Immunoassay as a Screening Tool for Triazine Herbicides in Streams

Dec 26, 1990 - 2 U.S. Geological Survey, U.S. Department of the Interior, Lawrence, KS ... were used for weed control in the production of corn and so...
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Comparison with Gas Chromatographic-Mass Spectrometry Methods 1

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D. AGoolsby ,E. M.Thurman ,M. L. Clark , and M. L. Pomes 1

U.S. Geological Survey, U.S. Department of the Interior, Denver, CO 80225 U.S. Geological Survey, U.S. Department of the Interior, Lawrence, KS 66049 U.S. Geological Survey, U.S. Department of the Interior, Iowa City, IA 53344

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Immunoassay and GC/MS (gas chromatography/mass spectrometry) analysis for triazine herbicides were compared in order to evaluate the potential of immunoassay as a screening tool. Water samples were collected at 146 sites on streams in a 10-state region of the midwestern United States during 1989 before and shortly after application of pre-emergent herbicides that were used for weed control in the production of corn and soybeans. The sites were sampled a third time in fall 1989 during a low streamflow period. The two methods compared well with a rank correlation coefficient of 0.90 for immunoassay results less than 5 ug/L (micrograms per liter). The presence or absence of triazine herbicides can be determined visually at concentrations of about 0.5 ug/L, and the detection limit can be lowered to about 0.2 ug/L with a spectrophotometer. The median pre-application and fall concentrations of triazine herbicides in the streams were determined by immunoassay to be less than 0.2 ug/L and 0.3 ug/L, respectively, compared with a post-application median of 3.4 ug/L. Immunoassay appears to be a rapid, reliable, and low-cost analytical screening method for detecting triazine herbicides in water.

