EPA 600/3-76-048
June 1976
Ecological Research Series
EFFECTS, UPTAKE, AND METABOLISM OF
METHOXYCHLOR, MIREX, AND 2,4-D IN SEAWEEDS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-76-048
June 1976
EFFECTS, UPTAKE, AND METABOLISM OF METHOXYCHLOR,
MIREX, AND 2,4-D IN SEAWEEDS
by
Harish C. Sikka, Gary L. Butler, and Clifford P. Rice
Syracuse University Research Corporation
Syracuse, New York 13210
Contract No. 68-03-0271
Project Officer
Gerald E. Walsh
Environmental Research Laboratory
Gulf Breeze, Florida 32561
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE, FLORIDA 32561
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DISCLAIMER
This report has been reviewed by the Office of Research and
Development, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products con-
stitute endorsement or recommendation for use.
ii
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ABSTRACT
This report presents the results of a study concerning effects, uptake,
and metabolism of mirex, methoxychlor, and 2,4-D in the seaweeds Ulva sp.,
Enteromorpha sp., and Rhodymenia sp. None of the pesticides, at concen-
trations corresponding to their maximum solubility in seawater, had any
significant effect on photosynthesis, protein, carbohydrate, lipid,
chlorophyll, carotenoid, or trace metal content of the algae. All three
algae removed substantial amounts of mirex and methoxychlor from the
medium, but uptake of 2,4-D was extremely low. The rate of uptake of
methoxychlor was considerably greater than that of mirex. Bioaccumula-
tion of methoxychlor was greater than that of mirex. Enteromorpha
accumulated considerably more mirex and methoxychlor than Ulva or
Rhodymenia.
Both Ulva and Enteromorpha failed to metabolize either mirex or 2,4-D.
Enteromorpha metabolized methoxychlor to a limited extent. After 7 days
of incubation with ^C-methoxychlor, a major portion of the ll*C in the
tissue and medium was present in unchanged methoxychlor. A small amount
of a llfC-metabolite, 2,2-bis (p_-methoxyphenyl)-l,l-dichloroethylene, was
detected in both the tissue and medium. In addition, medium contained
2,2-bis (£-hydroxyphenyl)-l,l,l-trichloroethane and four unidentified
minor llfC-metabolites. Unlike Enteromorpha, Ulva did not metabolize
methoxychlor.
This report was submitted in fulfillment of Contract No. 68-03-0271
by the Syracuse University Research Corporation under the sponsorship
of the U.S. Environmental Protection Agency. Work was completed as of
August 31, 1975.
iii
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CONTENTS
Page
Abstract ill
List of Tables vi
Acknowledgment vii
Sections
I Introduction 1
II Materials and Methods 3
III Results and Discussion 13
IV Conclusions 34
V Recommendations 35
VI References 36
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TABLES
No. Page
1 Photosynthesis in Seaweeds Treated with
Methoxychlor , Mirex, and 2,4-D ....... ........ 17
2 Protein Content of Seaweeds Treated with
Methoxychlor, Mirex, and 2,4-D .............. -
3 Total and Soluble Carbohydrate Content of Seaweeds
Treated with Methoxychlor, Mirex, and 2,4-D ........ 20
4 Pigment Content of Seaweeds Treated with
Methoxychlor, Mirex, and 2,4-D ......... ..... 21
5 Total Lipid Content of Seaweeds Treated with
Methoxychlor, Mirex, and 2,4-D ............ ... 21
6 Trace Metal Content of Seaweeds Treated with
Methoxychlor, Mirex, and 2,4-D ............... 22
7 Uptake of lt|C-Methoxychlor by Seaweeds ........... 23
8 Uptake of 11+C-Mirex by Seaweeds .............. 24
9 Uptake of 14C-2,4-D by Seaweeds ....... . ...... 25
10 Bioaccumulation of Methoxychlor, Mirex, and
2,4-D by Seaweeds .................... 26
vi
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ACKNOWLEDGMENT
The technical assistance of Robert Lynch is gratefully acknowledged.
vii
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SECTION I
INTRODUCTION
GENERAL
Mirex (dodecachlorooctahydro-l,3,4-metheno-2H-cyclobuta [cd] pentalene),
methoxychlor [2,2-bis(£-methoxyphenyl)-l,l,l-trichloroethane] and 2,4-D
(2,4-dichlorophenoxyacetic acid) are three commonly used pesticides.
