Photodegradation of Halogenated Xanthene Dyes - ACS Publications

4

Downloaded by UNIV OF MICHIGAN ANN ARBOR on February 18, 2015 | http://pubs.acs.org Publication Date: June 30, 1978 | doi: 10.1021/bk-1978-0073.ch004

Photodegradation of Halogenated Xanthene Dyes JAMES R. HEITZ Department of Biochemistry, Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, Mississippi State, MS 39762 W. W. WILSON Department of Chemistry, Mississippi State University, Mississippi State, MS 39762 At the outset, the work of several other of our colleagues who aided in the collection of the data used in this presenta tion should be acknowledged. They are Dr. Gajanan Pimprikar, Mr. Richard D. Vincent, Mr. John E. Fondren, Jr., Mr. William A. Peoples, II, and Mr. Kashinath Nag--all of Mississippi State University. Since this symposium is devoted to "Disposal and Decontam ination of Pesticides," it is appropriate to justify the inclu sion of xanthene dyes as pesticides before discussing our work on the degradation and detoxification of these molecules. Within the last six years, i t has been reported that when insects were fed certain dyes and subsequently exposed to visible light, a toxic reaction was observed (1-10). An example of this toxicity in insects caused by the synergistic effects of visi ble light and dyes is shown in Table I. Imported fire ants were field collected and maintained in the mound soil in the labora tory on a water diet for seven days. After that time, approxi mately 100 specimens were put into glass petri dishes. A piece of wet filter paper provided moisture. Aqueous sucrose solu tions, to which variable amounts of rose bengal had been added, were placed in plastic cups containing a small piece of cotton dental wicking. This served as the food source as well as the means of administering the dye to the insects. After incubating the fire ants in the petri dishes for 24 hours, they were exposed to 3800μW/cm visible light from two 40W General Electric cool white fluorescent lamps. This is approximately 10 percent of the light available on a sunny day. The data are presented as percent mortality ± one standard deviation at 1, 2, 3, 4, and 6 hrs of light exposure at four rose bengal concentrations ranging from 4.9 x 10 M down to 4.9 x 10 M. There is an inverse relationship between the feeding concentration and the LT value as well as the time of light exposure and the LD value. Later studies have shown that it is a superior technique to present the insect mortality data as a function not only of the 2

-3

-4

50

50

In Disposal and Decontamination of Pesticides; Kennedy, M.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.


30.5 (±16.4)

7.4 (±4.9)

3.7 (±2.6)

2.46

0.98

0.49 6.1 (±3.6)

14.5 (±7.1)

45.1 (±21.8)

64.7 (±9.2)

13.6 (±7.9)

23.6 (±4.3)

59.8 (±29.3)

88.7 (±8.9)

23.8 (±14.4)

34.3 (±7.0)

73.6 (±17.7)

94.8 (±3.7)

Exposure Time ( h r ) * 2 3 4

44.4 (±20.6)

60.7 (±7.4)

89.0 (±14.8)

99.7 (±0.7)

6

50

7.0

5.2

2.4

0.7

L T

b

.97

.98

.99

.90

r

5 0

1

LD c 5.5 3.5 (10-3 M) 2.5 1.8 0.24 r .96 .97 .98 .96 9 ^Data presented as percent mean m o r t a l i t y ± one standard d e v i a t i o n , d e t e r m i n e d by l i n e a r r e g r e s s i o n o f m o r t a l i t y data a t a known dose l e v e l . CDetermined by l i n e a r r e g r e s s i o n o f m o r t a l i t y data d u r i n g a known time i n t e r v a l . (Reproduced from Reference 3.)

42.1 (±7.6)

1

4.90

Rose Bengal (10-3 M )

M o r t a l i t y o f the Imported F i r e Ant as a F u n c t i o n o f Dye C o n c e n t r a t i o n and Time

Table I



4.

