Document zQV6zEjz96k9bvOOgyqNbMa4m

TOXICOLOOY AND APPLIED PHAPMACOLOOY 22, 628-640 (1972) Increase in the Toxicity of Organophosphorus Insecticides to House Flies Due to Polychlorinated Biphenyl Compounds1-3 -- T. W. Fuhremann and E. P. Lichtenstein Dtpartmrnt of Entomology, University of Wisconsin, Madison, Wisconsin 33706 . Received October 22, 1971 Increase in the Toxicity of Organophosphorus Insecticides to House Flies Due to Polychlorinated Biphenyl Compounds. Fuhremann, T. W. and Lichtenstein, E. P. (1077\ AppL Pharmacol. 22, 628-640. The polychlorinated biphenjd^PCBL^roclor* 1248--although nontoxic by itself to house flies at doses orHkTtf/fly--significantly increased the toxicity or the oxygen analogs of 5 organophosphorus insecticides after simul taneous topical application to houseflies. Both p,p'-DDT and p,p'-DDE, applied at sublethal doses, also increased the toxicity of paraoxon, as measured by an increase in the mortality of house flies from 2 to 60 and 73%, respectively. The PCB, applied simultaneously with parathion, how ever, decreased the toxicity of parathion to house flies, but increased mortalities when applied to flies 0.5 hr after the application of the insecticide. Aroclor* 1248 apparently inhibited the conversion of parathion to its toxic , activation product, paraoxon, and the breakdown of paraoxon into non toxic compounds. This was also indicated by the recovery of larger amounts of ,4C-paraoxon and smaller amounts of water soluble ,4C-product$ from flies treated simultaneously with Aroclor* 1248 plus paraoxon in com parison to those which had been treated with ,4C-paraoxon only. Utilizing the 10,000 g supernatant from house fly homogenates, the addition of Aroclor* 1248 with ,4C-paraoxon in vitro also resulted--after 2 hr incuba tion at 30DC--in higher recoveries of14C-paraoxon and in a reduction of the appearance of water soluble 14C-products. Identical results were obtained with subcellular fractions (10,000 supernatant) from flies treated with Aroclor* 1248 18 hr before homogenate preparation: no difference was observed in the degradation of paraoxon or the appearance of water soluble products with Aroclor* applied to the living flies or to the 10,000 g super natant of homogenates from untreated flies. Since PCBs are in the environ ment, their potential effects on biological systems, especially in combination ' with other synthetic chemicals, should not be disregarded. \ Polychlorinated biphenyl compounds (PCBs) produced in the U.S. by the Monsanto Chemical Co. under the name Arbcldr, are currently receiving the attention of ecologists, toxicologists and pesticide analysts because of their apparent ubiquity and coincidental occurrence with chlorinated insecticide residues in the environment 1 Reprint requests should be addressed to E. P. Lichtenstein. * From a dissertation submitted by the senior author in February 1972 in partial fulfilment of the requirements of the M.S. degree. Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison and by grant EP-00817. Contribution from the Wisconsin Agricul' tural Experiment Station as a collaborator under North Central Cooperative Research Project 96, \ "Environmental Implications of Pesticide Usage." . \ ' Presented at the National Institute of Environmental Health Sciences Meeting on Polychlorinated ' Biphenyls, Rougemont'. Norlh Carolina, December 20-21, 1971. ' Copyright O l*7i by Academic Prt*, Inc. All of reproduction In niiy form mined. 628 (Holm Peaka numbt 1969; A rock skin fi Util Aroch I ppm residu They e gistic e for "it that " chlorii elm le The insect! of PC I chlorii was fo and dl PCB c invest Chemi Inse methy and o ,4C-Pe purcht pared parath tilled v for I r --colorle which' MC-pai (TLC) I sol oxon v liquid and in The tainin ^lii.u niL.!ii|^pni**ppp USW 032027 PCB AND ORGANOPMOSPHORUS INSECTICIDES 629 (Holmes et a!., 1967; Widmark, 1967; Risebrough et al., 1968; Koeman et a!., 1969; Peakall and Lincer, 1970). Many PCB compounds can interfere with the analysis of a number or chlorinated hydrocarbon insecticides (Widmark, 1967; Lichtenstein et at., 1969; Reynolds, 1969, 1971). Litterst and Lichtenstein (1971) reported that the PCB, Aroclor 1254, exhibited a toxicity similar to that of DDT after human HeLa cells and skin fibroblasts in tissue culture had been exposed to this compound. Utilizing insects as test organisms, Deonier et al. (1946) reported that several Aroclor compounds were toxic to the larvae of Anopheles quadrlmaculatus (Say) at I ppm in water. Tsao et al. (1953) reported that Aroclor 5460 greatly increased the residual toxicity of lindane, but had no toxic effect by itself to Musca domestica (L.). They also reported "some indication that this chlorinated polyphenyl may have a syner gistic effect with lindane." "Chlorinated polyphenyls" were also recommended in 1953 for "improving lindane residues" (Hornstein and Sullivan, 1953). The authors stated that "lindane residues can be made to last longer and look better" if applied with chlorinated polyphenyls. Aroclor 5460 was later reported to exhibit toxicity to the elm leaf beetle Galerucella xanthomelaena (Schr.) (Duda, 1957). The biological interaction and synergistic effects of PCBs on DDT and dieldrin in insects was shown by Lichtenstein etal. (1969). These authors reported that the toxicity of PCBs to house flies and fruit f\its(Drosophila) decreased