and a purified 21-day fathead minnow extract (B) from the diphenyl-labeled exposure.

The molecular ion was M/E - 264

and the b a s e peak i n both s p e c t r a was M/E - 7 7 . A l a r g e p o r t i o n o f the 1 4 C - r e s i d u e s extraction

i n whole body f i s h

and m i l d a c i d or base h y d r o l y s i s .

These r e s i d u e s

tissues

was n o t r e c o v e r e d by s o l v e n t

appeared t o be a s s o c i a t e d

vlth

807

proteinaceous material.

The c o n c e n t r a t i o n s

of n o n - e x t r a c t e d

t o 55Z) i n t h e 14C:D-exposed f i s h and were n o t r e a d i l y phase.

Differences

disposition

appear to exist

of s u b s t i t u t e d

The d a t a from t h i s resulting earlier

i n " i n v i v o " b i o c h e m i c a l mechanisms r e l a t e d

and n o n - s u b s t i t u t e d

study indicate

work of Muir ( 7 ) .

phosphate) reactions

by f i s h .

indicates

high (8

to the

p r o d u c t s o f 2-IPDP. of t h e 2-IPDP may have o c c u r r e d ,

The p r e s e n c e of d i p h e n y l p h o s p h a t e c o n f i r m s t h e

C o n j u g a t i o n of p h e n o l s may be a m a j o r r o u t e of e x c r e t i o n

phosphate esters

and p r o t e i n a c e o u s m a t e r i a l s

hydrolysis

that enzymatic hydrolysis

i n p h e n o l i c s and d i p h e n y l p h o s p h a t e .

hydrolyzed triaryl

r e s i d u e s were p a r t i c u l a r l y

e x c r e t e d d u r i n g t h e 14-day e l i m i n a t i o n

for

The p r e s e n c e of 1 4 C - r e s i d u e s a s s o c i a t e d

with lipids

t h e o c c u r r e n c e of p h e n o l i c o r f r e e a c i d ( d i p h e n y l

with biogenics or ring destruction

and s u b s e q u e n t i n c o r p o r a t i o n

of

14C-fragments into biochemical pathways. ACKNOWLEDGEMENTS We t h a n k G i n g e r G i b s o n and Thomas Durham f o r sample p r o c e s s i n g ; implementing biological

Denny B u c k l e r f o r

e x p o s u r e s ; and Derek Muir f o r s u p p l y i n g 1 4 C - d i p h e n y l p h o s p h a t e and f o r

a l l o w i n g us t o u s e u n p u b l i s h e d i n f o r m a t i o n on t h e f a t e of t r i a r y l

phosphates.

REFERENCES

(1)

" A s s e s s m e n t o f t h e Need f o r L i m i t a t i o n on T r i a r y l

Midwest R e s e a r c h I n s t i t u t e

and T r i a l k y l / A r y l

f o r t h e U. S. E n v i r o n m e n t a l P r o t e c t i o n

P h o s p h a t e s " (1979)

Agency, W a s h i n g t o n , D.C., EPA

C o n t r a c t No. 6 8 - 0 1 - 4 3 1 3 . (2)

Mayer, F. L.

(1981)

Adams, W. J . ,

F i n l e y , M. T . , M i c h a e l , P. R., M e h r l e , P. M. & S a e g e r , V. W.

"Phosphate Ester Hydraulic Fluids:

An A q u a t i c E n v i r o n m e n t a l A s s e s s m e n t of P y d r a u l s 50E

and l l S E " A q u a t i c T o x i c o l o g y and Hazard A s s e s s m e n t :

(3)

F o u r t h C o n f e r e n c e , ASTM STP 737, 1 0 3 - 1 2 3 .

Saeger, V W., Hicks, 0., Kaley, R. G., Michael, P. R., Mieure, J. P., & Tucker, E. S.

(1979) E n v i r o n . S c i . T e c h n o l .

13, 8 4 0 - 8 4 4 .

(4) Lombardo, P and Ergy, I . J . (1979) J . A s s o c . O f f . A n a l . Chem. 62, 4 7 - 5 1 . (5)

H e s s e l b e r g , R. J . & J o h n s o n , J . L.

(6)

Schultz,

(7)

Muir, D. C. G. & G r i f t ,

(1972) B u l l . E n v i r o n . Contam. T o x i c o l . 7, 1 1 5 - 1 2 0 .

D. P. (1973) J . A g r i c . Food Chem. 21, 1 8 6 - 1 9 2 . N. P.

(1982) " D e t e r m i n a t i o n of T r i a l k y l / A r y l

D e g r a d a t i o n P r o d u c t s i n W a t e r , Sediment and F i s h , " Workshop, Ottawa, O n t a r i o , May 1 2 - 1 4 .

(Received in The N e t h e r l a n d s

iO February

P h o s p h a t e s and t h e i r

p r e s e n t e d a t t h e S i x t h Annual A.O.A.C. S p r i n g

1983)