Large quantities of pre-emergent herbicides are used annually in the corn (Zea mays L.) and soybean (Glycine max L.) producing g region of the midwestern United States. Approximately 1.5 x 10 kg (kilograms) are applied each year over a 10-state region (1). The herbicides are applied to cropland prior to or during planting of seed within about a 1-month period from late April through late May. Typical application rates for atrazine and alachlor, the This chapter not subject to U.S. copyright Published 1991 American Chemical Society Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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p r e d o m i n a n t h e r b i c i d e s u s e d i n the a r e a , range from about 0.8 t o 1.5 k g / h e c t a r e . P r e v i o u s s t u d i e s have shown t h a t r u n o f f from f i e l d s immediately a f t e r a p p l i c a t i o n r e s u l t s i n l a r g e c o n c e n t r a t i o n s of h e r b i c i d e s i n s u r f a c e water (2. 3 ) . T h i s f i n d i n g has been documented i n s e v e r a l s m a l l s c a l e s t u d i e s (4-10), b u t has n o t b e e n examined a t a m u l t i - s t a t e s c a l e t o d e t e r m i n e i f t h e h e r b i c i d e s cause r e g i o n a l w a t e r - q u a l i t y contamination. Large c o n c e n t r a t i o n s o f h e r b i c i d e s i n d r i n k i n g water, consumed o v e r l o n g p e r i o d s o f time c a n have a d v e r s e e f f e c t s on human h e a l t h ( 1 1 ) . Furthermore, c o n v e n t i o n a l water t r e a t m e n t p r a c t i c e s do n o t e f f e c t i v e l y remove h e r b i c i d e s , s u c h as a t r a z i n e ( 1 1 ) . I n o r d e r t o a d d r e s s t h e s e c o n c e r n s , a r e c o n n a i s s a n c e was c o n d u c t e d d u r i n g 1989 on a l a r g e number o f streams i n 10 S t a t e s ( I l l i n o i s , I n d i a n a , Iowa, Kansas, M i n n e s o t a , M i s s o u r i , Nebrasks, Ohio, South Dakota, and W i s c o n s i n ) i n the midwestern U n i t e d S t a t e s . O b j e c t i v e s o f the r e c o n n a i s s a n c e were t o : (1) Determine the u s e f u l n e s s o f immunoassay as a l o w - c o s t s c r e e n i n g t o o l f o r t r i a z i n e h e r b i c i d e s p r i o r t o the a n a l y s e s o f water samples by more c o s t l y GC/MS, and (2) d e t e r m i n e i f immunoassay i s a v i a b l e method t o a s s e s s the g e o g r a p h i c and s e a s o n a l d i s t r i b u t i o n o f h e r b i c i d e s i n streams i n a l a r g e r e g i o n o f the midwestern U n i t e d S t a t e s . The f o c u s o f t h i s paper w i l l be on the u s e f u l n e s s o f immunoassay as a screening tool. The s e a s o n a l and g e o g r a p h i c d i s t r i b u t i o n o f h e r b i c i d e s w i l l be a d d r e s s e d elsewhere. Experimental Design o f Reconnaissance. Sampling s i t e s were s e l e c t e d a t 150 U.S. G e o l o g i c a l Survey s t r e a m f l o w - g a g i n g s t a t i o n s by a s t r a t i f i e d