with an increasing degree of chlorination and appeared to be less toxic than dieldrin by a factor of at least 1000. It was found, however, that sublet hal doses of several PCBs increased the toxicity of DDT and dieldrin. As a continuation of this study, the effects and interactions of selected PCB compounds with some organophosphorus insecticides on and in house flies were investigated. METHODS Chemicals Insecticides used in this study were analytical grade oxygen analogs of azinphos- methyl, of Diazinon (diazoxon), of Dyfonate (dyfoxon), of malathion (malaoxon), and of parathion (paraoxon). Parathion, p,p'-DDE and p,p'-DDT were also used. l4C-Parathion, labeled in the 2,6 ring positions (specific activity 0.58 mCi/mmole) was purchased from the Tracerlab Co., Waltham, Massachusetts. MC-paraoxon was pre pared by oxidation of uC-parathion with bromine water. For this purpose, 5 mg MC- parathion (11.3 pCi) was placed in a 15 ml, glass-stoppered test tube with 10 ml dis tilled water and 5 drops of reagent grade bromine. The contents of the tube were shaken for I min. Nitrogen was bubbled through the solution until it become completely colorless. The solution was then extracted 5 times with 10 ml portions of chloroform, which were pooled and dried over anhydrous sodium sulfate. To verify the presence of MC-paraoxon, the,chloroform solution was subjected to thin layer chromatography (TLC) and autoradiography as described below. Isolates from the thin layer plate that had the same Rf value as reference grade para oxon were eluted with acetone and analyzed by gas-liquid chromatography (GLC) and liquid scintillation counting (LSQ. Results by GLC and LSC were in close agreement and indicated an 80% yield of ,4C-paraoxon. The j?CB compounds used jn this study were Aroclor 1248 and Aroclor 1268, con taining 48 and 68 % chlorine by weight, respectively. They had been obtained through ' .. \ '\ . -' \ DSW 032028 630 FUHREMANN AND LICHTENSTEIN the courtesy of Dr. R. F. Thomas, USDA, Beltsvilie, Maryland. NADPH was pur chased from Nutritional Biochemical Co., Cleveland, Ohio. Solvents used were redistilled acetone, chloroform, n-hexane and reagent grade, absolute; ethanol, methanol and diethyl ether. Insects and the Application of Chemicals Two strains of houseflies, Musca domestical L.) were used in this study :CSMA, 1948 strain (succptiblc to DDT) was obtained from the Wisconsin Alumni Research Founda tion, Madison, Wisconsin, and a second strain (P2/sel), resistant to DDT and naphtha lene vapors was obtained from Dr. M. Agosin, University of Georgia, Athens. Two microliters of acetone containing an appropriate amount of the test compound were topically applied with an Isco model M-420 microapplicator onto the abdominal stcrnites of 2-3 day old female house flies anesthetized with C02. After treatment, 3 replicates each consisting of 15 flies were held in glass jars for various time periods indicated for each experiment. Flies which had not revived after 30 min were disre garded in subsequent mortality counts. Mortality counts or further experimentation were then performed as outlined below. Extraction and Analysis Several extraction procedures, described separately with each experiment, were used in these studies. Gas-liquid chromatography was employed for quantitative and qualitative analyses ofinsecticides and their potential degradation products in the hexane or ether extraction phases. A Packard Model 7834 gas chromatograph equipped with a 1.22 m x 3 mm i.d. Pyrex glass column containing 5% Dow Corning 11 silicone grease coated on 60/80 mesh, acid washed, dimethyldichlorosilane treated, chromosorb W was maintained at a temperature of 180C and a helium carrier flow rate of 60 ml/min. A potassium chloride thermionic detector, especially sensitive to phosphorus, was modified and operated as described by GiufTrida et al. (1966). Thin layer chromatography was performed to evaluate qualitatively the hexane or ether phases of extracts obtained in these studies. These phases were concentrated to approximately 0.05 ml and analyzed by TLC and autoradiography as described by Lichtenstein and Fuhremann (1971). 1 Liquid scintillation counting was performed to determine the radiocarbon content of ' the various samples by utilizing a Packard model 3320 liquid scintillation spectro meter. One milliliter aliquots of hexane extracts of samples were placed in 14 ml of a '' toluene-based counting solution while 1 ml aliquots of water phases and ether or acetone extracts were counted in 14 ml of a dioxane-naphthalene based counting solution. Data were corrected for background, counter efficiency and dilutions and ex pressed as total dpm per extraction phase. s EXPERIMENTAL ' r Effects of'PCBs on the Toxicity of Insecticidal Compounds \ 1 To determine the effect of a PCB compound on the toxicity of organophosphonis in- \ Secticides, CSMA strain Musca domestica were treated as described with an insecticide, '\ ` * i/ \ ,\ . .i \ wit sisl 0.0 ma Fin a be wer dat eac ave me f ferr or< wci on l cor 10, 1 due ,CS or i a1 mo 1 flie: 12 per rec The I T to h OXOI 24 h T WO! on 124 gre P>P cot \ / mmmmmMm********* f- DSW 032029 STLCOPCB4015991 )PH was pur- eagent grade, : CSMA, 1948 :arch Founda* and naphthaAlhens. est compound the abdominal r treatment, 3 5 lime periods in were disreperimentation eriment, were ative analyses [her extraction