Mirex has been used for many years to control imported fire ants in
pasture lands in the southeastern United States (Alley, 1973). Methoxy-
chlor, a broad-spectrum insecticide, is a very likely biodegradable
replacement for DDT. Though chemically similar, it is less toxic to
fish, birds and mammals than DDT (Pimental, 1971). Recently, methoxy-
chlor has replaced DDT for several predominantly environmental uses,
such as controlling blackfly larvae in streams, elm bark beetles, and
fruit and garden pests (Burdick et al, 1968). 2,4-D is widely used to
control broadleaf weeds. Formulations of 2,4-D are also extensively used
to control aquatic plants, such as water hyacinth and Eurasian water-
milfoil (Lawrence, 1962).
These pesticides may enter the estuarine environment in a variety of
ways, including application to control objectionable flora and/or fauna,
disposal of wastes from pesticide manufacturing and formulation plants,
run-off from treated lands adjacent to estuaries, and drift from pesti-
cide application. The contamination of the estuarine environment with
pesticides and their metabolites is of great environmental concern
because of their potential toxicity to estuarine fauna and flora. To
evaluate the impact of pesticides in the estuarine environment, it is
important that we have a knowledge of their fate, and the effects that
they and their metabolites have on the biota.
Among the estuarine biota, seaweeds play a vital role in marine ecology.
They contribute to .the oxygen supply in the marine environment through
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photosynthesis and also play an important role in nutrient regeneration.
If pesticides adversely affect the growth of these algae, significant
changes may occur in the estuarine ecosystem.
The seaweeds may play a role in determining the fate of pesticides in
the estuarine environment. They may remove pesticides by adsorption
and/or absorption and may degrade them. The pesticides and their meta-
bolites which are accumulated by the seaweeds may be transferred to other
trophic levels and may constitute a health hazard. Therefore, identifi-
cation and measurement of residues of pesticides and their metabolites
are necessary to fully evaluate their impact on the marine environment.
A number of studies have been done to examine effects and metabolism of
pesticides in marine phytoplankton, but no information is available on
effects, uptake, and metabolism of pesticides in seaweeds. This project
was undertaken to investigate effects and fate of 2,4-D, mirex, and
methoxychlor in three species of commonly occurring seaweeds, Ulva
lactuca (Chlorophyta), Enteromorpha linza (Chlorophyta), and Rhodymenia
pseudopalmata (Rhodophyta). The overall objective of this investigation
was to provide information needed for establishing marine water-quality
standards for the above chemicals.
SPECIFIC OBJECTIVES
1. To study effects, if any, that 2,4-D, mirex, and methoxychlor have
on photosynthesis of Ulva lactuca, Enteromorpha linza and Rhodymenia
pseudopalmata at concentrations not exceeding pesticide solubility in
the growth medium.
2. To establish whether pesticides affect the chemical composition
(carbohydrate, protein, pigment, lipid and trace metal content) of
seaweeds.
3. To determine uptake and metabolism of the pesticides by the seaweeds.
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SECTION II
MATERIALS AND METHODS
PESTICIDES
Technical-grade pesticides were used to evaluate effects on seaweeds.
The sources and the purity of the chemicals were: 2,4-D, purity 97%
(The Dow Chemical Company); methoxychlor, purity 98% (E.E. duPont
de Nemours and Co.); and mirex, purity 98% (Allied Chemical Compnay).
Pesticides labeled with ltfC were used to study uptake and metabolism
by the seaweeds. Uniformly ring-labeled lt*C-2;4-D was purchased from
the California Bionuclear Corporation, Sun Valley, California.
14C-Uniformly ring-labeled methoxychlor and 1 **C-uniformly labeled mirex
were purchased from the Mallinckrodt Chemical Company, St. Louis, Missouri.
