Document 6bYxv5o9onnZe8J3a4bv0MdJE

JOHNSON AND LULVES: DBP AND DEHP IN HYDROSOIL 339 mined from these unknowns (Table 4). Phthalic acid is rapidly decarboxylated (Table 3), we postulate, to form either the 1,2-dihydroxy ben zene (catechol) or benzene. We believe the hydroxylated compound is the most probable product. Meikle (1972) indicated that when the carboxyl group is attached directly to the aromatic ring, the most common microbial attack on the molecule is via the oxidation decarboxylation pathway. Aerobic and anaerobic investigation of 14C-n-butyl phthalate and 14C-phthalic acid (both carboxyl-labelled) tend to support this bio degradation pathway (Table 3). TLC-autoradiograms of 14C-BP hydrosoil extracts reveal the disappearance of the monoester concomitant with the appearance of trace amounts of material at R, 0.09 (phthalic acid) and loss of radioactivity from the TLC plates. Autoradiograms of phthalic acid extracts showed no detectable degradation products, and furthermore, after 7 days incuba tion the hydrosoil extract contained less than 5% of the radioactivity that was recovered from the autoclave-killed control ether-extract. Radiores pirometry data showed rapid decarboxylation of phthalic acid in hydrosoil (Table 3). For ex ample, we recovered 40.1 and 75.8% of the 14Cphthalic acid radioactivity as trapped 14C02 after incubation in hydrosoil for 3 and 7 days, re spectively. Similarly, in hydrosoil, nearly 33% of the radioactivity of the monoester 14C-BP ap peared as 14C02 in 3 days and nearly twice that amount in the next 4 days. In general, the dis appearance of radioactivity recovered from TLCplates closely paralleled the recovery of 14C02 trapped in the respirometer samples. No radio activity was detected in the OV-225 Chromosorb W column from either 14C-phthalic acid or 14C-BP samples. There is extensive literature on the microbial destruction of the aromatic nucleus, leading to the ultimate utilization of the C-fragment by microorganisms (Evans 1963; Gibson 1968, 1972). Unfortunately, we were unable to obtain substantial evidence to suggest ring cleavage after ester hydrolysis because the phthalic acid moiety was only 14C-carbonyl labelled. Acknowledgments We thank J. Johnson, J. Hogan, F. Mayer Jr., R. Schoettger, and D. Stalling for their advice and assistance. American Public Health Association, American Water Works Association, and Water Pollu tion Control Federation. 1965. Standard methods for the examination of water and wastewater. New York.N.Y. p. 257-259. Corcoran, E. F. 1973. Gas-chromatographic detection of phthalic acid esters. Environ. Health Perspect. 3: 13-15. Evans, W. C. 1963. The microbiological degradation-of aromatic compounds. J. Gen. Microbiol. 32:177-184. Gibson, D. T. 1968. Microbial degradation of aromatic compounds. Science 161:1093-1097. 1972. Initial reactions in the degradation of aromatic hydrocarbons, p. 116-136. In Degradation of synthetic organic molecules in the biosphere. Proc. Conf. San Francisco, Calif. National Academy of Sci ences, Washington, D.C. Graham. P. R. 1973. Phthalate ester plasticizers -- why and how they are used. Environ. Health Perspect. 3: 3-12. Hall, A. 1971. A modem plastics special report in chemi cals and additives. Mod. Plast. 48: 58. Hites, R. A. 1973. Phthalates in the Charles and Mer rimack rivers. Environ. Health Perspect. 3:17-21. Holz, D. D,, F. L. Mayer Jr., and R. C. Tindle. 1972. A core-type sampler for pesticide studies. Prog. FishCult. 34:117-118. Kaufman, D. D., J. R. Plimmer, P. C. Kearney, J. Blake, and F. S. Guardia. 1968. Chemical versus microbial decomposition of amitrol in soil. Weed Sci. 16:266-272. Mathur, S. P. 1974. Phthalate esters in the environment: pollution or natural products? J. Environ. Qua). 