m x 3 mm i.d. aled on 60/80 maintained at A potassium modified and the hexane oi mccntratcd to i described by ion content of ation spectroI in 14 ml of a and ether or ased counting utions and ex- hosphorus inan insecticide, PCB AND ORGANOPHOSPHORUS INSECTICIDES 631 with Aroclor or simultaneously with both. For this purpose 3 replicates, each con sisting of 15 flies, were topically treated with either 0.02 fig of parathion or paraoxon, 0.04 fig of diazoxon, 0.08 fig of dyfoxon or azinphosmethy! oxygen analog or 0.30 fig of malaoxon per fly. In addition 45 flies were treated with 10 fig Aroclor 1248 per fly. Finally 45 flies were simultaneously treated with either one of the insecticides at the above mentioned concentrations, plus Aroclor 1248 (10 pg/fly). Mortality counts were performed after a holding period of 24 hr. These experiments, repeated on different dates, were conducted with paraoxon 6 times, with parathion 4 times and twice with each of the other compounds. The percent mortality of the insects was expressed as averages of the mortality counts from different tests with SD or deviations from the mean. Because the susceptibility of flics to toxicants varies over a certain period, dif ferences in mortalities observed due to the application of parathion or paraoxon only, or due to the simultaneous application of one of these insecticides with Aroclor 1248, were evaluated with the paired / test (Steel and Torrie, I960). In this way data obtained on different days between a control and one variable could be compared. Utilizing DDT resistant flies (P2/sel strain), identical tests as described above were conducted except that paraoxon was applied at 0.04 ftg/fly and Aroclor 1248 at lOpg/fly. To test the effects of a different PCB compound on CSMA flies, a similar test was con ducted with Aroclor 1268 (10 pg/fly) and with paraoxon (0.05 /ig/fly). To determine the LDSOof paraoxon with house flies as affected by Aroclor 1248, CSMA flies were treated as described with serial dilutions of paraoxon to which none or a standard amount (10 /xg/fly) of Aroclor 1248 was added. Flies were observed over a 10 hr period. Mortality data obtained after 4 hr were used to plot eye-fltted dose mortality curves. Todetcrmine whether PCBs have a residual effect on the toxicity of paraoxon, CSMA flies were treated as described with Aroclor 1268 (10 pg/fly) and held for 2 days in 12 x 12 in. screen cages. Control flies were treated with acetone only. After this holding period both groups of flies were treated with paraoxon (0.05 pg/fly). Mortalities were recorded after a 24 hr holding period. The Effects of Various Quantities of Aroclor 1248, DDT or DDE on the Toxicity of Paraoxon to House Flies To determine the effect of various levels of Aroclor 1248 on the toxicity of paraoxon to house flies, CSMA strain flics were treated simultaneously with 0.02 fig para oxon plus 0.1,0.5, 2, 4,6, 8 or 10 fig of Aroclor 1248 per fly. These flies were held for 24 hr, when mortalities were recorded. To determine whether DDT and DDE, which are structurally similar to the PCBs would also affect the toxicity of paraoxon to CSMA strain Musca domestica, 3 groups of flies were treated with various amounts of either p,p'~DDT, p,p'-DDE or Aroclor 1248. Another group of flies was treated with 0.02 fig paraoxon per fly, and 3 additional groups 6f flies were treated with 0.02 fig paraoxon per fly plus either Aroclor 1248, p,p'-DDTqr p,p'-DDE (Table 2). These flies were then held for 24 hr when mortality counts were'performed. \ /\ osw 032030 STLCOPCB4015992 632 FUHREMANN AND LICHTENSTEIN Mode of Action of Aroclor 9 1248 Since the toxic effects of parathion on insects is primarily due to the formation of paraoxon in the insect body (Metcalf and March, 1953), experiments were conducted to investigate the effects of Aroclor* 1248 on this process. The effect ofAroclor* 1248 on housefly mortalities after in vivo conversion ofparathion to paraoxon was determined by treating flies with parathion and--at intervals there after--with Aroclor* 1248. If PCB would primarily effect paraoxon, this efTect would only be noticeable at some interval after fly treatment with parathion, during which time paraoxon could have been formed in vivo. Tests were therefore conducted with CSMA flics which were treated with 0.02 pg of parathion per fly. Immediately there after, and also 2 or 4 hr later, groups of 45 flies were treated with 10 pg of Aroclor* 1248/fly or with acetone only (solvent control). Each group of 45 flies (15 flies per bio assayjar) was then held for 24 hr at which time mortality counts were performed. The distribution andpersistence ofparaoxon in houseflies as affected by Aroclor* 1248 was determined in another series of 5 separate experiments conducted at different dates. For this purpose, I group of 500 female CSMA house flies was topically treated with 0.02/xg(4.5 x 10'VCi) ,4C-paraoxon per fly and another group of 500 flies with 0.02 pg ,4C-paraoxon plus 10 pg Aroclor* 1248 per fly. Each group was then held for 2 hr in 1000 ml cheesecloth-covered Pyrex beakers. At the end of the holding period the radio carbon content and the amount ofparaoxon on the outer surfaces ofthe insect, the inside of the insect and on the inner glass surfaces of the beakers were determined. For this purpose, the flies of each group were anesthetized with C02 and then placed into 