ΗΕΓΤΖ AND WILSON

Halogenated Xanthene Dyes

37

d i e t a r y dye c o n c e n t r a t i o n ; b u t a l s o as a f u n c t i o n o f t h e t i s s u e dye l e v e l . The f i r s t t o x i c r e a c t i o n i n v o l v e d t h e s y n e r g i s t i c e f f e c t o f v i s i b l e l i g h t and dyes on t h e i n s e c t . A second dye induced t o x i c r e a c t i o n i n i n s e c t s was discovered i n our l a b o r a t o r y which occurred i n t h e absence o f l i g h t and was c o n s i d e r a b l y slower than t h e p r e v i o u s l y discussed l i g h t - c a t a l y z e d r e a c t i o n . Figure 1 shows the p r o b i t m o r t a l i t y f o r three i n s e c t species exposed t o a 5 χ 10"3M rose bengal food source i n t h e absence o f l i g h t . P r o b i t s a r e merely a l i n e a r t r a n s f o r m a t i o n o f the sigmoidal death curve. The feeding medium f o r t h e b o l l w e e v i l (X) was an a r t i f i c i a l d i e t e s s e n t i a l l y developed by L i n d i g and Malone (11). A one percent sucrose s o l u t i o n was used f o r the imported f i r e a n t (0). A two percent m i l k - s u c r o s e s o l u t i o n was used f o r the house f l y (·). P r e l i m i n a r y s t u d i e s o f the face f l y i n d i c a t e that the s u s c e p t i b i l i t y of t h i s insect c l o s e l y p a r a l l e l s that of the house f l y . There appears t o be a wide divergence i n t h e s u s c e p t i b i l i t y o f d i f f e r e n t species o f i n s e c t t o t h e l i g h t independent t o x i c mechanism. The mechanism which appears t o be o p e r a t i v e i n the l i g h t c a t a l y z e d r e a c t i o n i s shown i n F i g u r e 2. The dye i n the ground s i n g l e t s t a t e absorbs a photon o f v i s i b l e l i g h t (a) and i s e x c i t e d t o some higher s i n g l e t s t a t e . I f the dye was r a i s e d t o the second e x c i t e d s i n g l e t s t a t e o r some higher s t a t e , i t would g i v e o f f the excess energy as heat (h) and decay t o the f i r s t e x c i t e d s i n g l e t s t a t e . The l i f e t i m e o f t h e f i r s t e x c i t e d s i n g l e t s t a t e i s on the order o f nanoseconds· There a r e three main f a t e s o f t h e dye molecule i n t h i s s t a t e : 1) i t may g i v e o f f the excess energy as heat (h) and r e t u r n t o t h e ground s i n g l e t s t a t e ; 2) i t may g i v e o f f the excess energy as l i g h t ( f ) and r e t u r n t o the ground s i n g l e t s t a t e — t h i s i s d e f i n e d as fluorescence; 3) i t may go from a s i n g l e t s t a t e t o a t r i p l e t s t a t e by i n v e r t i n g the s p i n o f an e l e c t r o n — t h i s i s c a l l e d i n t e r s y s t e m c r o s s i n g . I f t h e dye moves t o t h e t r i p l e t s t a t e by intersystem c r o s s i n g , i t has reached a more s t a b l e s t a t e w i t h a l i f e t i m e greater than microseconds. I f one ignores t h e r e v e r s e intersystem c r o s s i n g back t o the f i r s t e x c i t e d s i n g l e t s t a t e , there a r e three main f a t e s o f t h e dye molecule i n t h i s s t a t e : 1) i t may g i v e o f f the excess energy as heat (h) and r e t u r n t o the ground s i n g l e t s t a t e ; 2) i t may g i v e o f f t h e energy as l i g h t (p) and r e t u r n t o t h e ground s i n g l e t s t a t e — t h i s i s d e f i n e d as phosphorescence; 3) i t may g i v e the energy t o a second molecule, i n t h i s case, oxygen. The dye thereby r e t u r n s t o t h e ground s i n g l e t s t a t e and the oxygen i s r a i s e d t o the f i r s t e x c i t e d s t a t e . T h i s use o f t h e dye molecule t o absorb l i g h t energy and t r a n s f e r i t t o oxygen t o form the very r e a c t i v e and t o x i c s i n g l e t oxygen i s i n t e g r a l to d y e - s e n s i t i z e d p h o t o o x i d a t i o n and probably t o t h e dye-induced t o x i c i t y o f v i s i b l e l i g h t i n i n s e c t s . Since movement o f t h e s i n g l e t dye t o t h e t r i p l e t dye i s c r i t i c a l to the t o x i c r e a c t i o n , t h e more phosphorescent t h e dye i s , the



