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The fate of the plasticizer, di-n-octyl phthalate (DOP) has been examined in 33-day
terrestrial-aquatic and three-day aquatic model ecosystems. The five organisms of
the two systems contained residues of DOP, demonstrating the propensity of this
lipoid soluble organic molecule to be concentrated from the water. The residues
in the organisms in the three-day system were higher than in the 33-day system with
the exception of the fish, indicating perhaps, that DOP can undergo some degrada-
tion before the fish is placed in the system on the 30th day. A half-life of five days
for DOP disappearance from the water was calculated from water samples taken
periodically. Further, effects of mixed function oxidase a~nd esterase inhibitors were
investigated on the metabolism of DOP by various selected organisms and tissues of
the two ecosystems.
The continual increase in the use of plastics in construction, food packaging and coat-
ing is demonstrated by the production of 4 to 5 X 109 pounds of vinyl chloride
and its polymers (PVC) in 1972 (Anon. 1973a). The annual production of PVC
materials in 1972 was 20% greater than it was in 1971, and over the ten-year period 1962-
1972 an average annual production increase of about 10.6% occurred (Anon. 1973a). The
production of useful articles from PVC requires the incorporation of plasticizers. The
largest group of plasticizers used in the United States are derivatives of phthalic acid, with
the octyl esters constituting greater than 50% of the phthalate plasticizers produced in
1969 (U. S. Tariff Commission 1971). The total production of octyl phthalate esters
reached 4.5 X 107 pounds in 1972 (Anon. 1973b).
The large-scale production and use of these plasticizers in the United States has had an
environmental impact, as phthalates have been found as pollutants in streams, rivers
(Mayer e t al. 1972, Hites and Bieman 1972) and fish taken from these bodies of water
(Mayer e t al. 1972). Therefore, it is necessary to derive information about the uptake by,
and the metabolism and distribution in, aquatic organisms of these widely produced in-
dustrial chemicals. The development of the model ecosystem (Metcalf e t al. 1971) es-
tablished a means for evaluation of the potential environmental fates of agricultural and
industrial chemicals in a terrestrial-aquatic ecosystem. In recent work (Metcalf e t al.
1973a) the fate of di-2-ethylhexyl phthalate (DEHP) was examined in this system and it
was demonstrated that mosquito larvae, the P h y s a snail, and the mosquito fish, G a m b u s i a
affinis Baird and Girard, accumulated DEHP 100,000, 21,000, and 130 times, respectively,
the concentration of DEHP in the water. Other recent studies (Sanders et al. 1973) also
report the accumulation by several aquatic invertebrates exposed to low levels of this
plasticizer. Therefore, in view of the unfavorable concentration from the aquatic environ-
ment of phthalate esters by a variety of aquatic organisms, it is requisite that further in-
formation be derived on the effect of chemical structure on environmental concentration
of phthalate esters by aquatic organisms. The data reported in this paper describe the fate
of di-n-octyl phthalate (DOP) in a three-day aquatic model ecosystem and in a 33-day
terrestrial-aquatic model ecosystem. In addition, the effects of esterase and multifunction
oxidase inhibitors on the uptake and metabolism of DOP by 4th-instar mosquito larvae,
Culex pipiens quinquefasciatus Say, and salt marsh caterpillar larvae, Estigmene acrea
(Drury), were examined as well as the in vitro metabolism of DOP by liver microsomes
from females of Gambusia affinis Baird and Girard.
The procedures for the operation of the model ecosystem (Metcalf et al. 1971) were
followed. Each ecosystem was treated with 5 mg of DOP and the experiment was repeated
two times. The initial extracts of the organisms were made with acetone. The residues
from the organisms after extraction with acetone were treated with 0.20 N (two ml)
hydrochloric acid for one hr at 70~ This procedure should release conjugated radio-
active material. Finally, after acid hydrolysis the residue was solubilized for scintillation
counting in Protosol| (New England Nuclear) for counting in Aquasol| (New England
Nuclear). To separate and identify metabolites, degradation products or both, the initial
acetone extracts of the organisms were spotted with suspected degradation products of
DOP on tic (thin layer chromatography) plates (0.250 mm silica gel GF-254, Brinkmann)
and developed in a solvent system consisting of benzene-acetone-petroleum ether-acetic
acid (50:5:25:1). Radioautograms were prepared (No-screen X-ray film, Eastman Kodak)
to facilitate the location of the metabolites and degradation products. The distribution of
metabolites was determined by scraping the spots from the plate for liquid scintillation
counting in a fluid containing PPO (7 g), POPOP (0.05 g), and naphthalene (120 g) in
dioxane (one L). Quenching was corrected through the use of an external standard.