250 ml glass stoppered Erlenmeyer flasks. To remove external residues with ace tone, each group or flies was shaken 3 times with 75 ml portions of acetone. These ace tone rinses were quantitatively decanted through a Btichner funnel, pooled and desig nated "External." The rinsed flies were then macerated with 50 g of anhydrous sodium sulfate in a Waring Blendor and homogenized 3 times with 100 ml of chloroform. The chloroform extracts of each group were dried over anhydrous sodium sulfate and evaporated on a flash evaporator at 35C. The residue was then redissolved in n-hexane and designated "Internal." ....... The chloroform extracted fly pulp was then dried overnight at 50C, its weight was determined and aliquots were combusted in an oxygen filled Schbniger flask (Kelly , etal., 1961). The liberated ,4COj was absorbed in 3 ml phenethylamine: methanol (1:2). , This solution was then dissolved in 14 ml of toluene-based counting solution for deter\ mination of the radiocarbon content by LSC. Results obtained from the combusted fly \ material were then designated as "Bound" radioactivity. Finally the residue on the inside walls of the beakers in which the treated flies had been held was removed by first scrubbing the beaker walls with 10 ml water (using the cheesecloth beaker covers) and then with five 25-ml portions of acetone. The water and acetone were combined and concentrated in a flash evaporator at 35C until most ofthe acetone was eliminated. The remaining water (and acetone) were then adjusted to volume, and aliquots were used for the determination of their radiocarbon content. Results obtained by this procedure were designated as "Excreted" radioactivity. It had been determined in preliminary experiments that, after adjusting the pH of the water to 1.5 with^ N HC1, extraction with n-hexane or diethyl ether did not remove any \ ' ! \. UiUJWMfflp .1.1 | ..... 111 nwy * QSW 032031 ion of therewould which with there* oclor* sr bio- 1248 dates. with 02 fig hr in radio inside placed th accse ace* desig- te in a roform d on a gnated ht was (Kelly 1(1:2). delerted fly es hod ng the er and of the ted to intent. of the /eany PCB AND ORGA NOPHOSPHORUS INSECTICIDES 633 radioactivity from the water. Paraoxon, added to aliquots of this water, however, was extracted with both hexane and diethyl ether. For additional analyses by TLC and auto radiography, the acidified water was evaporated to dryness on a flash evaporator at 40oC, and the residue was redissolved in methanol. Degradation ofparaoxon by homogenates ofhouse flies as affected hy Arocior* 1248. In the experiments described above, Arocior* 1248 and paraoxon had been applied to the living insect. To further elucidate the mode of action and/or the effects of Arocior 1248 on the persistence of paraoxon in houseflies, experiments were conducted in vitro. Flies were treated with Arocior* 1248, held for 18 hr, and paraoxon was added to the 10,000 g supernatant prepared from homogenates of these flies. Homogenates were also prepared from acetone treated (solvent control) flies, and both the Arocior and para oxon were added simultaneously to the 10,000 g supernatant prior to incubation. In preliminary experiments 400 female CSMA flies were each topically treated with 10 pg Arocior 1248 as described. Control flies were treated with acetone only. The 2 groups of flics were then held for 18 hr at room temperature. After that time the dead flies were discarded, while the living flics were anesthetized with C02 and frozen over dry ice to facilitate removal of the abdomens from the insect bodies; 300 abdomens from each group were used for enzyme preparation at 0.4C. The abdomens were homogenized in a 50 ml conical, ground glass tissue grinder (Bellco Glass, Vine land, New Jersey) with 30 ml grinding medium consisting of 0.15 M sodium phosphate buffer plus 0.25 m sucrose and 0.2% bovine serum albumin-fraction V (Grand Island Biological Co., Grand Island, New York). The pH was 7.3. The homogenates(IOabdomens/ml grinding medium) were then centrifuged at 1500 g for 2 min. After the floating material and substances adhering to the upper walls of the centrifuge tubes had been resuspended in the supernatant, this supernatant was centrifuged at 10,000g for 20 min in a Beckman model L2-50 ultracentrifuge. The resulting supernatant, along with re suspended floating and adhering substances served as the enzyme source. To demon strate that the degradation of paraoxon was an enzyme catalyzed reaction, tests were conducted both in the absence of insect homogenate and also with the 10,000 g super natant which previously had been heated for 10 min in boiling water to denature the protein. Replicate incubation mixtures consisted of 3 ml of boiled or unboiled enzyme preparation to which 300 fig NADPH (1,3 x 10~4 m) and 50 fig (0.113 pCi) of UCparaoxon contained in 10 fil ethanol were added. These incubation mixtures, contained in aluminum foil-covered 10 ml Erlenmeyer flasks, were then incubated for 4 hr in the dark in a 30C water bath rotary shaker (120 rpm). To terminate the reaction 5 ml of acetone was added to each mixture, which was then quantitatively transferred with 2 5-ml portions of water and 5 ml of acetone into a 60 ml separatory funnel. These incuba tion mixtures were then extracted with three 10-ml portions of n-hexane and then with 3 10-ml portions ofdiethyl ether resulting