3: 189-197. Mayer, F. L. Jr., and H. O. Sanders. 1972. Toxicology of phthalic acid esters in aquatic organisms. Environ. Health Perspect. 3: 153-157. Mayer, F. L. Jr., D. L. Stalling, and J. L. Johnson. 1972. Phthalate esters as an environmental contamin ant. Nature 238:411-413. Meikle, R. W. 1972. Decomposition: qualitative relation ship, p. 145-251. In C. A. I. Goring and J. W. Hamaker [ed.] Organic chemicals in the soil environ ment. Dekker, New York, N.Y. Metcalf, R. L., G. M. Booth, C. K. Sehuth, D. J. Hansen, and P.-Y. Lu. 1973. Uptake and fate of di-2-ethylhexy! phthalate in aquatic organisms and in a model ecosystem. Environ. Health Perspect. 4: 27-34. Ogner, G., and M. Schnitzer. 1970. Humic substances: Fulvic acid-diakyl phthalate complexes and their role in pollution. Science 170:317. Saeger, V. W., and E. S. Tucker. 1973. Phthalate esters undergo ready biodegradation. Plast. Eng. 29:45-49. Sanders, H. O., F. L. Mayer Jr., and D. F. Walsh. 1973. Toxicity, residue dynamics and reproductive effects of phthalate esters in aquatic invertebrates. Environ. Res. 6: 84-90. Stalling, D. L., J. W. Hogan, and J. L. Johnson. 1973. Phthalate ester residues -- their metabolism and analysis in fish. Environ. Health Perspect. 3:159-173. Tepper, L. B. 1973. Phthalic acid esters -- an overview. Environ. Health Perspect. 3:170-182. U.S. Tariff Commission. 1971. Report on plasticizers. Government Printing Office, Washington, D.C. DSW 271851 .in jjj STLCOPCB4062852 338 J. FISH. RES. BOARD CAN.. VOL. 32(3). 1975 Table 3. Aerobic degradation of 14C-di-n-butyl phthalate, 14C-di-2-ethylhexyl phthalate, l4C-mono-/ibutyl phthalate, and 14C-phthalic acid by freshwater hydrosoil in a radiorespirometer. 14C-labelted compound l4COj evolved (total dpm x 103)* 3 days 7 days 14 days 21 days 28 days Total dpm Recovered as ,4COj Introduced as 14C- compound" di-/t-butyl phthalate di-2-ethylhexyl phthalate mono-n-butyl phthalate phthalic acid Control' ND ND 12.4 97.3 0 24.3- 8.6 5.0 1.6 2.1 12.0 8.5 7.6 23.0 181.1 0 ND ND 0 ND ND ND ND 00 39.2 30.2 35.4 278.4 0 50.0 50.0 38.0 268.6 0 *14C02 trapped in monoethanol amine :ethylene glycol. Data represent average of duplicate samples. ND = not done. "Approximate value + 5.0%. "All heat-killed and NaNj controls showed <0.1% total dpm introduced as ,4C02. Decarboxylation in Hydrosoil We attributed the rapid loss of radioactivity from the hydrosoils to biodegradation of the labelled phthalates during incubation (Table 3). We speculate that the 14C-labelled portion of the phthalate molecule, the carbonyl group, is exposed to further degradation after initial hydrolysis of the ester-linkage. Subsequently, the labelled car bon is removed from the mono-phthalate (or phthalic acid, salicylate, or benzoate) moiety in the form of 14C02 by decarboxylation. To test this hypothesis, we inoculated 14C-n-butyl phtha late (BP) and 14C-phthalic acid into hydrosoils and incubated them in a radiorespirometer. Dur ing aerobic incubation, we detected no 14C-vola- tile compounds other than 14C02 escaping from the hydrosoil into the effluent gas and no radio activity in the toluene extract from the OV-225 column. Recovery of radioactivity from the C02-trapping solution closely paralleled the disappearance of 14C-label!ed material from TLC-autoradiograms of both 14C-DBP and 14C-DEHP (Table 3). Only trace amounts of 14C02 activity were recovered from heat-killed and NaN3 controls. We interpret these data to indicate that the re labelled material in the monoethanol amine solu tion is carbon-14 labelled carbon dioxide