Uptake and fate of Di-2-ethylhexyl phthalate in aquatic organisms and in a model ecosystem.

Di-2-ethylhexyl phthalate (DEHP), often referred to as dioctyl phthalate or DOP, is the most widely used plasticizer for vinyl plastics, and approximately 350 million pounds were produced in the United States during 1970. Altogether the production of phthalate ester plasticizers was 855 million pounds (1). The cumulative production of DEHP and related phthalate plasticizers in the United States since 1943 is in excess of 8000 million pounds (2). DEPH is an external plasticizer which softens resins without reacting with them chemically, and it may be present in concentrations up to 40% of the weight of the plastic as in the familiar laboratory tubing. As a result of the large production and wide distribution and destruction of plastics, DEHP has become uniquitous and has been found in milk (3), deep frying fat (4), and human blood plasma (5). DEHP has also begun to appear as a micropollutant in the tissues of a variety of organisms. Taborsky (6) isolated it from bovine pineal glands, and Nazir et al. (7) found it in mitochondria from the hearts of cattle, dogs, rabbit, and rat. DEHP has been

Di-2-ethylhexyl phthalate (DEHP), often referred to as dioctyl phthalate or DOP, is the most widely used plasticizer for vinyl plastics, and approximately 350 million pounds were produced in the United States during 1970. Altogether the production of phthalate ester plasticizers was 855 million pounds (1). The cumulative production of DEHP and related phthalate plasticizers in the United States since 1943 is in excess of 8000 million pounds (2). DEPH is an external plasticizer which softens resins without reacting with them chemically, and it may be present in concentrations up to 40% of the weight of the plastic as in the familiar laboratory tubing. As a result of the large production and wide distribution and destruction of plastics, DEHP has become uniquitous and has been found in milk (3), deep frying fat (4), and human blood plasma (5). DEHP has also begun to appear as a micropollutant in the tissues of a variety of organisms. Taborsky (6) isolated it from bovine pineal glands, and Nazir et al. (7) found it in mitochondria from the hearts of cattle, dogs, rabbit, and rat (8). Therefore DEHP has all the requisite properties to be classified as an environmental micropollutant (9). Our investigation was undertaken to study its metabolism and possible biomagnification in a variety of aquatic organisms and its ecological behavior in food chains of a laboratory model ecosystem (10).

Expeimentl Procedures
14C Carbonyl-labeled DEHP was prepared from 10 mg phthalic 7-1 4C-anhydride, 9.2 mCi/mmole (New England Nuclear), by heating it in a sealed tube for 25 hr at 1300C with 100 Ml of 2-ethylhexyl alcohol and a trace of phthalic acid. The product consisted of 78% DEHP and was purified by column chromatography on silica gel and by subse-quent preparative thin-layer chromatography (TLC) on silicic acid with a solvent system of benzene, Skellysolve B, acetone, and acetic acid in proportions of 65:25:25:5 by volume. The final product used in these experiments had a radiopurity of more than 99% and was diluted to a specific activity of 5.56 mCi/mmole or 31,400 dpm/pg. An additional sample of 14C-labeled DEHP was purchased (from New England Nuclear) with a specific activity of 1.64 mCi/mmole.
The degradation products of 14C-DEHP in the various animal and plant tissues were separated by TLC and autoradiographs were made on Eastman no-screen x-ray film. The identities of the various metabolites were determined by cochromatography with known standards; radioactive spots were removed from the TLC plates by elution in scintillation fluid and determined quantitatively by liquid scintillation counting.

Uptake Studies
These were carried out to detennine the uptake of DEHP directly from water by a variety of aquatic organisms and to study the metabolic transformations occurring over a short period. The organisms chosen were the water flea Daphnia magna, the mosquito larva Culex pipiens quinquefasciatus, the "fingernail" clam Sphaerium striatinum, the guppy Lebistes reticulatus, and the aquatic plant Elodea canadensis. Standard reference water (11) was made from glass-distilled water, and 2.5-liter portions were placed in 5-liter battery jars. The 14C-labeled DEHP was dissolved in 1 ml of acetone and added quantitatively to the battery jars at concentrations of 0.1 and 10 ppm. Groups of organisms were exposed to the DEHP for various intervals as indicated in the tables.

