Bul l. Chin. Malacol. Soc. 中兩貝話、 4: 43-46 (1977) 000061 DDT METABOLISM BY OTALA LACTEA (STYLOMMATOPHORA: HELICIDAE) K. H. WURZINGER * -h、 d JEos吧 phqM)VA-HUρLMtJIEm pLVTa川句dwu. hNbveYL皂、yku τ ptr0 冒 srta 訓。心 趴 V hr' 川 ‘們 3ll且 ‘而' C 戶， E&h nA U AβLU 叫 凹的紅mMs ‘‘ 戶 F LU QdvJ e ?h 成 司、》 l nu 叫 w 門 ， m , ­ ABSTRACT Digestive gland tissue of Olala laclea Mül1 er fed 8GCI-DDT at a concentration of 40I,g DDT jg body weight contained only radioactive DDT & DDD as revealed by silica gel thin layer chromatography and liquid scinti11 ation spectrometry. The ratio of DDD to DDT stayed fai r1 y constant at approximately 3: 1. INTRODUCTION Terrestrial gastropods, as members of natural communities, have become increasingly ex­ posed to numerous synthetic pesticides. As non-target organisms of many of these chemicals, snails and slugs are able to accumulate and concentrate various insecticides without any noti­ ceable deleterious effect at concentrations equal to or higher than the concentrations in their physical environment. The organochlorine insecticide 1, 1, l-trichloro, 2, 2-bis (p-chlorophenyl) ethane (DDT), is one of the chemicals accumlated by many soil organisms, in c1 uding terrestrial gastropods. The information in the literature on the bioaccumulation and bioconcentration of DDT and of its degradation products DDD (1, I-dichloro, 2, 2-bis (p-chlorophenyl) ethane) and DDE (1 , l-dichloro, 2, 2-bis (p-chlorophenyl) ethylene) by terrestrial gastropods and other soil organisms has been revie\ved and summarized by Thompson (1973). Little information is available on the metabolic fate of DDT 洞 ' ithin the gastropod body. From earlier studies on the helicine snails Cepaea and Ota旬 it was determined that 80% or more of the DDT residues \\'ithin the snail's body was localized in the digestive gland (Dindal & Wurzinger, 1971; Wurzinger & Dindal, 1975). However, the identity of the residues found in the tissues is not known. In order to determine what these residues are, the present study was undertaken examining digestive gland tissue of Otala lactea 必1í.i1l er using thin layer chro­ matography and liquid scintillation spectrometry. MATERIALS & METHODS The snails, Otala [actea Mi.i ller, were obtained from Ward's Natural Science Establishment, Rochester, New York, USA, and maintained as previously described (Wurzinger & Dind祉， 1975). Each animal received one feeding of radioactive p, p'-DDT on lettuce at a concentr- * Present address: Mollusk Di\'ision, Museum of Zoology, University of Michigan, Ann Arbor, Michigan, U. S. A. 48109 44 K' JI. WURZINGER ation of 40μg DDT jg body weigbt (with shelI) and were frozen at 一 18 0 C at various times after feeding. Ten animals not fed any radioative DDT served as controls. The radioactive DDT, purchased from the Radiochemical Centre, Amersbam, Buckingb­ amshire, England, was labeIIed with 36CI in the para, para' position. This position is of unique analytical significance because the ring structure of the molecule is not broken during me­ tabolism, therefore known DDT metabolites remain isotope labelled. Digestive gland tissue was excised from the animals, weighed and separated into several samples. One set of samples was analyzed for total radioactive DDT residues (DDT plus any metabolites produced) llsing liquid scintiIIation spectromelry (Wurzinger & Dindal, 1975). Olher samples were analyzed for individual DDT metabolites using thin layer chromatography as de­ scribed by Wurzinger (1973). Pre-coated Eastman Chromagram@ non-flourescence silica gel sheets and Eastman thin layer apparatus were used for aII thin layer chromatography. The tissue was homogenized with a Potter Elvehjehm type tissue horoogenizer with teflon pestle in 1 ml of 3% trichloroacetic acid and extracted with 1 ml hexane. The hexane extract was concentrated with a gentle ~tream of air to approximately 0.1 ml, spotted onto the precondi­ tioned Chromagram@ sheets and the n-heptane solvent front allowed to migrate 15 cm at room temperature. The Chromagram@ sheet was then air-dried and sprayed with a silver nitrate-2- phenoxyethanol chromogenic agent. The sprayed chromatogram was then dried at room tem司 perature for 5 minutes and at 75 0 C for 15 minutes. The pesticide spots became visible after exposure of the chromatogram to ultraviolet light. The chromogenic agent was prepared as fo11ows: to 1 ml of 10% aqueous silver nitrate add 10 ml of 2-phenoxyethanol, dilute this mixture to 200 ml with acetone, add one drop of 30% hydrogen peroxide and mix (Sherma, 1973). The identity of the pesticide spots from the hepatopancreas was determined by comparison of the Rf values of the unknowns to