random p r o c e d u r e d e s i g n e d t o ensure g e o g r a p h i c d i s t r i b u t i o n . D r a i n a g e - b a s i n a r e a s r e p r e s e n t e d by the s a m p l i n g s i t e s ranged from 2 2 l e s s t h a n 260 km (square k i l o m e t e r s ) t o more t h a n 1,800,000 km f o r a s i t e on the M i s s i s s i p p i R i v e r . The median d r a i n a g e a r e a was 2 1800 km . The a g g r e g a t e d r a i n a g e a r e a o f i n d i v i d u a l h y d r o l o g i c 2 u n i t s sampled was about 518,000 km . The streams were sampled t h r e e times d u r i n g 1989: (1) I n l a t e March and A p r i l b e f o r e h e r b i c i d e s were a p p l i e d t o the f i e l d s (145 immunoassay a n a l y s e s ) , (2) i n May and June d u r i n g r u n o f f from the f i r s t r a i n f a l l f o l l o w i n g h e r b i c i d e a p p l i c a t i o n (135 immunoassay a n a l y s e s ) , and (3) i n the f a l l (October-November) d u r i n g l o w - s t r e a m f l o w c o n d i t i o n s when most o f t h e water i n t h e streams was d e r i v e d from ground-water s o u r c e s (146 immunoassay a n a l y s e s ) . V e r t i c a l l y i n t e g r a t e d water samples were c o l l e c t e d a t t h r e e t o f i v e p o i n t s a c r o s s each stream. Samples were c o l l e c t e d and c o m p o s i t e d i n g l a s s c o n t a i n e r s and f i l t e r e d t h r o u g h g l a s s f i b e r f i l t e r s (GGF14230, Geotech, I n c . , Denver, C o l o . ) h a v i n g a p o r e d i a m e t e r o f a p p r o x i m a t e l y 1 micrometer i n o r d e r t o remove particulate material. The f i l t r a t e was c o l l e c t e d i n 125-mL ( m i l l i l i t e r ) g l a s s b o t t l e s t h a t had been h e a t e d t o 350°C ( d e g r e e s C e l s i u s ) to remove o r g a n i c m a t t e r . Samples were r e f r i g e r a t e d t o about 4°C u n t i l

analyzed.

Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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Immunoassay. A l l samples were a n a l y z e d f o r t r i a z i n e h e r b i c i d e s u s i n g RES-I-MUNE immunoassay k i t s (ImmunoSystems I n c . , S c a r b o r o u g h , Maine). The k i t s use p o l y c l o n a l a n t i b o d i e s c o a t e d on the w a l l s o f p o l y s t y r e n e t e s t tubes and an a t r a z i n e - e n z y m e c o n j u g a t e p r e p a r e d by c o v a l e n t l y b i n d i n g a t r a z i n e to h o r s e r a d i s h p e r o x i d a s e by a m o d i f i e d c a r b o d i i m i d e t e c h n i q u e (12-13). O t h e r r e a g e n t s i n the immunoassay k i t s i n c l u d e t h r e e s t a n d a r d s ( n e g a t i v e c o n t r o l , 0.1 ug/L a t r a z i n e s o l u t i o n and 1.0 ug/L a t r a z i n e s o l u t i o n ) , s u b s t r a t e , chromogen, and a " s t o p " s o l u t i o n o f 2.5 N (normal) s u l f u r i c a c i d . I n s t r u c t i o n s p r o v i d e d w i t h the k i t were f o l l o w e d . The k i t s were s t o r e d a t 4 t o 8°C