in a water-acetone phase and an organic solvent phase. The organic solvent phase was dried over anhydrous sodium sulfate and adjusted to volume. All fractions were then analyzed by LSC for their radiocarbon content, while the organic solvent phases were also analyzed by GLC, TLC and autoradiography. The final experiments were conducted at a different time with another group of flies. '[These tests were performed the same way as described above, but in addition 200 fig Arocior* 1248 in 1 Op.1 ethanol was added to each replicate homogenate of control flies (acetone pretreated) simultaneously with paraoxon just prior to incubation. This was \ "i \t DSW 032032  ;/ .i. < i ' 'V V , 1 i < 634 FUHREMANN AND LICHTENSTEIN I. i done to determine whether a time lapse after the in vivo application of Aroclor was necessary to afTect the degradation of paraoxon, or if the addition of this PCB to the prepared enzyme would cause a similar effect in vitro. Differences due to Aroclor* treatment were evaluated with the ftest (Steel and Torrie, I960). RESULTS AND DISCUSSION Effects of PCBs on the Toxicity of Insecticidal Compounds Data presented in Table 1 indicated that at a dose of 10 p.g/fly, Aroclor 1248 by itself was not toxic to female CSMA house flies. However, mortalities of flies due to paraoxon were increased from 5% (without Aroclor 1248) to 95% (with Aroclor 1248). Conversely, this PCB reduced the mortality due to parathion. Differences ob* served in mortalities due to Aroclor 1248 applied with paraoxon were significant at the 0.1 % level and with parathion at the 2% level. These data might, therefore, indicate that Aroclor 1248 inhibited the in vivo conversion of parathion to paraoxon and the detoxification of paraoxon itself. Mortalities observed after the application to flies of (' P f( a l ts A ri w P ci TABLE I Effect of the Polychlorinated Biphenyl, Aroclor 1248, on the Toxicity of Insecticidal Compounds after Topical Application to Musea Domestic<f Insecticide11 None Paraoxon Parathion Diazoxon Dyfoxon P=0-Azinphosmethyl Malaoxon pg/fly _ ____ 0.02 0.02 0.04 0.08 0.08 0.30 Aroclor 1248 None (control) 10 pg/fly (% mortality/24 hr) _ 5 5 16 5 28 2 4 3 50 12 29 19 0 95 6* 2 2r 86 4 68 16 96 2 83 6 % Control' _ 1900 13 313 1700 192 286 ' * Insecticide and/or Aroclor* applied topically to the ventral portion of the abdomen. * Total number of tests (each 3 times replicated--15 flies per replicate) were 6 for the control (None) \ and with paraoxon, 4 with parathion and 2 with each of Ihe remaining compounds. Results are expressed , as mean SD. * % Control - Mortality in % of mortality observed with insecticide but no Aroclor* (None-- cpntrol) Differences in mortality due to Aroclor* treatment are significant at the 0.1 % level (d), or the ,2 % level (e) as determined by the paired / test. p> T i: p ,, (he oxygen analogs of other commonly used organophosphorus insecticides were also substantially increased when applied in combination with Aroclor 1248. Since the effects, of this PCB were most pronounced with paraoxon, this compound was selected as a model for further study. Similar results were Obtained with another strain of Musca domestied (Pj/scI). Aroclor* r268, a PCB with a chlorine content of 68%, also increased fly mortalities \ DSW 032033 STLCOPCB4015995 :lor was ZB to the Aroclor * 1248 by cs due to Aroclor ences ob:Bnt at the indicate n and the to flies of PCB AND ORGANOPHOSPHORUS INSECTICIDES 635 (% dead within a 24 hr exposure period) due to paraoxon by a factor of 2.7 as com pared to flies that were treated with paraoxon only. When the effect of Aroclor 1248 on the LD50 of paraoxon was investigated, it was found that PCB increased the toxicity of paraoxon by a factor of 4 (Fig. 1), resulting in an LD50 of 0.013 ^g paraoxon/fly. Mortalities observed after paraoxon only had been applied did not further increase 2 hr after the insecticidal application. However, mor talities observed with the application of comparatively lower levels of paraoxon plus Arodor continued to increase up to about 10 hr, indicating a possible inhibition ofthe rapid detoxification of paraoxon. When Aroclor 1268 was applied 2 days prior to paraoxon treatment, mortalities were increased from 16% (acetone pretreated) to 81 % (Aroclor pretreated) 24 hr after paraoxon application. This would indicate a residual effect of PCBs on paraoxon toxi city to house flies. Control' 1900 13 313 1700 192 266 nlrol (None) ire expressed jw* {None-- k s4'c <r the j i w aih0> | Since the as selected (P*/el). nortalities 1 / i i ri Fio. I. Dose-mortality curves obtained 4 hr after application of paraoxon only or of paraoxon plus PCB (Aroclor* 1248) to Musca domefHat. The Effects of Various Quantities of Aroclor 1248, DDT or DDE on the Toxicity of Paraoxon to House Flies Results obtained after-paraoxon or paraoxon plus increasing amounts of Aroclor 1248 had been applied to house flies are presented in Fig. 2. Topical application of 0.02 pg paraoxon caused only a 6% mortality of house flies over a 24 hr period while the \ N AROCLOR - B4R/FLV Pro, 2. Effect of Increasing amounts of Aroclor* 1248 on the toxicity of a constant amount of paraon (0.02 pg/fly) simultaneously applied to female Musca dometttea. 