Model Ecosystem Study
A laboratory model ecosystem with a terrestrial-aquatic interface and a sevenelement food chain has been found very useful in estimating the potential environmental effects of DDT and other pesticides (10), particularly in regard to ecological magnification and biodegradability.
14C-DEHP was evaluated in this system by applying 5 mg of the carbonyl-labeled compound to Sorghum plants on the terrestrial end of the system. After 33 days, the various organisms in the system were homogenized in water, extracted with diethyl ether, and the concentrated extracts resolved by TLC on silicic acid by use of benzene, Skellysolve B, acetone, and acetic acid in the proportions 65:25:25:5 by volume. The radioactive spots were located by autoradiography and the parent compound and metabolites determined quantitatively by liquid scintillation counting.

PRsults and Discussion
Uptake Studies Autoradiograms of the homogenized extracts of the organisms exposed to 1 4C-DEHP at 10 ppm for 1, 2, and 7 days are shown in Figures 1 and 2; a quantitative estimation of the distribution of the radioactivity in the various spots is given in Table  1. The data demonstrate the steady decrease of DEHP in the guppy from 88.5% of total radioactivity after 1 day, to 37.1% after 2 days and 16.8% after 7 days. There was a commensurate increase in the polar metabolites at the bottom of the chromatograms, from 11.5% after 1 day to 34.2% after 2 days and to 80.6% after 7 days. One of the identified metabolites was phthalic acid, which comprised 23.8% of the radioactivity after 2 days and then decreased to 4.8% after 7 days. Small amounts of a metabolite which is apparently phthalic anhydride appeared after 2 and 7 days. Uncontaminated guppies fed Daphnia with a 1-day uptake of 14C0 labeled DEHP showed the same degradation pattem (  A detailed study was made of the rate of uptake of 14C-DEHP by Culex larvae, Daphnia, snail, Elodea, and guppy exposed to 0.1 ppm and 10 ppm over intervals from 1 hr to 48 hr. The distribution of 1 4C in the organisms at the various intervals is shown in Table 2.
From these uptake studies, the following conclusions can be drawn.

Model Ecosystem Study
During the 33-day period of the model ecosystem study, the concentration of 14 C in the aquatic phase reached a peak of 0.031 ppm at the fifth day after treatment and declined to 0.0077 ppm at the end of the experiment. This decline was clearly the result of the uptake of DEHP and its degradation products by the organisms of the model ecosystem as shown in Table 3. The distribu-      products found in the model ecosystem, the major degradative pathways seem to be through hydrolysis of the ester groups to gX produce MEHP, then phthalic acid, and then phthalic anhydride. tion of radioactivity was clearly indicated by the autoradiograph in Figure 5. At the conclusion of the experiment, the water contained 0.00034 ppm DEHP, the algae 18.32 ppm (53,890X ) the snails 7.30 ppm (21,480X ), the mosquito larvae 36.61 ppm (107,670X), and the fish 0.044 ppm (130X). Thus the model ecosystem experiment was in good agreement with the uptake studies in showing that the mosquito larvae had the highest bioconcentration factor, and the fish the least. The autoradiographs (Fig. 5) showed the presence of almost nothing but DEHP in algae and mosquito larvae, indicating that these organisms have little degradative capacity. The snail produced substantial amounts of mono-2-ethylhexyl phthalate (MEHP), phthalic anhydride, and phthalic acid. The fish were more active in metabolism, and about half the total amount of 14C found in their bodies was a compound, Rf 0.65, which cochromatographed with phthalic anhydride and is presumed to be that compound reformed from phthalic acid.
It is interesting to compare the model ecosystem studies of DEHP with that of DDT conducted under identical conditions (10).
The experiments reported above demonstrate that DEHP is a microchemical environmental pollutant which is rapidly biomagnified by a variety of plants and animals in an aquatic system. DEHP is biodegraded very slowly in algae, Daphnia, mosquito larvae, snails, and clams and more rapidly in fish by hydrolysis at the ester bonds to form monoethylhexyl phthalate, phthalic acid, phthalic anhydride, and a variety of polar metabolites and conjugates. However, DEHP closely resembles DDT in rate of uptake and storage, and it obviously partitions strongly in the lipids of plants and animals and is concentrated through food chains. The biomagnification of DEHP together with its teratogenic properties and its enormous rate of production and ubiquitous use indicate the need for much further study of its environmental distribution and fate. Present data suggest the need for restrictions on the use and waste disposal of DEHP.