38

DISPOSAL AND DECONTAMINATION OF PESTÏODES

more t o x i c i t should be. W i t h i n t h e xanthene s e r i e s o f dyes, t h i s has been observed. Decreasing t o x i c i t y g e n e r a l l y corresponds w i t h decreasing halogen content. F i g u r e 3 shows t h e s t r u c t u r e o f the xanthene dyes most s t u d i e d t o date. Rose bengal, t h e b e s t s e n s i t i z e r t o date i n a l l cases, c o n t a i n s i o d i n e a t A and c h l o r i n e a t B. P h l o x i n B, c u r r e n t l y being t e s t e d on t h e imported f i r e ant, c o n t a i n s bromine a t A and c h l o r i n e a t B. E r y t h r o s i n B, c u r r e n t l y under t e s t w i t h two Musca s p e c i e s , c o n t a i n s i o d i n e a t A and hydrogen a t B. E o s i n y e l l o w i s h c o n t a i n s bromine a t A and hydrogen a t B. The two dyes shown i n e f f e c t i v e i n every study t o date a r e f l u o r e s c e i n , c o n t a i n i n g hydrogen a t both A and Β and rhodamine B, c o n t a i n i n g hydrogen a t A and Β and w i t h two wing oxygens r e p l a c e d by d i e t h y l a m i n o groups. T h i s i n s p e c t i o n o f the dye s t r u c t u r e s a l s o may serve t o e x p l a i n t h e observed s i m i l a r i t y between t h e light-dependent and l i g h t - i n d e p e n d e n t mechanisms. Increased halogen content may i n c r e a s e t h e t o x i c i t y o f t h e light-dependent mechanism by i n c r e a s i n g t h e t r i p l e t s t a t e p o p u l a t i o n s . Increased halogen content may i n c r e a s e the t o x i c i t y o f t h e l i g h t - i n d e p e n d e n t mechanism s i m i l a r t o an o r g a n o c h l o r i n e mechanism. There would be two e n t i r e l y d i f f e r e n t mechanisms, b u t i n e x t r i c a b l y l i n k e d together through the halogen content o f t h e dye molecules themselves. Having shown t h e p e s t i c i d a l p o t e n t i a l o f t h e xanthene dyes, i t now becomes important t o i n v e s t i g a t e t h e degradation o f t h e dyes once they a r e i n t r o d u c e d i n t o t h e environment. When n i t r o g e n gas was bubbled through a rose bengal s o l u t i o n t o decrease as f a r as p o s s i b l e t h e oxygen c o n c e n t r a t i o n i n the s o l u t i o n , then s e a l e d and p l a c e d i n s u n l i g h t f o r two months, a s t r a w - c o l o r e d s o l u t i o n was generated. I f t h e rose bengal s o l u t i o n was n o t deoxygenated and was i n s t e a d l e f t open t o t h e environment and f u l l s u n l i g h t f o r two months, a c l e a r s o l u t i o n was generated. The i n c r e a s e d a v a i l a b i l i t y o f oxygen i n t h e l a t t e r case probably f a c i l i t a t e d t h e d e c o l o r i z a t i o n r e a c t i o n . The e x t e n t o f the d e c o l o r i z a t i o n i s shown i n F i g u r e 4. The v i s i b l e a b s o r p t i o n spectrum o f rose bengal between 400nm and 600nm i s d e p i c t e d by curve B. Upon photodegradation, the a b s o r p t i o n disappears completely (curve A ) . A t t h i s p o i n t t h e q u e s t i o n remained as t o t h e complexity o f the photodegradation r e a c t i o n . A f t e r a p a r t i a l l y complete photodegradation r e a c t i o n h i g h performance l i q u i d chromatography was used t o i n v e s t i g a t e the composition o f the s o l u t i o n . F i g u r e 5 shows pure rose bengal on t h e l e f t and t h e p a r t i a l l y degraded rose bengal on the r i g h t . These t r a c e s , and t h e ones t h a t f o l l o w , were generated u s i n g a Waters M6000A pump, a U6K i n j e c t o r , a yBondapak r e v e r s e phase C^g column, and a 440 U V - v i s i b l e d e t e c t o r . The d e t e c t o r was s e t a t 546nm. The samples were e l u t e d w i t h a 70 percent methanol-30 percent 0.01M ammonium a c e t a t e b u f f e r . I t i s obvious t h a t as t h e rose bengal photodegrades, a m u l t i p l i c i t y o f products appears. F u r t h e r , due t o t h e decrease i n r e l a t i v e