the Rf values of standards that had been run on the same chromatogram. The standards used and their Rf values are listed in Table 1. Table 1. R f values of DDT residue and metabolite standards (n-Heptane solvent system) Compound Rf o,p'-DDT 0.54 p,p'-DDT 0.47 DDD 0.29 DDE 0.63 Kelthane ~ 0.06 DDA O FoIIowing identifìcation of the various DDT metabolites and residues, the spots were cut from the Chromagram'ID sheets and their radioactivity determined by liquid scintiIIation spec­ trometry in a Packard Tri-Carb Liquid ScintiIIation Spectrometer Model 3375. The scintiIIa­ tion solution was composed of 5 gj liter PPO (2, 5-diphenyloxazole) and 0.3 gj liter dimethyl POPOP (1 , 4 bis[2(4-methyl-5-phenyloxazolyl)] benzene) in scintillation grade toluene (Wurzinger & Dindal, 1975). DDT Metabolism by Otala lactea (Stylommatophora: Helicidae) 45 RESULTS The Rf values of the DDT residues and DDT metabolite standards used in this study are presented in Table 1. Pesticide residues and metabolites recovered from the digestive gland of Otala were DDT, DDD and DDE. All three of these compounds were recovered from the experimental as well as from the non-exposed control animals. Total radioacitve DDT residues (DDT plus any metabolite produced) in the digestive gland ranged from approximately 100 parts per million (ppm) to more than 250 ppm (data from Wurzinger & Dindal, 1975). More than 96% of the total radioactivity was recovered as DDD and DDT (Table 2). DDD residues comprised 21 to 29% and DDT residues comprised 67 to 77% of the total radioactivity in the digestive gland. The remainder of the radioactivity was recovered in the smear of material near the origin of the chromatogral"fl. Radioactivity in this region varied from 0 to 3.7% of the radioactivity recoyered, with most samples measuring 0 radioactivity. No radioactivity was found in the spots corresponding to DDE. Table 2. Total radioacti\'e DDT residue (DDT and metabolites) and iudividual residues and metabolites in Otala lactea digestive gland after a single feeding of radioactive DDT (mean 土 standald error). Individual residue and metabolites (%) Time after Amount of DDT feeding Residue* (ppm) DDT DDD DDE 6 hours 100.5 土 12.9 71 .4:i: 13.1 27.7 :i: 14.0 。 12 hours 93.9 士 15.1 67.3 士 9.0 29.0 土 3 .4 。 24 hours 178.0 土 2 1. 8 70.1 士 6.1 28.6 士 6.5 O 48 hours 237.9 土 55.0 69.5 土 8.0 27.9 士 8 .4 O 4 days 177.0 士 39.3 76.8 土 4.1 21.4:i: 4.1 O 5 days 278 .4土 73.6 70. 5:i: 11. 6 26. 9:i: 11. 3 O 6 days 283. 7:i: 18.7 77.6 士 8.6 21. 4:i: 7.9 O 8 days 134.1 :i: 16.6 76.5 士 2.3 23.2 士 2.1 。 10 days 105.0 :i: 16.8 75 .4士 2.5 24.6 :i: 2.5 。 * Data from \Vurzinger & Dinda1, 1975. DISCUSSION The metabolic fate of DDT in invertebrates other than insects is poorIy known. The evidence on DDT metabolism in soil invertebrates is largely circumstantial and mainly derived from comparisons of residues in the soil and in the organisms. Such circumstantial evidence indicates that DDT is dehydrochlorinated to DDE by oligochaetes (Thompson, 1973). This route of DDT degradation does not seem to be operational in terrestrial gastropods. Slugs from fields that had been sprayed only with DDT contained more DDD than DDE (Davis, 1968; Davis & French, 1969). Slugs kept in the presence of or fed DDT produced mainly DDD (reviewed by Thompson, 1973). This is in agreement with the results obtained from Otala. Although both DDD and DDE were detected in the digestive gland by thin layer chromatography, the spots corresponding to DDE contained no radioactivity, indicating 46 K. H. WURZJNGER that this metabolite had not been produced from the radioactive DDT fed to the snails. The experimental as well as the non-exposed control animals contained DDT, DDD and DDE in­ dicating that they had ingested these chemicals at some time prior to this study. Since the animals exposed to the radioacti\'e DDT contained no radioactive DDE, this metabolite was presumably ingested pre-formed. As in mammalian liver, the DDT metabolite found in the snail digestive gland is DDD. The ratio of DDD to DDT was approximately 1 to 3 throughout the period of this study. This constancy in the ratio indicates an equilibrium condition which might be the result of conversion of DDD to some other substance, which is excreted from the hepatopancreas. Alternatively, if this other metabolite is a polar compound, it would not have been extracted with the methods used in this study. The possibility that the DDD in Otala digestive gland is a product of bacterial metabolism needs to be investigated since gastropods have a direct connection between the lumen of the digestive tract and the digestive gland tubules.