'I wwmrm' mm****** m iwpwwpyii! . I* Wfppppu y .w ipw DSW 032034 636 FUHREMANN AND LICHTENSTEIN addition of increasing amounts of Aroclor 1248 with a constant amount of paraoxon (0.02 ^g/fly) increased (he toxicity of paraoxon in a nearly linear mode. Two fig of this PCB were sufficient to increase fly mortality due to paraoxon to 90%. The effect of Aroclor compounds in increasing the toxicity of paraoxon to house flies also occurs with structurally related chlorinated aromatics (Table 2). p,p'-DDT, which is essentially nontoxic to house flies at 0.05 /ig/fly, increased the mortalities due to para oxon. p,p'~DDE, which was nontoxic to flies even at a level of 5 ^g/fly, increased the fly mortality due to 0.02 fig of paraoxon when applied at a level ofO. I fig DDE per fly. These data illustrate the potential effects and interactions of environmental chemicals in bio logical systems. DDE, a widely distributed metabolite of DDT in the environment was at least as effective as its parent compound in increasing the toxicity of paraoxon to flies. TABLE 2 Effects of the Chlorinated Aromatics, Aroclor 1248, />,/>'-DDT or p,p'-DDE, on the Toxicity of Paraoxon to Musca Domesticcf Paraoxon (M/fty> None -- Chlorinated aromatic (jig/fly) Aroclor** p,p'~DDT p,p'~DDE 0.1 1.0 5.0 0.05 0.1 0.1 1.0 % Mortality of house flles/24 ht* 5.0 None 0.02 --0 0 0 6 16 88 100 2 17 60 55 220 73 86 90 * Paraoxon and/or chlorinated aromatics applied topically to the ventral portion of the abdomen. * Average of 3 replicates, each consisting of 15 flies. Mode of Action of Aroclor 1248 The role of Aroclor in decreasing the toxicity of parathion and in increasing that of paraoxbn (Table 1) indicated possible effects on enzymatic processes. If PCBs inhibit enzymatic degradation of these insecticides, it could explain the divergent effects with parathion and paraoxon. It is generally accepted that the toxicity of parathion is due to 1 its in vivo conversion to paraoxon (Metcalf and March, 1953). Assuming that the PCB ' inhibited both the enzymatic conversion of parathion to paraoxon and the degradation ' of paraoxon itself, this would explain the above mentioned findings. The effect ofAroclor* 1248 on housefly mortalities after in vivo conversion ofparathion to paraoxon. Results obtained in experiments where flies had been treated with parathion (0.02 fig/f\y) and simultaneously with Aroclor 1248 (10 fig/f\y) demonstrated that the PCB reduced the toxicity of parathion as indicated by a decrease in mortality from 18 to 0%. However, when flies were treated with Aroclor 30 min after the parathion ^application, fly mortalities were 4 times greater than With control flies treated with acetone (solvent controls) 30 min after the application of parathion. No further sub\ stantia! increase in fly mortalities was observed when Aroclor* was applied 2 or 4 hr \ after fly treatment with parathion. Since parathion is converted to paraoxon during the \ holding period, the subsequent treatment with Aroclor* possibly inhibited the degr; mort Th sena para> that i were respt was extra althc Appi parai Pi Norw A roc T 1248 *D of 4rE exteri - wa i.i. S% (h w the i able parav mate water authc with r Rest had cretr of p frac to 3) mm* mu ji. unwif DSW 032035 STLCOPCB4015997 faoxon of (his i ISC flics , which 3 paraIhc fly , These lin b/ont was xon to 0 90 men. hat of nhibit Is with ilucto t PCB lation ilhion ithion at the )m IB thion with ' sub- r 4 hr sg the 1 the PCB AND OROANOPHOSPHORUS INSECTICIDES 637 degradation of paraoxon and thereby increased its toxic action, resulting in increased mortalities. The distribution and persistence of ,4C-paraoxon on and in house flies, due to the pre sence of Aroclor* 1248, is presented in Table 3. In the presence of Aroclor* 1248, paraoxon was more persistent. This was demonstrated by GLC analyses which showed that in the absence of Aroclor* (None-Table 3) only 7 and 6% of the applied paraoxon were recovered from the outside ("External") and the inside ("Internal") of the flies, respectively. In the presence of Aroclor* 1248, however, 18% of the applied paraoxon was recovered from the fly rinsings ("External") and 16% was recovered from fly extracts ("internal"). Data based on radiocarbon content also indicated similar results, although ,4C-compounds other than paraoxon are included in these values. TABLE 3 Effects of Aroclor* 1248 on the Fate of ,4C-Paraoxon in Musca domestica (in Vivo) APP'ied* plus Recovered* in percent of applied from housefly fractions External' Internal' Bound' Excreted' Total None Aroclor* ,4C-DPM Paraoxon ,4C-DPM Paraoxon 15 6 72 21 8 18 9' 17 5 64 26 54 16 8f 31 -- 52 -- 36 6 ND 18 5' ND' 71 13 70 34 'Topicallyapplied "C-paraoxon(0.02w4.5 x 10"'fiCl/fly)or ,4C-paraoxonplus 10pgAroclor* 1248 per fly. * Data were obtained 2 hr after house fly treatment, 500 flies/trealment per test. Results are averages of 4-5 tests SD. Paraoxon determined in by GLC. ' External - Acetone rinses of whole flies; Internal - chloroform extracts of homogenized flies after external rinses; Bound - nonextractable l4C determined by combustion of extracted flies; Excreted - water-acetone rinses of fly holding jars after the 2 hr holding period. Differences in recoveries due to Aroclor* treatment are significant at the I % (d), 0.1 % (e) or 5% (f) level as determined by paired t test. * No paraoxon was detected. Water soluble products of the originally applied ,4C-paraoxon were obtained from the inside surface of the holding jars as described. These l4C-products were not