in the form of 14C-carbamate; the evolution of 14C02 is due to the direct enzymic activity of the hydro soil microflora on the 14C-phthalates. Table 4. R/ values of phthalic acid esters and potential degradation products. Compound Rj value'" n-butyl salicylate Di-2-ethylhexyl phthalate Di-n-butyl phthalate Benzoic acid Salicylic acid Mono-2-ethylhexyl phthalate Mono-n-butyl phthalate p-hydroxybenzoic acid 3-hydroxyphthalic acid Phthalic acid 0.90 0.85 0.81 0.60 0.51 0.36 0.33 0.20 0.16 0.09 Solvent system = petroleum ether:diethyl ether:acetic acid, 77:20:3 (vol/vol/vol) TLC plates were developed 3 times. "Values are the mean of five replicates. PAE Degradation Pathway We propose a pathway for the biodegradation of DBP and DEHP by microflora in freshwater hydrosoil. The proposed scheme is predicated on TLC-autoradiography evidence. Under either aerobic or anaerobic conditions, the diesters are initially hydrolyzed at the ester linkage to form the phthalic acid half-ester and the corresponding alcohol. We believe this monoester is subsequently degraded, primarily to phthalic acid, by con tinuous esterase activity in the hydrosoil. We suspect that the exposed carboxyl group of the monoester undergoes decarboxylation (either oxidative or reductive) to form the benzene or salicylate derivatives. The R/ values of authentic standards of benzoates and salicylates, however, exceed the R/ values of 0.25 and 0.15 deter mined f acid is postulat zene (c droxylat product. carboxy ring, th< molecul' pathway ,4C-n-bt carboxy; degrada grams < disappe: the appe 0.09 (p from thi acid exi products tion the of the r autoclav pirometi phthalic ample, v phthalic incubatit spectivel the radi peared a amount appearar plates cl trapped activity ' W colum samples. There destructi. the ultir microore 1972). I substanti after esf moiety w We tha Schoettge: assistance. Americai> Wate TION ( DSW 271852 STLCOPCB4062853 Recovered radioactive compound after day(s) incubation JOHNSON AND LULVES: DBP AND DEHP IN HYDROSOIL /* r- sn r> o VO VO O t- cm so +1 +! +1 +| O VO v O ri +1 +1 +1 +1 SO Os o ri Sf-O 0r0r cn VO -- o VO N.. * o' o H -H O O -H *H O no t- rC4i <s fi o o b <o o N +HH +1 +1 H VO m +1 wtx *-> o <si ci oo +I+IOO -H -H -onN oo Os m +1 +J +1 +1 ^ m \ BaD +J 00 IX vo m* 8=3+' 5. > oo 0\v>nso^ Eoo g O SSoo-'Ov-o m+1 O+1G\ o -+----+1 /> r** 2 oo H oo Tt --' +1 +1 +1 +1 +1 vo n n n o ~8 * xai CLO g = n u ~s tf-s- 2C O W> Cd S *0 *o .2 `o c ed cd tn o O o +1 +1 +1 -H -H +! nnn*-Oin dso1 T <s c4 oo <n m * O o vn * +1 +lf" +IH oo O so m *SE gK C= Cjjd ivno 5 B*+M Ci > a .! gOS-- 1 2 ' 00. -Ocd fLc* *JC '*o*', * o 2 fgol |I 8 1 3 R B 8 ft.S U a 8|cd at o cd *ed O"3f fi. a-- 33 ^,, = :t2_k c 1" "I IIS o-S 6 g 8|.s gDSDDSo cd a Eo g CQ a 88-TMa at c5 _ t> Id o *rOa CO --a 3oC _C>kk3>C^* wciScc . 3 C ? .O gg -c9 Oe jc* j= .S a ea Cv"0 Cd o| "5oOu1 *nou5 Cl iRgs E e 11 3 2 a. .f eSx co Q cod ^JS O-- b C A u js. ei cd < 337 DSW 271853 I STLCOPCB4062854 336 J. FISH. RES. BOARD CAN., VOL. 32(3). 1975 Table 1. Biodegradation of ,4C-di-n-butyl phthalate and l4C-di-2-ethylhexyl phthalate in freshwater hydrosoil. Incubation (days) Percent recovery of radioactivity from hydrosoil ------------ :--------------------- Aerobic Anaerobic lAC-di-n-butyl phthalate 1 95" 5 3` 7 5` 14 8` 30 Heat-killed control4 3` 100 14C-di-2-ethylhexyl phthalate? 