The procedures for the three-day model aquatic ecosystem (Metcalf et aL 1973b)have
not been described before, and, therefore, the following is a detailed outline of the design
246 J.R. Sanborn et al.
and operation of the system. The main unit of the system is a three-L, three-neck, round-
bottom flask equipped with (1) a microwire condenser for the reduction of losses of
volatile compounds, (2) an inlet tube for air to aerate the system, and (3) an outlet tube
into two traps employed for the collection of volatile compounds. The consecutive traps
contain acetonitrile (100 ml) to trap pollutants or their degradation products that may
be volatile and 0.50 N sodium hydroxide (100 ml) for collecting radioactive carbon
dioxide. The flask contains two L of standard reference water (Freeman 1953) which
provides suitable nutrients for the organisms and enhances the reproducibility of the
results. The type and number (where necessary) of organisms added to the system con-
stitute a freshwater food chain: phytoplankton; zooplankton; green filamentous alga,
Oedogonium cardiacum (Huss.); Physa snails Wittrock (6-8); 4th-instar mosquito larvae,
Culex pipiens quinquefasciatus Say (,x, 500); and the mosquito fish, Gambusia affinis
Baird and Girard (3). The entire system is placed in an environmental chamber held at
26~ with a 12-hr photoperiod at 2,000 footcandles of light. The ecosystem experiment
is initiated by placing all organisms, except the fish, in the flask for a one-day acclimation;
then a radiolabeled industrial pollutant is added at a level normally found in rivers and
lakes (for di-n-octyl phthalate, 0.30 mg). After 24 hr, 50 mosquito larvae and 100
Daphnia are removed for analysis and three Gambusia affinis Baird and Girard are added.
Twenty-four hr later the experiment is terminated and the water and organisms are
analyzed for radioactive metabolites by the same procedures employed for the 33-day
ecosystem.
Microsomal studies
The livers from five female Gambusia affinis Baird and Girard (total 261 mg) were
removed and ground in a Potter Elvehjem tissue grinder in cold 0.10 M phosphate buffer,
pH 7.80. The suspension was centrifuged ten min at 12,000 X G at 4~ The pellet was
discarded and the supernatant then was centrifuged 60 rain at 105,000 X G for one hr in
a Beckmann L350 ultracentrifuge. The supernatant was discarded and the microsomal
pellet was resuspended in eight ml of the same phosphate buffer. This suspension (one ml)
was added to four scintillation vials, and two vials were incubated with paraoxon
(4x 10-s M) at 22~ for 15 min. This procedure should inhibit non-oxidative esteratic
hydrolysis of the phthalate ester (Abemathy and Casida 1973). After incubation with
paraoxon, 100/ag of unlabeled DOP and 5.5/~g of 14C carbonyl labeled DOP were added
to each vial. Then 1.6 mg of NADPH, 3.6 mg of glucose-6-phosphate and one unit of
glucose-6-phosphate dehydrogenase in three ml of phosphate buffer were added to each
vial. The microsomal catalyzed reactions were allowed to proceed at 22~ for 30 min
and then were quenched with one ml of acetone and extracted with anhydrous ether
(4x2 ml). The ether extracts were dried over sodium sulfate, concentrated, and spotted
on a tic plate for analysis of the metabolite distribution.
Uptake and metabolism of DOP by mosquito larvae and salt marsh caterpillar larvae
alone and in the presence of tri-o-cresyl phosphate (TOCP) and piperonyl butoxide (PB).
The experiment for the uptake and metabolism of DOP by Culex larvae in the presence
of PB or TOCP was initiated by acclimatizing three groups of 200 larvae each in one L
of standard reference water (Freeman 1953) for one day. To the three jars were added
Uptake of Di-n-octyle Phthalate by Aquatic Organisms 247
0.10 ppm of DOP for the control, 0.10 ppm of DOP + 0.50 ppm TOCP and 0.10 ppm +
0.50 ppm PB in the two remaining jars. At the end of 24 hr the larvae were ground up in
acetonitrile and the extracts were spotted on tic plates for assessment of metabolite dis-
tribution by radioautography.
For the salt marsh caterpillar experiments, six 4th-instar Estigmene acrea (Drury) larvae
were employed. Two were used as controls and two each were given food with TOCP or
PB added to it. Each larva was placed in a separate petri dish and fed rehydrated dehydrated
potato chips (Pillsbury, Hungry Jack Hash Brown Potatoes| for 24 hr to clear the gut of
pigments from the previous diet. After the gut was cleared, ten/al of a 5% acetone solu-
tion of PB or TOCP was evaporated on a wet potato chip, and the chip was given to the
caterpillars. Twelve hr after the first chip was given, another inhibitor-impregnated chip
was given to the caterpillar. Twenty-four hr after the first chip was given, a fresh chip with
100 /ag of 14C DOP was placed in the dish for caterpillar consumption. Both feces
(methanol) and the whole caterpillar (acetone) were ground up after 24 hr, and the solvent
extracts were spotted on tic plates for evaluation of metabolite distribution.
Tankl Tankll
Spot No. Rf a H2 0 b T-H 20c Total H2 0 T-H 2 0 Total
Ib 0.75 . . . . 09 -
tration factors for these organisms could be in error from 2.25 times for the fish to 55.5
times for Daphnia if DOP concentrations were based on total 14C.