PESTICIDE SOLUBILITY DETERMINATION
An excess of each pesticide was added to artificial seawater medium
having a salinity of 33-34 ppt. (For medium composition, refer to page 6.)
After 24 hours of shaking on a wrist shaker, the mixture was filtered
through a 0.45 u porosity glass fiber filter. The concentration of mirex
or methoxychlor in the filtrate was determined by gas-chromatography,
whereas the 2,4-D concentration was determined spectrophotometrically.
To analyze the filtrate for mirex and methoxychlor, aliquots were
extracted successively three times with equal volumes of pesticide-grade
•
petroleum ether. The first two extracts were combined, and the third
was analyzed separately. The extracts were concentrated by being
evaporated in a Kuderna-Danish apparatus and analyzed for pesticides
using a Microtek-Model MT-220 gas-chromatograph equipped with a 63Ni
electron capture detector. Operating temperatures for the various
components were: inlet, 210°C; oven, 205°C; and detector, 290°C. The
chromatographic column, an 800 cm x 4 mm (i.d.) glass U-tube, was packed
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with an equal-weight mixture of 1.5% OV-17 and 1.95% QF-1 on 80-100 mesh
size Supelcoport. The carrier gas (nitrogen) flowed at a rate of 50 ml/min.
The amount of pesticide in the extract was determined from peak area
measurements which were compared with those of standard mirex or methoxy-
chlor solutions. Mirex or methoxychlor could not be detected in the
third extract of each set.
The concentration of 2,4-D in the filtrate was determined spectrophoto-
metrically by measuring absorbance at 271 nm in a Gary 14 spectrophoto-
meter. A standard curve relating optical density at 271 nm to the con-
centration of 2,4-D was used to determine the concentration of the
herbicide in the unknown sample.
SOURCE OF SEAWEEDS
It was considered desirable to use seaweeds grown in the laboratory because
their age and culture conditions were known. Such organisms were
expected to be more uniform in their physiological response than field-
collected seaweed. When it was not possible to culture a sufficient
amount of algal material in the laboratory, seaweeds collected from the
field and maintained in the laboratory were used for experimental work.
It may be mentioned that algae collected from the field at different
times of the year may vary in their physiological conditions, which may
alter their response to toxicants. Also, seaweeds obtained from estuarine
areas may contain pollutants present where the plants were collected. In
such cases, problems arising from the interaction of the test chemicals
with other pollutants may arise.
Cultures of Ulva lactuca, Enteromorpha linza, and Rhodymenia
pseudopalmata were obtained from the University of Indiana Culture
Collection of Algae. Field-collected Ulva sp., Enteromorpha sp. and
Rhodymenia sp. were purchased from the Northeast Marine Specimens
Company, Inc., Woods Hole, Massachusetts. The latter were maintained
in aerated seawater in a cold room at 4°C under a light intensity of
about 150 lux.
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CULTURE CONDITIONS
Although the culture of unicellular marine phytoplankton has been studied
in detail (Guillard and Ryther, 1962; McLachlan, 1964; Mclachlan, 1973;
Provasoli et al, 1957) there is little information available on methods
for culturing seaweeds. Since the proposed studies required the use
of a relatively large amount of algal tissue, techniques for culturing
seaweeds were developed to obtain sufficient plant material. Favorable
conditions for growing or maintaining the organisms were determined by
varying the factors most likely to affect the culturing process: (1)
size of inoculum; (2) composition of the growth medium; and (3) light
and temperature.
Inoculum
Vegetative Propagation - The algae used were obtained from the field or
grown from stock cultures. Prior to being used, field-collected plants
were maintained in aerated seawater in a cold room at 4°C under a light
intensity of approximately 150 lux. To culture the organisms, sections
of the algae were transferred to the appropriate growth medium contained
in 80 x 100 mm Pyrex dishes and grown in a growth chamber under conditions
of light and temperature described on page 6.
Propagation From Zoospores - Under laboratory conditions, Ulva and
Enteromorpha may be induced to form reproductive structures (zoospores)
which may then be used for establishing cultures. Zoospores were
obtained by filtering one-month-old cultures of the algae through a
double layer of cheesecloth. The number of zoospores per ml of culture
was estimated using a hemocytometer. Approximately 2,000 spores were
added to each culture dish (80 x 100 mm Pyrex dish) containing a
sterilized growth medium and were incubated in the growth chamber,
*
Approximately 5% of the added zoospores germinated.