extract able with n-hexane or diethyl ether at a pH of 1.5, although added amounts of l4Cparaoxon could be fully extracted by this method. This indicated that the "excreted" materials were not paraoxon. In addition, analyses by TLC and autoradiography of the water soluble products indicated a radioactive spot with the same Rf value as that of authentic amino paraoxon. This, however, could not be confirmed by spraying the plate with color reagents specific for amino compounds (Lichtenstein and Fuhremann, 1971). Results obtained by LSC showed that the amount of water soluble I4C-products which had been "excreted" by flies treated with paraoxon only was twice the amount "ex creted" by thosi flies treated with l4C-paraoxon plus Aroclor* 1248. The total amount \ of paraoxon (measured by GLC) recovered from the "External" and "Internal" ^ fractions was 13 % of the applied dotage in the absence of Aroclor* 1248, but amounted i to 38 % in its presence) (Table 3). These data demonstrate that the presence of Aroclor* ' /' "l DSW 032036 STLCOPCB4015998 \ 638 FUHREMANN AND LICHTENSTEIN 1248 on or in house flies prevented the breakdown of ,4C-paraoxon into unidentified water soluble products, thereby causing greater insect mortalities. The total recoveries of ,4C-compounds from control and Aroclor treated flies were identical (70 and 71 %) (Table 3). The unaccountable radioactivity may have volatilized from the surface of the treated flies or been lost during the process of individual treat ment of 500 flies and subsequent experimental procedures. Degradation ofparaoxon by homogenates of house flies as affected by Aroclor 1248. Preliminary studies with l4C-paraoxon treated house fly homogenates indicated that no degradation of paraoxon occurred in the absence of a fly homogenate or with boiled fly homogenates. In both cases, all the paraoxon and radiocarbon added to the incubation mixtures was recovered as determined by GLC and LSC, respectively. With unboiled homogenates from flics treated 18 hr previously with acetone (controls), however, only 17 I % of the applied paraoxon and 31 3% of the applied radiocarbon were re covered from the organic extraction phases of the incubation mixtures. This indicated that degradation of paraoxon to water soluble compounds had occurred with non- boiled homogenates. When homogenates from Aroclor treated flies were used, more paraoxon (27 5% of applied) and more radiocarbon (41 0% of applied) were re covered from the organic solvent phases, indicating that Aroclor inhibited the degrada tion of paraoxon. The water phase, however, contained less radiocarbon when incubation mixtures from Aroclor treated flies were used, ascompared to incubation mixtures from acetone treated flies (controls). This appears to be additional evidence of reduced activity of homogenates from Aroclor treated house flies. In the final series of experiments, Aroclor 1248 had been applied either to houseflies ("in vivo," Table 4) which were held for 18 hr prior to the preparation oT homogenates, or to homogenates ("in vitro", Table 4) prepared from acetone (control) treated house flics. Results in Table 4 demonstrate that in the absence of an enzyme preparation or with the boiled 10,000 g supernatant from the fly homogenate nearly all the paraoxon and radiocarbon applied to the incubation mixtures was recovered in the hexane: ether extracts. However, with homogenates from untreated flies (controls), only 33% of the applied paraoxon and 36 % of the applied radiocarbon were recovered from the organic extraction phases of incubation mixtures. When either Aroclor was added with para oxon to homogenates from untreated flies, or when homogenates were prepared from . Aroclor treated flies, results were similar (Table 4) to each other. The amount of para oxon recovered from homogenates containing Aroclor 1248 was 1.6 times greater than amounts recovered from homogenates without Aroclor. The water phases derived ' from samples where no homogenate was present or from boiled homogenates did not contain appreciable radiocarbon; however, the water phases derived from unboiled homogenates of untreated flies contained 56% of the totally applied radiocarbon (Table 4). When Aroclor was present in the homogenates, the radiocarbon content of the water phases was only 40-43% of the total application. These data indicate that Aroclor 1248 increased the persistence of paraoxon and decreased the formation of water soluble metabolites. However, no difference was observed in the degradation of paraoxon oY the appearance of water soluble products when either Aroclor was applied to the living house flies or when Aroclor was added to the 10,000 g super \ natant of homogenates from untreated flies. ' V / \ r \ 1 >' \ ' S." m'Uftyffl' .'Mf. i.u DSW 032037 Enzy Norn Unlr be Untr Aroc (ir A roc I- rP (C4KI untre 'C from '/ supc: r ' * t- A wen lion "am final tain: Si 18 1 (con bein Pr appr tenst' Th Deo la Due lii STLCOPCB4015999 'identified flics were volatilized iual treat- for* 1248. cd that no boiled fly leubation unboiled ever, only i were reindicated vith nonsed, more were redegrada- mixturcs n acetone ictivity of houseflies ogenates, ted house ration or paraoxon me: ether 1% of the c organic vith paraired from l of para:ater than s derived :s did not unboiled iocarboh ontent of icate that nation of dation of lor* was g super- PCB AND ORGANOPHOSPHORUS INSECTICIDES 639 TABLE 4 Effect of Aroclor 1248 on the in Vitro Degradation of "C-Paraoxon with House Fly Homogenates Enzyme source None Untreated Hies,* boiled Untreated flies' (CK) Aroclor treated (in vivo) flies-'1 Aroclor in vitro* Recovered as % of applied from extraction phases Hexane: ether MC* P--O* %CKr Water ,4C %CK` Total ,4C 99 2 99 1 95 2 97 0 288 294 . 