and

the c o n t e n t s were a l l o w e d

t o warm t o

about 23°C p r i o r t o making the a n a l y s i s . The samples and s t a n d a r d s o l u t i o n s (160 m i c r o l i t e r s o r 4 d r o p s ) were p l a c e d i n s e p a r a t e t e s t t u b e s , i m m e d i a t e l y f o l l o w e d by 4 drops o f the enzyme c o n j u g a t e . The t e s t tube was g e n t l y mixed f o r 2-3 seconds, and t h e n a l l o w e d t o incubate f o r 5 minutes. A t r a z i n e i n the sample and the a t r a z i n e enzyme c o n j u g a t e compete f o r a l i m i t e d number o f a n t i b o d y b i n d i n g s i t e s on the t e s t tube. A f t e r the i n c u b a t i o n p e r i o d , the u n r e a c t e d m o l e c u l e s were washed away by r i n s i n g the t e s t tube f o u r times w i t h reagent water. S u b s t r a t e (4 d r o p s ) and chromogen (4 d r o p s ) were added, g e n t l y mixed i n the tube, and a l l o w e d to i n c u b a t e f o r 2 minutes. The p r e s e n c e o f bound a t r a z i n e - e n z y m e c o n j u g a t e i n the t e s t tube c o n v e r t s the s u b s t r a t e to a compound t h a t c a u s e s the chromogen to t u r n b l u e . Because the number o f a n t i b o d y b i n d i n g s i t e s and the number o f a t r a z i n e - e n z y m e c o n j u g a t e m o l e c u l e s r e m a i n about c o n s t a n t , a sample c o n t a i n i n g a s m a l l c o n c e n t r a t i o n o f a t r a z i n e a l l o w s many a t r a z i n e - e n z y m e c o n j u g a t e m o l e c u l e s t o be bound by the a n t i b o d y . Therefore, a small concentration of a t r a z i n e i n the water sample p r o d u c e s a d a r k - b l u e s o l u t i o n . C o n v e r s e l y , the more a t r a z i n e p r e s e n t i n the water sample the l i g h t e r the c o l o r . Thus, c o n c e n t r a t i o n i s i n v e r s e l y p r o p o r t i o n a l to c o l o r . The r e a c t i o n was a r r e s t e d by a d d i n g 1 drop o f the 2.5 N s u l f u r i c a c i d s o l u t i o n , w h i c h h a l t s the b l u e c o l o r development and c a u s e s the r e a c t i o n s o l u t i o n t o t u r n y e l l o w . A v i s u a l comparison between the samples and the n e g a t i v e c o n t r o l t h e n was made t o d e t e r m i n e i f t r i a z i n e h e r b i c i d e s were p r e s e n t o r a b s e n t and the r e s u l t was r e c o r d e d . Because o f the r a p i d r e a c t i o n k i n e t i c s , o n l y f o u r t o f i v e samples, one s t a n d a r d , and a n e g a t i v e c o n t r o l c o u l d be a n a l y z e d i n one r u n . A f t e r the v i s u a l c o m p a r i s o n was made, 0.50 mL o f d e i o n i z e d w a t e r was added to each tube t o p r o v i d e a d d i t i o n a l volume, and the s o l u t i o n was t r a n s f e r r e d i n t o a 1 cm ( c e n t i m e t e r ) c u v e t t e . The OD ( o p t i c a l d e n s i t y ) o f the samples, s t a n d a r d s , and n e g a t i v e c o n t r o l were r e a d u s i n g a M i l t o n Roy S p e c t r o n i c 401 single-beam spectrophotometer ( L i n d e n , New J e r s e y ) t h a t had been z e r o e d a g a i n s t a d e i o n i z e d water b l a n k a t 450 nm (nanometers). The OD o f each s t a n d a r d and sample was n o r m a l i z e d f o r c o l o r v a r i a t i o n s i n the n e g a t i v e c o n t r o l by d i v i d i n g the ODs by the OD o f the n e g a t i v e c o n t r o l and m u l t i p l y i n g by 100 t o o b t a i n p e r c e n t NC ( p e r c e n t o f negative control). A w o r k i n g c u r v e was p r e p a r e d f o r each o f the t h r e e s a m p l i n g p e r i o d s by p l o t t i n g the c o n c e n t r a t i o n o f the s t a n d a r d s as a f u n c t i o n o f the p e r c e n t NC on s e m i - l o g paper f o r a l l measurements made on s t a n d a r d s d u r i n g the s a m p l i n g p e r i o d . A b e s t f i t l i n e was drawn t h r o u g h the median p e r c e n t NC f o r each s t a n d a r d (see f i g . 1 f o r example). The c o n c e n t r a t i o n f o r each sample ( i n

Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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F i g u r e 1. Immunoassay w o r k i n g c u r v e f o r r e l a t i o n between t r i a z i n e h e r b i c i d e c o n c e n t r a t i o n and o p t i c a l d e n s i t y f o r p o s t a p p l i c a t i o n sampling p e r i o d .

Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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ug/L) was o b t a i n e d by r e a d i n g the c o n c e n t r a t i o n c o r r e s p o n d i n g t o the p e r c e n t NC f o r t h a t sample. F o r q u a l i t y - a s s u r a n c e p u r p o s e s , about 8 p e r c e n t o f a l l samples were s u b m i t t e d f o r immunoassay a n a l y s i s as b l i n d d u p l i c a t e s . In a d d i t i o n , about 25 p e r c e n t o f the samples ( m o s t l y from the p r e - and p o s t - a p p l i c a t i o n p e r i o d s ) were a n a l y z e d i n a s e c o n d l a b o r a t o r y by immunoassay a n a l y s i s . The p r o c e d u r e u s e d by the s e c o n d l a b o r a t o r y was i d e n t i c a l t o t h a t d e s c r i b e d above e x c e p t t h a t a d i f f e r e n t i a l photometer ( A r t e l , I n c . , Windham, Maine) was u s e d i n s t e a d o f the M i l t o n Roy s p e c t r o p h o t o m e t e r . These q u a l i t y - a s s u r a n c e samples p r o v i d e d a means t o e v a l u a t e the v a r i a t i o n i n r e s u l t s w i t h i n a l a b o r a t o r y and between l a b o r a t o r i e s . Thurman and o t h e r s (14) have measured c r o s s r e a c t i v i t y i n the immunoassay f o r 12 common t r i a z i n e h e r b i c i d e s and metabolites r e l a t i v e to a t r a z i n e a t 1 ug/L c o n c e n t r a t i o n s f o r each h e r b i c i d e . The immunoassay r e s p o n s e was e x p r e s s e d as ug/L o f a t r a z i n e as shown i n f i g u r e 2. The r e s p o n s e was g r e a t e s t f o r a t r a z i n e (1.0 u g / L ) . Ametryn's c r o s s r e a c t i v i t y was e q u i v a l e n t t o 0.6 ug/L o f a t r a z i n e ; p r o m e t r y n and p r o p a z i n e , 0.5 ug/L; and so f o r t h ( f i g . 2 ) . The c r o s s r e a c t i v i t y o f each t r i a z i n e i s r e l a t e d t o the e x t e n t o f b i n d i n g t h a t o c c u r s between the a n t i b o d y c o a t e d on the t e s t tube and the s t r u c t u r e o f the c r o s s - r e a c t i v e m o l e c u l e . The b i n d i n g was s t r o n g e s t f o r compounds t h a t have s t r u c t u r e s most c l o s e l y r e s e m b l i n g the a t r a z i n e m o l e c u l e , t h a t i s , a 4 - e t h y l a m i n o and a 6i s o p r o p y l a m i n o group. F o r example, a 1 ug/L s o l u t i o n o f ametryn ( f i g . 2) has a c r o s s r e a c t i v i t y e q u i v a l e n t t o 0.6 ug/L o f a t r a z i n e . L i k e w i s e a s l i g h t change on the amino group from e t h y l t o i s o p r o p y l ( p r o p a z i n e ) e l i c i t s a r e s p o n s e e q u i v a l e n t t o 0.5 ug/L o f a t r a z i n e . S i m a z i n e , w i t h 2 e t h y l groups, has a c r o s s r e a c t i v i t y e q u i v a l e n t t o 0.2 ug/L o f a t r a z i n e . S u b s t i t u t i o n o f h y d r o g e n f o r e i t h e r the e t h y l ( d e s e t h y l a t r a z i n e ) o r the i s o p r o p y l ( d e s i s o p r o p y l a t r a z i n e ) l o w e r s the c r o s s r e a c t i v i t y t o l e s s t h a n 0.1 ug/L o f a t r a z i n e , o r e s s e n t i a l l y no r e s p o n s e . Because the immunizing h a p t e n (an a t r a z i n e - l i k e s t r u c t u r e ) was bound a t the 2 p o s i t i o n , i t appears t h a t the r e l a t i v e r e s p o n s e o f the immunoassay i s r e l a t e d t o a n t i b o d y r e c o g n i t i o n and b i n d i n g t o the a l k y l s i d e c h a i n s on the triazine ring. Only hydroxyatrazine d e v i a t e s from t h i s p a t t e r n i n c r o s s reactivity. Because i t has the same a l k y l s t r u c t u r e as a t r a z i n e , one w o u l d p r e d i c t a c r o s s r e a c t i v i t y s i m i l a r to ametryn (about 0.6 ug/L as a t r a z i n e ) . However, h y d r o x y a t r a z i n e gave a r e s p o n s e o f l e s s t h a n 0.1 ug/L as a t r a z i n e . A p o s s i b l e e x p l a n a t i o n i s t h a t the h y d r o x y l group d e c r e a s e s the b i n d i n g energy a t the s p e c i f i c antibody r e c o g n i t i o n s i t e . The d i d e a l k y l a t r a z i n e was nonreactive ( f i g . 2). N e i t h e r a l a c h l o r nor m e t o l a c h l o r c r o s s r e a c t e d w i t h a t r a z i n e , which i s c o n s i s t e n t w i t h r e s u l t s o f a p r e v i o u s study

(12). The immunoassay a l s o was examined f o r i n t e r f e r e n c e by n a t u r a l l y o c c u r r i n g humic and f u l v i c a c i d s (14), w h i c h a c c o u n t f o r the m a j o r i t y o f d i s s o l v e d o r g a n i c c a r b o n i n n a t u r a l w a t e r s ( 1 5 ) . A t r a z i n e was measured i n water samples t h a t c o n t a i n e d from 5 t o 100 mg/L o f humic and f u l v i c a c i d from the Suwannee R i v e r (standardr e f e r e n c e s u r f a c e - w a t e r sample) (16) and humic and f u l v i c a c i d s from B i s c a y n e a q u i f e r n e a r Miami, F l o r i d a ( 1 7 ) . F o r a l l samples, t h e r e was no d i f f e r e n c e between the immunoassay r e s p o n s e i n the p r e s e n c e and absence o f the humic m a t e r i a l . These d a t a i n d i c a t e

Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

8. GOOLSBYETAL.

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Triazine Herbicides in Streams

CI N

C)

H3C-CH-CH3

N

CHg-CHg

1 ng/L Atrazine scHg

CI

SCHg N

H

H

> ^ N - ^ N < H3C-CH-CH3 CHg-CHg

0.6 μ g / L Ametryn

H

>N^N^Sl