7 100" 14 30 Heat-killed control4 53' 41` 100 100b 69 59' 39' 2' 100 100` 100b 100" 100 Data represent the mean value of triplicate samples. Precision = 85 5%. Values rounded to nearest 1.0. bNo significant difference existed between control (P<0.05, Fisher's "r"). `Significant difference between control (P<0.05, Fisher's d30-day incubation. Degradation products were 2% of total radio activity recovered. under anaerobic conditions, whereas only 3% of the extractable labelled DBP and degradation products remained after 5 days of aerobic incuba tion. Degradation in anaerobic hydrosoils did not reach the aerobic level until the 30-day sampling. Ester hydrolysis required twice as much time in anaerobic as in aerobic hydrosoil, and decar boxylation time was about 6 times longer under anaerobic conditions. DEHP was completely re sistant to microbial attack under anaerobic condi tions. After 30 days, we observed no significant loss of 14C-DEHP activity in hydrosoils overlayed with nitrogen (Table 1). Identification of Degradation Products In TLC-autoradiography analyses of the 14CDBP and 14C-DEHP hydrosoil extracts we re peatedly found radioactivity in DBP extracts at R, 0.82, 0.33, 0.25, 0.15, 0.09, and 0.00 (Table 2). The spots at Rr 0.82, 0.33, and 0.09 (Table 4), when cochromatographed with nonradio-labelled authentic standards, were similar to the spots of di n-butyl phthalate, n-butyl phthalate, and phthalic acid, respectively. The spots at R, 0.25, 0.15, and 0.00 did not correspond to known degradation prod ucts (Table 4). Quantitation of the radioactivity at each R^ value by liquid scintillation spectrometry indicated that the major degradation product of the 1-day sample was the monoester (46.3%). Small amounts of compounds more polar than the monoester (6% of the total recovered radio activity) were at Rr 0.25, 0.09, and 0.00. Rr 0.09 corresponded to phthalic acid; Rf 0.25 and 0.00 were unidentified compounds. Significantly, recovery of radioactive material showed that samples incubated for 1 day con tained 95% of the total radioactivity found in the control. This recovery decreased in the 5-, 7-, 14-, and 30-day samples. In comparison with the control, only 2.9-7.9% of the radioactivity introduced into these samples was recovered (Table 2). Qualitative analyses of these extracts by TLC-autoradiography revealed the parent di ester, the monoester, phthalic acid, and a trace amount of an unknown or unknowns at the origin in an approximate ratio of 76:18:3:1% (Table 2). The slower rate of PAE degradation under anaerobic than under aerobic conditions is clearly reflected in the type and quantities of the radio active components extracted from hydrosoils after various incubation intervals (Table 1, 2). At least four compounds were detected in anaerobic DBP samples: the monoester, phthalic acid, and two unknowns at R/ 0.25 and 0.00. The compound or compounds at R; 0.15 was not seen in any of the autoradiograms taken from anaerobic extracts. Although n-butyl phthalate was the major degra dation product in either the aerobic or anaerobic samples after 1 day of incubation, the degradation rate was more than 1.5 times faster under aerobic conditions (32% vs. 50%). Qualitative compari son of aerobic with anaerobic phthalate degrada tion after the first 24-h incubation period is at best tenuous because of the rapid loss of radio activity from the samples. We did, however, observe an increase in the monoester after long incubation and an apparent lack of accumulation of the more polar degradation products in anaerobic samples (Table 2). TLC-autoradiographic analysis of aerobic DEHP extracts revealed radioactive spots at R; 0.85 and 0.36. Cochromatographic data with authentic standards suggest that the products were di-2-ethyIhexyl phthalate and the monoester, 2-ethyIhexyl phthalate. Liquid scintillation quan titation showed only 2% of the total recovered radioactivity as the monoester. We found no other compound in the aerobic