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Composition of the Growth Medium
Both synthetic and natural seawater media were tested for their ability
to support adequate growth of the alge. The media were those of
Erdschreiber (Frfyn, 1934), Guillard and Khyther (1962), ASP-6 (Provasoli,
1963; Provasoli et al, 1957), and an enriched synthetic seawater medium.
Synthetic seawater was prepared by dissolving 40 gm of Rila Marine Mix
(Rila Products, Teaneck, N.J.) in a liter of distilled water. The
fresh synthetic seawter was aged in a glass aquarium with a metal frame
containing thalli of field-collected Ulva. After one week, the seawater
was removed and stirred with activated charcoal to remove dissolved
organic matter. The medium was filtered, the salinity was adjusted to
33-34 ppt and the pH to 7.8. After autoclaving, each liter of the
medium was supplemented with the following (Ott, 1973): NaNO,, 200 mg;
sodium glycerophosphate, 25 mg; FeS0^.7H20, 4.98 mg; ZnSO,,7H20, 8.82 mg;
MnCl2.4H20, 1.44 mg; Mo03, 0.71 mg; Co(N03)2.6H20, 0.49 mg; H3B03,
11.4 mg; Na2EDTA, 56.6 mg; FeCNH^CSO^.eHjO, 7.0 mg; biotin, 1 yg;
vitamin B12, 1 pg; thiamin HC1, 200 yg.
Light and Temperature
Ulva and Enteromorpha were grown under controlled conditions in a growth
chamber, i.e., 14 hrs of light per day at 2150 lux and at 18°C + 1°.
These conditions proved more favorable than a light intensity of 5400 lux
and a temperature of 2Q°C. Rhodymenia was cultured under a light
intensity of 540 lux.
TREATMENT OF SEAWEEDS
Ulva
Only field-collected Ulva was used and experiments were begun less than
48 hrs after the tissue was received. Discs (5 cm in diameter) were
cut from the Ulva thalli, rinsed in the growth medium and suspended in
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a pesticide-saturated medium. At the end of the treatment period,
smaller discs were cut from the larger discs for measuring physiological
effects.
Enteromorpha
Preliminary experiments were conducted using individual germlings that
were obtained by scraping them from the walls of a culture dish and
transferring them to the growth medium. Attached germlings were used,
however, in later studies because they grew faster and remained healthy
longer. Enteromorpha germlings 3-4 weeks old were used for experimental
work. Culture dishes containing germlings were removed from the growth
chamber and the medium was replaced by one containing the pesticides.
The treated cultures were then returned to the growth chamber.
Rhodymenia
Culture dishes containing Rhodymenia thalli were removed from the growth
chamber. The thalli were rinsed with sterile growth medium and resus-
pended in a pesticide-saturated medium.
PHYSIOLOGICAL EFFECTS OF PESTICIDES
In these studies, the tissue was incubated in saturated solutions of
pesticides prepared as described above.
Photosynthesis
Photosynthetic activities of the algae were determined by measuring
llfC02 fixation by the tissue. To make this measurement, algal tissue
was incubated in 25 ml of growth medium containing 0.90 y moles of
NaH^COa (1,200,000 dpm) for 30 min at 20°C under a light intensity of
2150 lux. The tissue was then removed from the medium, washed free of
ll|C-sodium bicarbonate with medium, blotted dry, and analyzed for the
amount of 14C fixed. To analyze for 1£*C the tissue was homogenized in
a glass tissue-homogenizer with a liquid-scintillation solution
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containing PPO, POPOP, Triton-X, and toluene. The radioactivity of the
homogenate was measured in a Nuclear Chicago liquid scintillation counter.
In preliminary studies, photosynthesis was also measured by determining
oxygen evolution by the tissue. Small sections of algal tissue were
incubated in the growth medium containing 0.025M sodium bicarbonate and
oxygen evolution was measured polarographically at 20°C at a light
intensity of 2150 lux using a Yellow Springs Instrument Co. oxygen electrode.