3 0 30 5 5 102 2 102 1 36 2 55 2' 33 3 -+ 100 54 1' 164 56 I 100 40 2' 71 93 3 94 1 52 2* 52 1* 158 43 0 76 95 ir 2 * "C-dpm determined by LSC. Results are averages of replicated tests. * Paraoxon determined by GLC. Results are averages of replicated tests. ' Paraoxon in % of the amount recovered from homogenates of controls: untreated flies (CK - 100%). 4 ,4C-dpm in % of radiocarbon recovered from the water phase of homogenates of controls: untreated flies (CK * 100%). ' Control flics, topically treated with acetone only 18 hr before preparation of the 10,000* supernatant from fly homogenates. * Aroclor* 1248 was topically applied to house flies 18 hr before the preparation of the 10,000 g supernatant from fly homogenates. * Differences due to Aroclor treatment are significant at the I or 5% level (/ test). `Aroclor* 1248 was added directly to the 10,000* supernatant from fly homogenates. After analyses of the organic extracts by TLC and autoradiography, radioactive spots were observed that corresponded to paraoxon and to aminoparaoxon. Color confirma tion using reagents specific for amino compounds gave a positive reaction for the `'aminoparaoxon" spot. Because of interfering substances in the water phase after the final ether extraction, no TLC could be performed on these samples although they con tained substantial radioactivity. Since flies--used for the preparation of the 10,000 g supernatant--had been treated 18 hr previously with Aroclor 1248 in acetone (2 p.l/fly) or with acetone only (controls), solvent effects on these processes in living house fly populations are currently being investigated. Preliminary in vitro tests with subcellular components of rat liver did riot show any appreciable inhibitory effects of Aroclor 1248 on the degradation of paraoxon (Lich tenstein et at., unpublished). ACKNOWLEDGMENT The authors wish to express their appreciation to T. T. Liang for his technical assistance. REFERENCES ." Dbonier, C. C., Jones, H. A., and Incho, H. H. (1946). Organic compounds effective against larvae of Anopheles quadrimacuiatus. J. Econ. EntomoL 39, 459-462. . Duda, J. (1957). The use of chlorinated polyphenyla to increase the effective insecticidal life of lindane. J. Eeorti Entomol. 50, 218-219. / DSW 032038 STLCOPCB4016000 . * I '1 ii I > i V 'l I vv *,v 640 PUHREMANN AND LICHTENSTEIN Giuffrida, L., Ives, N. F., and Bostwick, D. C. (1966). Gas chromatography of pesticides-- Improvements in the use of special ionization detection systems. J. Ass. Offic. Anal. Chem. 49, 8-21. Holmes, D. G, Simmons, J. H., and Tatton, J. O. G. (1967). Chlorinated hydrocarbons in British wildlife. Nature (London) 216, 227-229. Hornstein, I., and Sullivan, W. N. (1953). The role of chlorinated polyphenyls in Improving lindane residues. J. Econ. Entomol. 46, 937-940. Kelly, R. G., Pests, E. A., Gordon, S., and Buyske, D. A. (1961). Determination of MCand 'H in biological samples by SchOniger combustion and liquid scintillation techniques. Anal. Blochem. 2, 267-273. Koeman, J. H., Ten Noever de Brauw, M. C., and de Vos, R. H. (1969). Chlorinated biphenyls in fish, mussels and birds from the river Rhine and the Netherlands coastal area. Nature (London) 221, 1126-1128. Lichtenstein, E. P., and Fuhremann.T. W. (1971). Activity of an NADPH-dependent nitro reductase in house flies. Science 172, 589-591. Lichtenstein, E. P., Schulz, K. R., Fuhremann, T. W,, and Liano, T. T. (1969). Biological interaction between plasticizers and insecticides. J. Econ. Entomol. 62, 761-765. Litterst, C. L., and Lichtenstein, E. P. (1971). Effects and interactions of environmental chemicals on human cells in tissue culture. Arch. Environ. Health 22, 454-459. Metcalf, R. L., and March, R. B. (1953). Further studies on the mode of action of organic thionophosphate insecticides. Ann. Entomol. Soc. Amer. 46, 63-74. Peakall, D. B., and Lincer, J. L. (1970). Polychlorinated biphenyls--Another long life widespread chemical in the environment. Bioscience 20, 958-964. Reynolds, L. M. (1969), Polychlorobiphenyls (PCBs) and their interference with pesticide residue analysis. Bull. Environ. Contam. Toxicol. 4, 128-143. Reynolds, L. M. (1971). Pesticide residue analysis in the presence of polychlorobiphenyls (PCBs). Residue Rev. 34, 27-57. Risebrouoh, R. W., Rieche, P., Peakall, D. B., Herman, S. G., and Kirven, M. N. (1968). Polychlorinated biphenyls in the global ecosystem. Nature {London) 220, 1098-1102. Steel, R. G. D., and Torrie, J. H. (1960). Principles and Procedures of Statistics, pp, 67-87. McGraw-Hill, New York. Tsao, C. H., Sullivan, W. N., and Hornstein, I. (1953). A comparison of evaporation rates and toxicity to house flies of lindane and lindane-chlorinated polyphenyl deposits./. Econ. Entomol. 46, 882-884. Widmark, G. (1967). Possible interference by chlorinated biphenyls. /. Ass. OJfic. Anal. Chem. 50, 1069. \ \ -/ .. m ijiijmuwpi^pili OSw 032G39 TO Sp pa: da i alb He pre spe of rat: que (I c saf( alb the < alb do: tol of prt Cop All t STLCOPCB4016001