ΗΕΓΓΖ AND WILSON

0

10

5 TIME (DAYS)

Figure 1. Light-independent mortality as a function of time of exposure to rose bengal in the food supply of the boll weevil (x), the imported fire ant (o), and the house fly (·)

2

FT

T,

_ ^

,h s|(s

1Λ

ROSE BENGAL

OXYGEN

Figure 2. Scheme suggested for the dye-sensitized photooxidation opera tive with the substituted xanthene series of dyes. (See text for explana tion of symbols. Reproduced from Reference 10).

Figure 3. The mo lecular structure of the xanthene dyes studied in this work. (See text for expla nation of the substituents A and B. Reproduced from Reference 5.)



40

DISPOSAL AND DECONTAMINATION OF PESTICIDES

Figure 4. The visible absorbance spec trum of a rose bengal solution which had been photodegradea by sunlight is shown by Trace A. Trace Β is the spectrum of the rose bengal solution before exposure to sunlight.

Figure 5. High-performance liquid chromatographic trace of purified rose bengal (left) and partially photodegraaed rose bengal (right) observed with a 546-nm absorbance

400

500 WAVELENGTH (nm)

TIME


600

4.

ΗΕΓΤΖ AND WILSON

41



1

r e t e n t i o n ( k ) o f the p r o d u c t s , one can p o s t u l a t e t h a t these products are more p o l a r than rose bengal. F i n a l l y , t h e f a c t t h a t these products are a b l e t o be d e t e c t e d a t 546nm and t h e f a c t t h a t completely photodegraded rose bengal has no v i s i b l e a b s o r p t i o n , i n d i c a t e s t h a t each o f these products i s an interme d i a t e i n the o v e r a l l r e a c t i o n . Although rose bengal and the photodegraded products do n o t absorb i n the u l t r a v i o l e t as s t r o n g l y as i n the v i s i b l e , the UV t r a c e s o f the l i q u i d chromatographic s e p a r a t i o n are shown i n F i g u r e 6. Pure rose bengal i s shown on the l e f t and p a r t i a l l y photodegraded rose bengal on the r i g h t . A g a i n , i t may be seen t h a t a l l o f the products formed d u r i n g t h i s r e a c t i o n are more p o l a r ; t h a t i s , a lower k , than rose bengal i t s e l f . In F i g u r e 7, the two p a r t i a l l y degraded r o s e bengal t r a c e s are presented together w i t h the 280nm a b s o r p t i o n t r a c e above t h e 546nm a b s o r p t i o n t r a c e . The p o s i t i o n o f rose bengal on these t r a c e s would correspond w i t h the s t r o n g doublet on the 546nm t r a c e . The p u r i f i c a t i o n and i d e n t i f i c a t i o n o f these i n t e r m e d i a t e compounds i s c u r r e n t l y an area o f prime c o n s i d e r a t i o n i n our laboratory. The r a t e o f photodegradation o f the xanthene dyes has a l s o been s t u d i e d . F i g u r e 8 shows the decrease i n absorbance a t 546nm o f s o l u t i o n s o f rose bengal as a f u n c t i o n o f i l l u m i n a t i o n time a t 5 l i g h t i n t e n s i t i e s between lmE/m «sec and 6mE/m . s e c . The l o g a r i t h m o f the absorbance i s l i n e a r l y dependent on t h e time o f i l l u m i n a t i o n and i s i n d i c a t i v e o f a f i r s t order r a t e o f r e a c t i o n . The pseudo f i r s t order r a t e constant (k^) can be c a l c u l a t e d f o r each r e a c t i o n by the method o f h a l f - l i v e s . I f the k i v a l u e s are p l o t t e d as a f u n c t i o n o f the l i g h t i n t e n s i t y used t o generate those v a l u e s , as shown i n F i g u r e 9, one o b t a i n s a l i n e a r r e l a t i o n s h i p . The slope o f t h a t l i n e i s k # a pseudo second order r a t e c o n s t a n t , which p r o v i d e s the b a s i s f o r compari son o f the s u s c e p t i b i l i t y t o photodegradation o f the v a r i o u s dyes (Table I I ) . By t h i s c r i t e r i o n , e r y t h r o s i n Β i s the most r e a d i l y photodegradable w i t h e o s i n y e l l o w i s h and rose bengal c l o s e behind. The two dyes t e s t e d which c o n t a i n no halogen, rhodamine Β and f l u o r e s c e i n , are most r e s i s t a n t t o photodegrada t i o n . I t appears t h a t halogen on the upper r i n g system f a c i l i t a t e s the r e a c t i o n w h i l e halogen presence on the lower r i n g r e t a r d s the photodegradation r e a c t i o n . I t may a l s o be mentioned t h a t phosphorescent dyes g e n e r a l l y photodegrade w h i l e f l u o r e s c e n t dyes do not. A t low dye c o n c e n t r a t i o n s , the r e a c t i o n i s c l e a r l y f i r s t order (Figure 10). As the i n i t i a l c o n c e n t r a t i o n i n c r e a s e s , t h e top l i n e f o r i n s t a n c e , t h e i n i t i a l p o r t i o n o f the r e a c t i o n i s slower than the f i r s t order r e a c t i o n observed a t lower concentra t i o n s . When the o v e r a l l r e a c t i o n i s observed a t v e r y h i g h c o n c e n t r a t i o n s , a d d i t i o n o f the two r e a c t i o n s y i e l d e d apparent zero order data. 1