Proteins
Protein content was determined according to the method of Strickland
and Parsons (1965). The tissue was boiled for 1 min in 1 ml of distilled
water and homogenized with 80% ethanol in a glass tissue-homogenizer.
The homogenate was centrifuged, the supernatant was removed, and the pellet
was extracted again with 80% ethanol. After the second extraction, the
residue was suspended in 4 ml of 6N HC1 and heated at 100°C for 4 hrs.
The acid extract was centrifuged, the supernatant adjusted to a pH of 1
with NaOH, and the final volume made up to 10 ml v;ith distilled water.
One ml of this protein extract was mixed with 1 ml of 2,4-hexanedione
reagent (1 ml of 2,5-hexanedione in 50 ml of 0.5M NaoCO-.H^O) and the
mixture was heated for 40 min at 100°C. The solution was then cooled
rapidly, made up to 9 ml with 95% ethanol, and one ml of Ehrlich's
reagent (800 mg dimethylaminobenzaldehyde dissolved in 30 ml of 95%
ethanol and 30 ml of concentrated HC1) was added. After the reaction
proceeded for 30 min at room temperature, the absorption was read at
530 nm. The concentration of proteins was calculated from a standard
curve prepared from bovine serum albumin.
Carbohydrates
The algae were analyzed for their water-soluble and total carbohydrates
content by the method of Ashwell (1957). The soluble carbohydrates were
extracted by homogenizing fresh tissue in 1-2 ml of distilled water.
The homogenate was boiled for one hour, centrifuged, and the supernatant
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made up to 10 ml with distilled water. One ml of this extract was added
to 9 ml of a 0.2% solution of anthrone dissolved in concentrated sulfuric
acid, and the mixture heated for 15 min at 90°C. After cooling the
solution to room temperature, its absorbance was measured at 625 nm, and
the concentration of soluble carbohydrates was calculated from a standard
curve perpared from glucose. For determination of total carbohydrates,
the tissue was digested with 60% H2SO, for 30 min. It was then centri-
fuged, and the supernatant was made up to 10 ml with 60% H-SO,. One ml
of this was added to 9 ml of anthrone reagent, and the absorbance was
measured at 625 nm.
Pigments
The procedure described by Strain et al (1971) was used to extract
pigments from the algae. The tissue was softened by placing it in
boiling distilled water for 1 min. It was then ground in a small volume
of acetone and made up to a volume of 5-10 ml with 80% acetone. The
homogenate was centrifuged, and the absorbance of the clear supernatant
was measured spectrophotometrically in a Gary Model 14 spectrophotometer
at 645, 663 and 480 nm. The methods of Kirk and Allen (1965), Kirk (1968),
and Davies (1965) were used to calculate the concentrations of chloro-
phyll (a_+ b) and caroteneids, respectively.
Lipids
The amount of total lipids in the tissue was determined as described by
Radin (1969). The tissue was lyopholyzed, weighed, and homogenized in
a glass tissue homogenizer in chloroform:methanol (2:1). The homogenate
was passed through a glass-fiber filter, the filter was washed with
chloroform, and the residue was discarded. The filtrate was place'd in
a previously weighed aluminum weighing pan, the extract was evaporated
almost to dryness on a hot plate and dried for about 18 hrs in an oven
at 100°C. The pans were cooled in a dessicator, and the weight of the
lipid residue was determined.
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Trace Metals
The algal tissue was lyopholized and ground to a fine powder in a mortar
and pestle. One gram of this tissue was digested with 5 ml of HNO_ and
2 ml of 70% HC10 . The digestate was evaporated to 3 ml, diluted to
20 ml with deionized, distilled water and passed through acid-washed
filter paper. The filter paper was washed with water, and the filtrate
was diluted to 50 ml with deionized water. A reagent blank was also
prepared using the procedures described above, except that the tissue
sample was excluded. A Perkin Elmer Model 303 atomic absorption spectro-
photometer was used to analyze the extracts. To analyze for magnesium,
a portion of the extract was diluted with a 1% (w/v) lanthanum solution,
and the determinations were made against standard blanks containing
a similar concentration of lanthanum. The other trace metals (Cu, Mn,
Zn and Fe) were analyzed using an undiluted extract. Concentrations of
the trace metals in the test solutions were determined from standard
curves prepared for each metal.
