The environmental fate of DDT

0045-6555/78/1201-0995~02.00/0

Chemosphere No. 12, pp 995 - 998. © Pergamon Press Ltd. 1978. Printed in Great Britain.

THE ENVIRONMENTAL FATE OF DDT A.S.M. MAREI], J.M.E. QUIRKE, G. RINALDI2, J.A. ZORO3 and G. EGLINTON Organic Geochemistry Unit, School of Chemistry, University of Bristol, BRISTOL BS8 ITS, England. Present addresses: l 2 3

Pesticide Chemistry Division, Plant Protection Department, Faculty of Agriculture, University of Alexandria, Alexandria, Egypt. Department of Geology, Indiana University, Bloomington, Indiana 47401, United States of America. Home Office, Forensic Science Laboratory, Priory House, Gooch Street North, Birmingham B5 6QQ, England.

(Received

14 D e c e m b e r

1978)

DDT is degraded when dissolved in bloodI and sewage sludge2'3'4'5'6. The degradation products have not, however, been identified. Zoro et al.b have also reported unidentified products from the room temperature incubation of DDT w i t h ~ t i n with a reducing agent (sodium dithionite) and sodium carbonate. The purpose of the present study is tp carry out the reaction of DDT with hematin in conditions similar to those used by Zoro et al. b and to identify and quantitate the products. Additionally, the compounds produced w h e ~ is incubated with blood have been investigated in order to determine whether the proteins in hemoglobin modify the role of the heme in the degradation of DDT. The products of the degradation of DDT with sewage sludge were also investigated. MATERIALS AND METHODS Chemicals. DDT (l,l,l-trichloro-2,2-bis(p-chlorophenyl)-ethane), DDD (l,l-dichloro-2,2bis(p-chlorophenyl)-ethane), DDE (l,l-dichloro-2,2-bis(p-chlorophenyl)-ethylene), DBP (4,4'dichlorobenzophenone) and DDA (bis(p-chlorophenyl)-acetic acid) analytical grade were purchased from Aldrich Chemicals Ltd. DDCN(bis(p-chlorophenyl)-acetonitrile) was prepared by the method of Cooke and Zoro7 and DDMU(l-chloro-2,2-bis(p-chlorophenyl)-ethylene) was prepared by the method of Chen and Cheng8. Hematin was purchased from Sigma Chemicals Ltd. Sewagesludge (2.5% solids) was obtained from an anaerobic digestor at Avonmouth Foul Water Treatment Works, Bristol. Thin Layer Chromatography ( t . l . c . ) was carried out on 200mm x 200mm x O.5mm GF254 silica gel (Merck Ltd.) plates, pre-eluted with ethyl acetate prior to reactivation at llOOC for 2 hours. Gas Liquid Chromatography (g.l.c.) was carried out on a Perkin Elmer F-ll model equipped with a 63Ni electron capture detector, an on-column all-glass injection system and a 6 foot x 0.25 inch 'silanized' glass column with a packing of 1.5% OV-17 and 1.95% QF-I mixed phase on Diatoport S, 80-I00 mash support. The chromatograms were obtained at 185°C in the isothermal mode, with nitrogen as the carrier gas. The flow rate was 75mi minute-l. The sa~le was applied in methylene chloride, and the retention times of the peaks compared with those of standards (Table l ) . Electron Impact Mass Spectrometry was carried out using a Varian Mat CH-7 mass spectrometer with an ion source temperature of 180~C. The sample (ca. 4~g) in methylene chloride was applied to a gold,crucible: the direct insertion probe was ~ e r a t u r e programmed from 25oc - lOOOCat 18°C mn . Spectra were obtalned every 8 seconds, uslng a data system prevlously described9. •

-

|

•

.-

993

--

_

.