extracts. After 30 days, hydrosoils incubated anaerobically showed no detectable DEHP degradation. DSW 271854 I_ STLCOPCB4062855 JOHNSON AND LULVES: DBP AND DEHP IN HYDROSOIL 335 (BBL). All hydrosoil samples contained 5% organic soil sample during incubation. Hydrosoils containing carbon (dry weight) (American Public Health As the two "C-diesters were sampled at 7-, 14-, 21-, and sociation 1971). Ten grams (wet weight) of hydro 28-day intervals for '*CO, production. Controls con soil were placed into a 50-ml Delong flask and sisted of both autoclave-killed and NaN* samples. covered with 20 ml of pond water. The flask content Air was passed at a flow rate of 5 ml/min into the was then inoculated with 100 #d of acetone containing experimental chamber after being scrubbed with 2 N the "C-labelled phthalate. Cultures were adjusted to NaOH to remove COi. The effluent gas from the contain about 1 mg/liter labelled phthalate. After incubation chamber was first passed through a 50.4- inoculation, the aerobic groups were incubated in a X 6.35-mm column containing OV-225 coated the water bath rotary shaker (180 rpm, 1 in orbit) and Chromosorb W HP and finally bubbled through a -ncy the anaerobic groups were placed in an anaerobic monoethanol amine:ethylene glycol-COt (3:7 vol/ uiral jar (Torsion Co.), evacuated under vacuum, and vol) trapping solution. The "COi was trapped as a overlayed with nitrogen. All samples were incubated carbamate. To investigate the efficacy of this system, Jeg- ,li-2'Litvl ;ind : of .! in at 22 C. We tested for spontaneous degradation (abiotic) of the phthalates by including an autoclavekilled control. The control sample was autoclaved at 15 lb pressure and 121 C for 20 min. We added 250 mg/liter sodium azide to some hydrosoil samples as a chemical means of inhibiting microbial activity (Kaufman et al. 1968). Hydrosoil was maintained we evaporated "C-labelled phthalic acid, DBP, DEHP, and BP in a small flask, connected it to the respirometer, and gently warmed the flask to vaporize the materials. We found only trace amounts of radio activity in the COrtrapping solution. The major portion of the radioactivity recovered was confined to the Chromosorb W column. Triplicate, 1-ml samples .on- under aerobic or anaerobic conditions and sampled of the "COj-mono-ethanol amine solution were added :crs. in triplicate at 1-, 5-, 7-, and 30-day intervals. Con to 10 ml of a dioxane-fluor mixture composed of 4 g was trols consisted of acetone and autoclave-killed hydro: of 2,5-diphenyloxazole (PPO), 200 mg of 1,4 bis-2- >ito- soil. All samples were extracted on the day of (4-methyI-5-phenyloxyazolyl)-benzene (POPOP), 100 sed collection. Each hydrosoil sample was initially acidi ml of methanol, and 60 g of naphthalene in 1 liter fied to pH 1-2 with concentrated HjPO, and extracted of dioxane. Scintillation samples were counted with 3 times with an equal volume of diethyl ether. The the Beckman 200-L liquid scintillation system and ether extracts were centrifuged for 10 min at 10,000 corrected for quench and background radiation. X g to remove particulate matter and break emul i .53 sions, combined, dried over anhydrous NaSO,, and Statistical Inference carefully evaporated to about 1 ml at room tem ,Ci/ perature under a hood. Extraction efficiency of the Mean comparisons were made according to Fisher's ere phthalates from hydrosoil was routinely 85 5%; test; significance was taken at P < 0.05. All data IBP -MV. 'vim I he ;he'.