UPTAKE AND METABOLISM OF MIREX, METHOXYCHLOR AND 2,4-D BY SEAWEEDS
These studies were done using ^C-uniformly ring-labeled methoxychlor
(sp. activity 4.03 mCi/mM), ^C-uniformly ring-labeled 2,4-D (sp.
activity 10.5 mCi/mM) and 14C-uniformly labeled mirex (sp. activity
5.76 mCi/mM).
Uptake Studies
The algal tissue was incubated in the Rila medium containing ll*C-labeled
methoxychlor, mirex, or 2,4-D. ll*C-pesticides were added as ethanolic
solutions so that the final concentration of ethanol in the medium was
0.005%. The cultures were incubated on a reciprocating shaker under
controlled environmental conditions described previously. At intervals
over a 6-day period following treatment, the tissue from the cultures
was sampled, washed, weighed and analyzed for total radioactivity. To
10
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determine the amount of 1£tC in the tissue, it was homogenized with scin-
tillation fluid in a glass tissue homogenizer; the homogenate was then
transferred to scintillation vials and counted for radioactivity.
Metabolism Studies
To study transformation of pesticides by the algae, both medium and
tissue were analyzed for the parent compound and possible metabolites.
Mir ex - The tissue was homogenized with acetone, the homogenate was
centrifuged, and the extract was decanted. The residue was re-extracted
with acetone, and the extracts were combined. The combination was
concentrated under a stream of nitrogen and chromatographed on thin-
layer silica-gel plates in the following solvent system: (i) hexane-
acetone (9:1), and (ii) heptane (Mehendle et al, 1972; Jones and Hodges,
1974). The chromatograms were scanned for radioactivity in a Nuclear
Chicago Actigraph.
The incubation medium was extracted three times with hexane. The three
extracts were combined, and the amount of radioactivity in the aqueous
and organic phases was determined. The hexane extract was concentrated,
and an aliquot was chromatographed on a thin-layer silica gel plate as
described above.
Methoxychlor - The tissue was homogenized with acetone in a glass tissue
homogenizer, the homogenate was centrifuged, and the extract was decanted.
The residue was then extracted with 80% acetone in the same manner. The
extracts were combined, and acetone in the extract was removed under
vacuum to give an aqueous solution. The latter was acidified to a pH of
2, extracted with ethyl ether, and the amounts of 1I+C in the ether and
aqueous phases were determined. The ether layer was concentrated and
chromatographed on thin-layer silica gel plates in the following solvent
systems: (i) benzene-acetic acid (9:1), (ii) petroleum ether-ethyl ether
(9:1), (iii) hexane-acetone (8:2) and (iv) petroleum ether-chloroform-
methanol (3:2:1). The chromatograms were scanned for radioactivity on a
Nuclear Chicago Actigraph.
11
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To analyze the medium for methoxychlor and its metabolites, it was
acidified to a pH of approximately 2 and then extracted three times with
ethyl ether. The ether extracts were combined, and the amounts of lHC
in the ether extract and the remaining aqueous fraction were determined.
The ether extract was subjected to thin-layer chromatography as described
above.
Synthesis of model metabolites of methoxychlor - The following model
metabolites were synthesized as described by Kapoor et al (1970) for
comparison with unknown metabolites produced by Enteroroorpha; (i)
2,2-bis (p_-methoxyphenyl)-l,l-dichloroethylene was prepared by refluxing
3.45 g technical analytical grade methoxychlor (99.2%) in 100 ml of
ethanol that contained 0.75 g KOH; it was then recrystallized from
ethanol; (li) 2,2-bis (p_-hydroxyphenyl)-l,l,l-trichloroethane was pre-
pared by condensing 10.8 g of phenol with 7.4 g of anhydrous chloral in
250 ml of chloroform to which was added 7.4 g of anhydrous aluminum
chloride at 4°C. Stirring continued at 20°C for 30 min and for a further
8 hrs at room temperature. The product was crystallized from methylene
chloride with a trace of ethanol.