2

2

2


42

DISPOSAL AND DECONTAMINATION OF PESTODES

h< _J

Figure 6. High-performance liquid chromatographic trace of purified rose bengal (left) and partially photode graded rose bengal (right) observed with a 280-nm absorbance detector

TIME

< oc

2 k—

5

Figure 7. Direct comparison of partially photodegraded rosebengal according to 280-nm absorbance (top) and 546-nm absorbance (bottom)

TIME


4.

ΗΕΓΤΖ AND WILSON 0.30

43


f


0.10

0.03

60 120 ILLUMINATION TIME (MIN)

180

Figure 8. Semi-log plot showing the de crease in absorbance of a rose bengal solution as a function of illumination time. Each trace represents data taken at a different intensity: (·), 1.0; (O), 2.0; (m), 3.5; (Π), 5.0; (x), 6.0; mE/m 'sec. 2

4

Figure 9. The rate constant k plotted as a function of the inci dent light intensity t

I 2 3 4 5 6 LIGHT INTENSITY (mE/m sec) 2

40

120

80

ILLUMINATION TIME

(MIN)

Figure 10. Semi-log plot showing the de crease in absorbance of rose bengal solu tions as a function of illumination time. Each trace represents data taken at a dif ferent rose bengal concentration: (x), 3.3 X 1(T*M; (O), 6.6 X 10'M; (·), 13.2 X JO M.


e


44

F i g u r e 11 shows t h i s e f f e c t from a d i f f e r e n t v i e w p o i n t . I f k^, c a l c u l a t e d from the h a l f - l i f e , i s p l o t t e d a g a i n s t t h e i n i t i a l rose bengal c o n c e n t r a t i o n , t h e r e a c t i o n r a t e i s found t o decrease as t h e rose bengal c o n c e n t r a t i o n i n c r e a s e s t o about 6 χ 1 0 " % , a t which p o i n t t h e r a t e becomes independent o f dye c o n c e n t r a t i o n . Below 4 χ 10" M, as shown i n t h e i n s e t , t h e r a t e i s a l s o indepen dent o f rose bengal c o n c e n t r a t i o n . I t may be hypothesized t h a t e i t h e r s u b s t r a t e quenching o f the e x c i t e d s t a t e by the dye may cause t h e decrease i n r e a c t i o n r a t e o r e l s e l i g h t i s r a t e l i m i t i n g a t c o n c e n t r a t i o n s higher than 6 χ 10" M.