-.tiuas esti:ade re30.one all data are based on this accuracy value. Auto radiography of thin-layer chromatograms indicate both "C-DBP and 14C-DEHP were 99+% pure. Chromatography-Autoradiography The ether extract was spotted by means of a Camag Linomat automatic TLC spotter on 0.2-mm pre coated silica gel thin-layer chromatography plates (Brinkman, EM Reagents, Silica Gel, F-254) con taining a fluorescent indicator. The TLC plates were developed 3 times in petroleum ether:diethyl ether: represent the mean value of samples removed from triplicate incubation vessels (x se). Results and Discussion Freshwater hydrosoil degraded both 14C-DBP and 14C-DEHP (Tables 1, 2, 3). Since autoclavekilled or NaNs controls did not significantly degrade either ester, we attributed the degradation of the two phalates to the enzymic action of microorganisms indigenous to freshwater hydro J ret acetic acid (77:20:3) (vol/vol/vol) (Stalling et al. soil. ires 1973). We used no-screen medical X-ray film to We found marked differences between the con visualize radioactive products by autoradiography and ditions and rates of biodegradation of the two and :aide ,'ling shortwave ultraviolet light (2537A) to detect nonlabelled standards and controls. The quantity of each radio-labelled material separated on the chromatogram was determined by scraping silica gel from the specific radioactive Rr area into a scintillation vial containing 15 ml of toIuene:fluor (Fluoralloy, esters. Forty-Six percent of the radio-labelled DBP in hydrosoil (Table 2) was degraded aerobically to mono-n-butyl phthalate within 24 h and nearly 98% (Table 1) of all radioactivity disappeared after 5 days. Under the same conditions, nearly "out ' cm and ante S Beckman Instrument Co.) and then counting the mixture with a Beckman 200-L liquid scintillation counter set for ,4C-/3-particle energy spectrum. Results were corrected for quench and background radiation. 14 days were required for 53% of the DEHPlabelled material to disappear (Table 2). Anaerobiosis slowed biodegradation of both esters. Although nearly 98% of the radio-labelled 'Oil, : c. Radiospirometry DBP disappeared during 30 days of incubation in the hydrosoil, the retarding influence of anaero ible We constructed a radiospirometer to detect pos biosis was evident. Nearly 70% of the DBP ..car sible "CO* evolution from the "C-phthalate hydro radioactivity remained after 5 days of incubation DSW 271855 STLCOPCB4062856 334 J. FISH. RES. BOARD CAN.. VOL. 32(3). 1975 se produit a un rythme raJenti d'un sixieme, alors que nous n'avons pas detecte de degradation de DEHP. La chromatographic en couche mince, de meme que la radiorespiratometrie, suggerent que les esters, apres hydrolyse initiale, subissent une decarboxylation, probablement vers la molecule 1,2-dihydroxybenzene. Received September 16,1974 Accepted December 2,1974 Reju le 16 septembre 1974 Accepte le 2 decembre 1974 Reports of phthalic acid esters (PAEs) in soil (Ogner and Schnitzer 1970), water (Corcoran 1973; Hites 1973), and fish (Mayer et al. 1972) have appeared in the recent literature. Mathur (1974) has only recently reviewed the topic of phthalate esters in the environment. During 1972, an estimated 1 billion pounds of 20 different phthalates were manufactured in the United States (Hall 1971). Although PAEs, the esters of benzene ortho dicarboxylic acid, have a broad spectrum of uses ranging from defoaming agents to perfume vehicles in cosmetic production (U.S. Tariff Commission 1971), they are utilized chiefly as plasticizers with the poly (vinyl chloride) polymers (PVCs). Used in conjunction with PVCs, plasticizers impart flexibility, workability, and extensibility to the product (Graham 1973). In as much as PAE plasticizers have a many faceted market in automotive, construction, clothing, home furnishing, medical, and