The data in Table III for the distribution of DOP in the three-day aquatic ecosystem
follow a pattern similar to that outlined for the 33-day system through the concentration
of DOP is much higher in all organisms except the fish. The Daphnia contained the most
DOP at 32.52 ppm, mosquito contained 18.30 ppm, and the snail, fish, and algae had
much less than these two organisms. The approximate magnification factors (EM) for
this three-day ecosystem are 660 for algae, 9,426 for Daphnia, 1.16 for fish, 5,300 for
mosquito, and 438 for the snail. Comparison of the Ecological Magnification factors for
the 33- and 3-day ecosystems reveals that, with the exception of Daphnia (0.28), all were
greater than one. The ratio of EM values (33/3) are 1.7 for mosquito, 4.2 for algae, 32.0
for snail and 8,100 for fish. The increased values for accumulation of DOP by the organ-
isms at the 33-day ecosystem clearly demonstrate the persistence of this material in an
aquatic system and its unfavorable accumulation by aquatic organisms.
In vivo metabolism of DOP by Culex and Estigmene acrea larvae. In an effort to clarify
the modification of DOP by selected organisms or tissues of these ecosystems, the
metabolism of DOP by the caterpillars, mosquito larvae, and liver microsomes from the
fish, was examined. In addition, the effect of metabolic inhibitors PB and TOCP on the
metabolism of DOP by these organisms was examined in an effort to separate oxidative
and esterase metabolism. The data for the/netabolism of DOP by Culex and saltmarsh
caterpillar larvae is tabulated in Table IV. The most interesting aspect of the data for the
mosquito and salt marsh larvae is that both piperonyl butoxide (PB) and tri-o-cresyl
phosphate (TOCP) reduce the formation of the mono octyl phthalate (MOP) about two-
fold in both insects. The similarity in the values for the formation of MOP-whether the
inhibitor is PB, a mixed-function oxidase inhibitor, or TOCP, an esterase inhibitor that is
252 J. R. Sanborn et al.
80 O
0 [~ C-O-nCSH17
70 -C-O-nC8H17
II
0
60 O
~ ,C-O--nC8 H17
*-50 ~C-OH
P_. II
x O
LU
40 O
ILl II
e-----e ~ C - O H
N30 C--OH
II
O
20
~ Unknown
10 Polar material
at origin
0 3 6 9 12 15 18 21 24 27 30
Time (days)
M i c r o s o m a l studies
The data for metabolism of DOP by hepatic microsomes from Gambusia affinis are
presented in Table V. Prior inhibition of the microsomal suspension with paraoxon
(15 min 10-s M) has a pronounced effect on the esteratic metabolism of DOP to MOP,
as this transformation is reduced about 23 times the number of uninhibited microsomes.
Uptake of Di-n-octyle Phthalate by Aquatic Organisms 253
Control PB b TOCpe
Compound Rfa (%14C) (%14C) (%14C)
Control PB TOCP
Compound Rfa (%14C) (%14C) (%14C)
This experiment indicates that the preferred metabolic pathway of DOP for these fiver
microsomes is non-oxidative ester cleavage, as apparently no oxidative metabolism occurs.
Perhaps this microsomal preparation, which epoxidizes aldrin at about the same rate as
trout liver microsomes (Krieger and Lee 1973) is not sufficiently active to introduce an
OH group either on the aromatic ring or on one of the hydrocarbon chains of the octyl
alcohol. Therefore, the more facile, non-oxidative hydrolysis of one of the alcohols pre-
dominates to yield MOP.
Conclusion
From the data presented in this paper and in other research reports it appears that
more information is needed on the fates, in aquatic organisms, of phthalate and other
widely used plasticizers. Though these materials appear to have a low degree of chronic
toxicity to aquatic organisms (Sanders et al. 1973), their long-term effects on the repro-
ductive potentials of aquatic organisms have not been established. Before such repro-
ductive studies can be rationally initiated, it is essential that information be obtained such
as that derived in this study, which describes the uptake and metabolic susceptibility of
aquatic organisms. Once these primary data are obtained, those materials which accumu-
late adversely and do not undergo metabolism readily should be examined in terms of
chronic effects on fish and other aquatic organisms. The necessity for long-term studies
on fish and other aquatic organisms is paramount, as these organisms constitute an im-
portant source of food as well as a multimiUion dollar recreational industry. Ideally, any
organic chemical produced in large quantities, such as plasticizers, should undergo careful
screening so that it does not become an undesirable environmental pollutant.
Uptake of Di-n-octyle Phthalate by Aquatic Organisms 255
Acknowledgments
The authors want to thank M. Kathryn McClendon for her skillful assistance during
this work. This investigation was supported in part by the Illinois Natural History Survey,
the Illinois Agricultural Experiment Station, Regional Project NC 96, U. S. Environ-
mental Protection Agency Grant No. EPA 800736, United States Department of the
Interior Grant No. 14-31-000-3879, and a grant to RLM from United States Department
of the Interior No. 14-31-0001-3273, Project No. B-050-Ill.
References