994

No. 12

Identification of the compounds was carried out by comparison of the mass spectra with those of the relevant standards (Table l ) . Incubation of DDT with Hematin. DDT (35mg) in ethanol (l.5ml) was added to a solution of hematin (6mg) in sodium carbonate (O.IM, lOml), sodium dithionite (250mg), and ethanol (lOml); some of the DDT precipitated from the solution. The mixture (pH 8.5) was stoppered and set aside in the dark, at room temperature, for 7 days; a precipitate was s t i l l present. After acidification to pH 6 by careful addition of dilute hydrochloric acid, the mixture was extracted with ether (25mi x 4). The combined extracts were washed with water, dried over sodium sulphate, f i l t e r e d and evaporated to dryness. The residue was dissolved in ether and subjected to t . l . c . in hexane:chloroform:methanol (27:2:1): 6 bands were obtained, each of which was scraped o f f the plate, and the products eluted with chloroform. The products from the incubation were ident i f i e d by comparison of their Rf values, and mass spectra with those of standards. Quantitation of the products was effected by g . l . c , analyses of total mixtures from the incubations, and the identities of the components were confirmed by coinjection with the appropriate standards. The retention times, Rf values and mass spectrometric data for the standards are shown in Table I. Ammonia (0.88, O.Iml) was added to the original hematin solution in a second, otherwise identical reaction which gave 7 t . l . c , bands which were identified as above. Incubation of DDT with Blood. Fresh cattle blood (15ml), treated with sodium oxalate (45mg) to prevent i t clotting, was added to a solution of sodium dithionite (250mg), aqueous sodium carbonate (O,IM, lOml) and ethanol (lOml), and incubated with DDT (35mg) in the manner described for the reaction of DDT with hematin. On acidification, the blood coagulated to a solid mass which was treated in an ultrasonic bath three times for l O minutes with diethyl ether/ hexane (20ml, l : l ) . The extracts were combined, washed with water, dried over sodium sulphate and evaporated to dryness. The residue was subjected to t . l . c . , as described in the DDT/hematin incubation, and 6 products were obtained. The incubation was repeated with the addition of ammonia (0.88, O.Iml) to the blood before addition of DDT and 7 products were obtained from t . l . c . The Rf values corresponded to those of the incubation of DDT with hematin and ammonia. In two further experiments, cattle blood and DDT were incubated with sewage sludge (2.5% solids, 25mi) both in the presence of ammonia (0.88, O.Iml) and in i t s absence; the reaction was carried out in a similar manner to those described above, and both incubations yielded 7 t . l . c , products. Incubation of DDT with Heated Blood. Cattle blood (15ml) was heated to 80oc for 30 minutes to release hemoglobin. SOdiUmcarbonate (O.IM, lOml) and Tween 80 detergent (25mg) were added to the cold blood, followed by sodium dithionite (250mg) and DDT (35mg) dissolved in ethanol ( l l . 5 m l ) . The mixture was stoppered and set aside in the dark for 7 days, and then processed as described in the fresh blood experiment: 6 products were obtained from t . l . c . The heated blood incubation was repeated with the addition of ammonia (0.88, O.Iml), with the addition of sewage sludge (2.5% solids, 25mi) and with the combined addition of ammonia (0.88, O.Iml) and sewage sludge (2.5% solids, 25mi). In each case, 7 products were obtained from t . l . c . Incubation of DDT with Sewage Sludge. DDT (35mg) was dissolved in ethanol (l.Sml) and incubated with sewage sludge (Z.5% solids, 25mi) in the presence of sodium dithionite (250mg) in an aqueous solution of sodium carbonate (O.IM, lOml) and ethanol (lOml) in the dark for 7 days, and then processed as described for the incubation of DDT with hematin. 7 Products were obtained from t . l . c . The experiment was repeated in an identical manner except that ammonia (0.88, O.Iml) was added to the sewage sludge before addition of DDT; 7 products were obtained from t . l . c . The Incubation of DDT in the Absence of Hematin, Blood or sewage Slud~e. DDT (3Stag) was dissolved in ethanol (l.5ml) and added to an aqueous solution of sodium carbonate (O.IM, lOml), ethanol (lOml), sodium dithionite (250rag) and ammonia (0.88, O.Iml). The mixture was stoppered, set aside for 7 days and processed as described in the incubation of DDT with hematin; 2 products were obtained from t . l . c . , which yielded mass spectra corresponding to DDT and DDA. Preparation of Samples for Quantitation b~/ ).l.c. All the experiments described above were repeated, and in each case, the total mixtu~) was treated with boron trifluoride and methanol, using the procedure of ~tcalfe and Schmitz mu in order to convert DDA to its methyl ester. The

No. I?

995

o

rH •"

H

Od v

o

,~D

H O

H H

OJ

(kl

Od

C'~

~

o

~ °

0

o

n

0

~

0

",.D

°.

4.J

~.~ I1) m H

~

m

~

~

0

o

~

3

~ g

~ ~

m

O.rl

~

°

~

0

°

°

° ~

0

0

.

.