packaging industries (U.S. Tariff Commission 1971), their appearance in aquatic organisms and habitats seems inevitable. Even though PAE residues are apparently ubiquitous in the environment, the acute and chronic effect on man and wildlife remains es sentially unknown -- "an etiology in search of a disease" (Tepper 1973). Fragmentary evidence suggests potential deleterious effects on freshwater biota. Sanders et al. (1973) and Mayer and Sanders (1972) reported reproductive inhibition and bio accumulation in the filter-feeding cladoceran Daphnia magna. Metcalf et al. (1973) also found rapid accumulation of phthalates in a variety of plants and animals in a model ecosystem. The fate of PAEs in the aquatic environment remains largely a mystery. Saeger and Tucker (1973) demonstrated that all PAEs tested underwent complete aerobic degradation, at rates comparable with those of a readily degradable linear alkyl benzene sulfonate, in the presence of activated sewage sludge and river water. Unfortunately, no data are available concerning degradation under the anaerobic conditions that frequently attend hydrosoils in the natural environment. A number of questions concerning the ultimate fate of PAEs remain unanswered. Do PAEs rapidly undergo complete biodegradation in the aquatic environment to harmless elemental fragments or are they resistant to microbial attack? Are the current PAE residues a reflection of persistency or an equilibrium between input and natural decomposer activity? In this investigation, we report on the biodeg radation of two widely used plasticizers, di-2ethylhexyl phthalate (DEHP) and di-n-butyl phthalate (DBP) by unidentified aerobic and anaerobic microorganisms found in hydrosoil of freshwater ponds. The studies were performed in the laboratory under aerobic and anaerobic con ditions, with 14C-labelled phthalic acid esters. The microbial degradation of phthalates was determined by thin-layer chromatography-auto radiography and measurement of 14C02 released from labelled phthalates. Materials and Methods Chemicals "C-carbonyI-labeIIed di-n-butyl phthalate (1.53 mCi/mM). di-2-ethylhexyl phthalate (1.64 mCi/ mM) and phthalic acid (2.74 mCi/mM) were purchased from Mallinckrodt Co. Standards of DBP and DEHP were obtained from Monsanto Company. Chemicals listed in Table 4 were purchased from Chem Service or Eastman Kodak Chemical Co. The "C-n-butyl phthalate (monoester, BP) was synthe sized from "C-DBP by Bacillus subtilis (Fish-Pesti cide Research Laboratory Collection). Acetone was used as the solvent carrier for all compounds investi gated (<0.01% vol/vol). Plate counts were made of control samples containing 0.1% (vol/vol) re distilled, analytical grade acetone at 1-, 7-, and 30day intervals. We found no evidence that acetone influenced the viability of the microflora in hydro soil or induced any radical pH changes in the cultures during incubation. Culturing and Extraction Methods Hydrosoil samples were taken from 0.04- and 0.10-ha freshwater ponds located at the Fish-Pesticide Research Laboratory. We used a core-type sampling device (Holz et al. 1972) to collect pond bottom material along the shoreline at a water depth of about 1 m. We removed the hydrosoil to a depth of 5 cm in the core sample at the mud-water interface and screened it through a 40-mesh wire sieve to eliminate macroinvertebrates. Plate counts of the hydrosoil, incubated both aerobically and anaerobically at 22 C, showed an average population of 5 X 10* viable organisms per milliliter on brain-heart infusion agar (BBL). carbon sociatior soil wei covered was ther the ''C-I contain inoculati water bt the anat jar (To overlaye at 