6

5

Table I I R e l a t i v e Rates o f H y d r o l y s i s Erythrosin Β Eosin Yellowish Rose Bengal Phloxin Β Fluorescein Rhodamine Β

12 10 10 5 1 0

Another q u e s t i o n o f c r i t i c a l importance t o t h i s study i s whether t h e dyes a r e d e t o x i f i e d d u r i n g t h e photodegradation process. Table I I I shows t h a t completely photodegraded rose bengal was completely i n e f f e c t i v e i n e l i c i t i n g the l i g h t - c a t a l y z e d t o x i c r e a c t i o n which was induced by t h e presence o f undegraded rose bengal. Both rose bengal s o l u t i o n s were made up a t 7 χ 10" M; one was photodegraded i n s u n l i g h t and t h e other l e f t i n t h e dark. Both s o l u t i o n s were concentrated t o an e f f e c t i v e l e v e l o f 5 χ 10" M b e f o r e f e e d i n g t o t h e house f l i e s f o r 24 h r s i n t h e dark. A f t e r 21 h r s exposure t o the f l u o r e s c e n t l i g h t , 95 percent m o r t a l i t y was observed due t o undegraded rose bengal compared w i t h zero m o r t a l i t y i n both photodegraded r o s e bengal and c o n t r o l p o p u l a t i o n s . A second experiment where t h e rose bengal c o n c e n t r a t i o n s were 2.5 χ 10" M, showed s i m i l a r r e s u l t s . F l i e s t r e a t e d w i t h undegraded rose bengal were a l l dead w i t h i n 5 hrs o f l i g h t exposure. There were no dead f l i e s i n e i t h e r t h e degraded rose bengal o r c o n t r o l p o p u l a t i o n s . These r e s u l t s were not a l l t h a t s u r p r i s i n g s i n c e the photodegraded rose bengal does not absorb v i s i b l e l i g h t . Table IV, however, shows a key e x p e r i ment. The photodegraded dye was a l s o i n c a p a b l e o f e l i c i t i n g t h e l i g h t - i n d e p e n d e n t t o x i c r e a c t i o n caused by undegraded rose bengal. Upon f e e d i n g on 2.5 χ 1 0 " % undegraded rose bengal i n the dark, m o r t a l i t y was observed a f t e r 72 h r s and by 96 h r s , 90 percent o f the f l i e s were dead. There was no t o x i c i t y i n t h e other two p o p u l a t i o n s . 6

4

3


4.

ΗΕΓΓΖ AND WDLSON


45

Table I I I


T o x i c i t y t o House F l i e s o f Photodegraded and Undegraded Rose Bengal i n t h e L i g h t

Treatment

0

Control Rose Bengal* Photodegraded Undegraded

0

a

Time o f I l l u m i n a t i o n (hr) 19 17 15 12 3

21

0

0

0

0

0

0

0 0

0 40

0 55

0 75

0 75

0 95

5

a

b

0 0

D a t a presented as percent m o r t a l i t y where the c o n t r o l was one chamber o f 10 f l i e s and t h e dye-treated were two chambers o f 10 f l i e s each. I n i t i a l rose bengal s o l u t i o n s were 7 χ 10""% i n d i s t i l l e d water. Photodegraded samples were exposed t o s u n l i g h t f o r 3 days, undegraded samples were kept i n t h e dark f o r same time p e r i o d . Dye samples were concentrated t o 5 χ 1 0 ~ % f o r adminis tration to f l i e s .

Table IV T o x i c i t y t o House F l i e s o f Photodegraded and Undegraded Rose Bengal i n t h e Dark

Treatment Control Rose Bengal* Photodegraded Undegraded

Time o f I l l u m i n a t i o n (hr) 84 72 36 60

0 0

a

96

0

0

0

0

0

0 0

0 0

0 0

0 30

0 90

3

0 0

a

D a t a presented as p e r c e n t m o r t a l i t y where each treatment was one chamber o f 10 f l i e s each. ^ I n i t i a l rose bengal s o l u t i o n s were 7 χ 1 0 " % i n d i s t i l l e d water. Photodegraded samples were exposed t o s u n l i g h t f o r 3 days, undegraded samples were kept i n t h e dark f o r same time p e r i o d . Dye samples were concentrated t o 2.5 χ 1 0 " % f o r a d m i n i s t r a t i o n to f l i e s .



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Photodegradation of Halogenated Xanthene Dyes - ACS Publications

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