~

0

r-I O

° ~

rH

.r~ . ~

+~

•

.~

~ ~ e

o

~z~

+~ +~ O

O

o

-I~

.el +I

O

~o~.~

0

u'x

....1-

ko

KO

o~

o~

S S S S S S I

I

I

I

0"1

~.O

",,.O

I ~

~

0

S c~

I

I

0,1

0.1

I 0

S c~S S S S S S

0 0 r~

o

996

No. 12

quantity of each component present was deduced from peak areas using response factors obtained by injections of reference compounds. RESULTS AND DISCUSSION The results of the incubations are listed in Table 2. All the products from each incubation were i d e n t i f i e d by their mass spectra, confirmation was obtained by co-injection of the appropriate standard compound on g . l . c . The DDT contained a trace of DDE (ca. 0.2%), which was taken into account for quantitation of DDE in the incubations. Sodium d i t h i o n i t e was included in the incubations to reduce iron ( I l l ) porphyrins to iron ( I I ) porphyrins. Aqueous sodium carbonate solution was included to dissolve the hematin, which is insoluble in water and in ethanol. Ammoniawas included in some incubations as a source of nitrogen to determine whether degradative products, such as DDCN, were formed. The incubation of DDT with sodium d i t h i o n i t e , aqueous sodium carbonate solution and ammonia yielded DDT and DDA. The l a t t e r is produced by hydrolysis of the trichloromethyl group of DDT; this well-known process occurs under mild conditions. DDT undergoes dehydrochlorination to DDE in strongly basic (pH 13) conditions; this reaction does not take place in moderat e l y basic (pH lO) conditionstl. DDA was the major product of the incubation of DDT with hematin, but DDD, DBP, DDE and DDMUwere also formed. The major product of the incubation of DDT with hematin and an~nia was DDD; DDCNwas also produced, and the concentration of DBP was higher than in the incubation without ammonia. The incubation of DDT with fresh blood, both with and without ammonia, yielded the same metabolites as were detected in the corresponding incubations of DDT with hematin; however, in each case, DDD was the major product. Once again, the concentration of DBP increased when ammonia was present. These incubations may indicate that DBP is formed, at least in part, from DDCN, possibly by an autoxidation processl2, which could occur during the processing of the metabolites after the incubations. The incubations of DDT and heated blood, with and without ammonia, yielded similar results to the corresponding incubations of DDT and fresh blood. This suggests that heme is the active site in blood for the degradation of DDT, since the protein moietyIRf hemaglobin is denatured on heating. This is in agreement with the s$~dies of Wade and Castro ~ and Castro and Bartnicki 14, and with the postulation of Miskus et al. ~ . The incubation of DDT with sewage sludge yielded DDCN, whether ammonia ~as added or not. The results of these incubations are similar to the results of Jensen et al. ~, which showed that DDD, DDE, DDMU, DDCNand DBP were produced by incubation of DDT with sewage sludge; DDA was not detected in Jensen's investigation, presumably because the products had not been esterified prior to g . l . c . The incubations of DDT with sewage sludge and blood, whether heated or fresh, produced DDD as the major product, with some DDCN. From these studies, 6 metabolites of DDT, DDD, DDE, DDMU, DDA, DBP and DDCNwere detected on incubation of DDT with sewage sludge and also with fresh blood and heated blood when ammonia was included. DDD and DDE are the most widespread degradation products of DDT in the natural environment, DDMU, DBP and DDA also occur to a lesser extent and the distributions of a l l these con~ounds have been reviewed recentlyl6,17,18. DDCNhas been detected in lake sediment5. In a l l the incubations of DDT with sewage_sludge and blood, DDD was the major product, which is in agreement with the theory of Fries 16. DDE, DDMUand DBP were also present in a l l the above incubations in r e l a t i v e l y small amounts. The results indicate DDA and DDCNmay be widespread in the environment. Both substances may elude detection owing to the r e l a t i v e l y low response of electron capture detectors to esterified DDA and DDCNl~; also, the polar carboxyl and n i t r i l e groups render the compounds more soluble in water than the other degradation products of DDT and hence they w i l l probably be diluted in aqueous environments.