22 ( (abiotic; killed cc 15 lb pr mg/liter a chem (Kaufm: under a> in triplk trols cot soil. Al collectio fled to p 3 times ether ex x g to sions, c< carefull) perature phthalat< all data radiogra both "C Chroma The e Linomat coated (Brinkm taining i develops acetic at 1973). visualize shortwa' labelled radio-lal was det specific containii Beckmai mixture counter were coi Radiosp We c sible '*( l DSW 271856 STLCOPCB4062857 Journal of^ Fisheries Research Board otCanada Volume 32, No. 3, March 1975 Journal dei office des recherches sunes pecheries du Canada Volume 32, n 3, mars 1975 Biodegradation of Di-zi-Butyl Phthalate and Di-2-Ethylhexyl Phthalate in Freshwater Hydrosoil1 B. Thomas Johnson and William Lulves Fish-Pesticide Research Laboratory, Bureau ofSport Fisheries and Wildlife U.S. Department ofthe Interior, Columbia, Mo. 65201, USA Johnson, B. T., and W. Lulves. 1975. Biodegradation of di-n-butyl phthalate and di-2-ethylhexyl phthalate in freshwater hydrosoil. J. Fish. Res. Board Can. 32:333-339. The phthalic acid esters di-2-ethylhexyl phthalate (DEHP) and di-n-butyl phthalate (DBP) which are used as plasticizers and recovered in routine chemical analysis offreshwater fish, were incorporated into freshwater hydrosoil in the laboratory. Samples containing about 1 mg/liter of these two esters, MC (carbonyl) labelled, were incubated aerobically and anaerobically for 1,5,7, 14, and 30 days. Differences in the rates and conditions of degradation of the two esters were marked. Under aerobiosis, 53% of the radio-labelled DBP was degraded within 24 h, and 98% within 5 days; DEHP, in contrast, was only 50% degraded after 14 days. Under anaerobiosis degradation of both esters was retarded. DBP was degraded only one-sixth as fast in hydrosoil overlayed with nitrogen whereas degradation of DEHP was not detected. Our evidence from both thin-layer chromatography and radiorespirometry suggests that the esters undergo decarboxyla tion after initial hydrolysis, probably to the 1,2-dihydroxybenzene molecule. Johnson, B. T., and W. Lulves. 1975. Biodegradation of di-n-butyl phthalate and di-2ethylhexyl phthalate in freshwater hydrosoil. J. Fish. Res. Board Can. 32: 333-339. Les esters di-2-ethylhexyl phthalate (DEHP) et di-n-butylphthalate (DBP) de l'acide phthalique, utilises comme plastifiants et recuperes lors d'analyses chimiques routinieres de poissons d'eau douce, ont etc incorpores a un hydrosol d'eau douce au laboratoire. Des echantillons contenant environ 1 mg/litre de ces deux esters, marques au l4C (carbonyl), ont ete incubes dans des conditions aerobies et anaerobies durant 1,5,7,14 et 30jours. La vitesse et les conditions de degradation des deux esters accusent des differences marquees. Dans des condi tions aerobies, 53% du DBP marque est degrade en dedans de 24 h et 98% en dedans de 5jours: par contre, DEHP n'est degrade que de 50% au bout de 14 jours. Dans des conditions anaerobies. la degradation des deux esters est retardee. Dans un hydrosol enduit d'azote, la degradation du DBP 'Trade names referred to in this paper do not imply endorsement of commercial products. Printed in Canada (J3569) Imprim6 au Canada (J3569) 333 DSW 271857 STLCOPCB4062858 Reprinted from Journal of the Fisheries Research Board of Canada Reimpression du ~5 Journal dei office des recherches sunespecheries du Canada Biodegradation of Di-n-Butyl Phthalate and Di-2-Ethylhexyl Phthalate in Freshwater Hydrosoil B. Thomas Johnson and William Lulves \ Volume 32 Number 3 1975 Pages 333-339 ^ Environment I ~ Canada Fisheries and Marine Service Environnement Canada Service des pftches et des sciences de la mer V.-' DSW 271858 STLCOPCB4062859