I~o. 12

997

0 OQ

~=~

0 0 c o 0 0 O 0 ~-- OX~X3 k O ..~ 0"~ CxJ C~ b'x 0"30d Od ~

o~I

r~ r-~ "~r ~ (XJ Oq

0 ~:~

•

,OJ

-um

,OJr-l~O.[mOJeq

0

,--I

tD r--I

°~ ,0 0 t,Q ~-~ 0

~t~ {',~0"~ ~eL.~ U'~ '~0 0~I 0"~ 0~I __~"._~" 0 ,-~ r-I

-~I

o~ o ~~

°H

o

~ o

,-I °M

~-

%

OX Um r-I OJ C~.-~" OJ ~-I ,-I 0.I OJ C'~ OJ o

~

~ o • ,--I I~ I~ OJ OJ ,'-I,-I ~

~ o

.~

oM

e~

C~ ,-I ,-I ,--I r-I ,-'I r-I

m

~0

t~

~

e-I - p c! 0 , 1 ~ " -~" m - - - l ' - - d " .hi" ~ ' - - ' ~ ~ " .-~ eq

N?

~::~,~

~ +

.2 0

,.--I ,.--I

• ~.o

+ I + I + I + I + I +

0 ,-.-I

I

I

I

I

1

+

+

+

+

+

~.,~

+

-~

I I 1 + + 1 1 1 1 + +

i<.°

°

I

l

I + + 1 1 1 1 + + 1 1

÷

+

l

l

l

l

l

l

l

i

l

m

tf

+Jo

!o~ Z

l

998

No. 12

CONCLUSIONS These in vitro experiments indicate that catalytic amounts of iron (II) porphyrins, at physiologica-a'T~n the presence of a reducing agent, can degrade DDT. The system used provides a model for the degradation of DDT in reducing sediments, which may contain small amounts of biologically-derived iron (II) porphyrins. The experiments indicate that DDD should be present in such sediments, together with small amounts of DDMU, DDE, DBP and DDCN. DDA may also be present, either as a result of degradation of DDT by iron (II) porphyrins or from hydrolysis of DDT. The experiments also indicate that iron (II) porphyrins should play a major role in the degradation of DDT in blood and tissues. Vitamin Bl~ may effect degradation of DDT in sewage sludge, in view of its structural similarity to the iron porphyrins, and its relative abundance18. ACKNOWLEDGEMENTS We thank the Natural Environment Research Council for a research grant (GR3/1257) and also for providing mass spectrometric f a c i l i t i e s (GR3/643) and the Nuffield Foundation for data processing f a c i l i t i e s . We thank Peter Nolan, Avonmouth Foul Water Treatment Works, Bristol, for the g i f t of sewage sludge. One of us (A.S.M.M.) wishes to thank the World Health Organisation for a research grant (M8/181/4/M.143). REFERENCES I.

Ecobichon, D.J., P.W. Saschenbrecker Science 1967, 156, 633.

2. 3. 4.

Hill,D.W., P.L. McCarty J.Water Pollut.Contra.Fed. 1967, 39, 1259. Hal vorsen, H., M. Ishaque, J. Solomon, O.W. Grussendorf Can.J.Microbiol. 1971, 17, 585. Albone, E.S., G. Eglinton, N.C. Evans, M.M. Rhead Nature 1972, 240, 420.

5. 6. 7. 8. 9. lO. II. 12. 13. 14. 15. 16. 17. 18. 19.

Jensen, S., R. G~the, M.-O. Kindstedt Nature 1972, 240, 423. Zoro, J.A., J.M. Hunter, G. Eglinton, G.C. Ware Nature 1974, 247, 235. Cooke, A.S., J.A. Zoro Bull.of Environ.Contam.Toxicol. 1975, 13, 233. Chen, Y.-L., H.M. Cheng Scientific Pest Control Boty-Kagaku 1965, 30, 51. Eglinton, G., B.R.T. Simoneit, J.A. Zoro Proc.Ro~.Soc.Lond.Ser.B. 1975, 189, 415. Metcalfe, L.D., A.A. Schmitz Anal.Chem. 1961, 33, 363. Smith, S., J.F. Parr J.A~ric.Food Chem. 1972, 20, 829. Kharasch, M.S., G. Sosnovsky Tetrahedron 1958, 3, 97. Wade, R.S., C.E. Castro J.Amer.Chem.Soc. 1973, 9_55,231. Castro, C.E., E.W. Bartnicki Biochem. 1975, 4._[, 498. Miskus, R.P., D.P. Blair, J.E. Casida J.ABric.Food Chem. 1965, L3, 481. Fries, G.F. Amer.Chem.Soc.Adv.Chem.Ser. 1972, I l l , 256. Fishbein, L. J.Chromato~. 1974, 9__88,177. Stotter, D.A. J.Inor~.Nucl.Chem. 1977, 3_99,721. Abou-Donia, M.B. Anal.Lett. (Received in UK 7 December 1978)

1974, Z, 313.