2,4-D - The tissue was homogenized with methanol, the homogenate was
centrifuged, and the extract was decanted. The residue was then extracted
with 80% methanol. The extracts were combined, and methanol in the
extract was removed under vacuum. The remaining aqueous solution was
acidified to a pH of approximately 2 and extracted with ethyl ether.
The ether extract was concentrated in a rotary evaporator under reduced
pressure and chromatographed on thin-layer silica gel plates in the
following solvent systems described by Hamilton et al (1971): (i)
chloroform, and (ii) n-butyl alcohol-benzene-water (1:9:10). The
chromatograms were then scanned for radioactivity on a Nuclear Chicago
Actigraph.
The medium was acidified to a pH of approximately 2 and extracted twice
with ether. The ether extracts were pooled and the amounts of 14C-in the
ether extract and the aqueous phase were determined. The ether extract
was concentrated under a stream of nitrogen and chromatographed on thin-
layer silica gel plates as described above.
12
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SECTION III
RESULTS AND DISCUSSION
CULTURE CONDITIONS
Major problems encountered in these studies were: (i) an extremely slow
growth rate of seaweeds cultured under laboratory conditions, and (ii)
maintenance of field-collected algae in a healthy state in the labora-
tory for longer than one week. A considerable amount of time was spent
determining culture conditions favorable for growing or maintaining the
algae. The findings of these exploratory studies are described below.
Medium
Sections of thalli from the three algae appeared to grow better in the
artificial seawater supplemented with trace elements and vitamins than
on Guillard and Ryther's medium 'f1 or on Provasoli's ASP-6 medium.
Therefore, the artificial seawater medium was used in all subsequent
studies. We believe that a synthetic medium with a well-defined com-
position is preferable for studies on effects of pollutants because
seasonal and geographical variations in natural seawater affect certain
physiological processes which may alter the response of organisms to
pollutants. Furthermore, artificial seawater medium permits one to
study only the effect of the pollutant under consideration, since under
these conditions the possibility of interaction with other pollutants
in natural waters is excluded.
Source of Inoculum
Although we were able to propagate both Ulva and Enteromorpha
vegetatively from sections of thalli, growth was quite slow and did not
yield sufficient plant material to study effects and metabolism of the
pesticides. In an attempt to obtain a larger amount of tissue, we
investigated the possibility of culturing the algae from zoospores. In
the case of Enteromorpha, we were consistently able to obtain sufficient
plant material when it was propagated from zoospores.
13
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It was possible to induce Ulva to produce zoospores, but the germlings
did not grow well. Since growth of Ulva, when propagated vegetatively
or from zoospores, was not satisfactory, thalli collected from the field
were used.
We were able to culture Rhodymenia vegetatively from sections of thalli
obtained from the Indiana Culture Collection, but growth of the algae
thus propagated was extremely slow. Rhodymenia collected from the ocean
was not satisfactory for these studies since the tissue deteriorated
rapidly, even when maintained in aerated seawater in a cold room at 4°C.
Loss of pigments was noticed 24-48 hrs after the tissue was transferred
to fresh growth medium in the growth chamber.
Based on the results of exploratory studies, the experiments were done
under the following culture conditions:
Culture Medium: Artificially enriched seawater supplemented with
trace elements and vitamins.
Temperature: 18°C + 1°
Experimental Plant Material:
Enteromorpha: Germlings from zoospores
Ulva: Sections of field-collected thalli
Rhodymenla: Sections of laboratory-grown thalli
ANALYSIS OF SYNTHETIC SEAWATER MEDIUM FOR 2,4-D, MIREX, METHOXYCHLOR,
DDT, DDD, DDE, DIELDRIN, TRACE METALS, AND ORGANIC MATTER
Prior to addition of trace elements and vitamins, the medium was analyzed
for pesticides, trace metals, and organic matter. The concentrations
of pesticides in the growth medium were below the limits of detection.
Since the medium consisted of synthetic seawater and was filtered
through charcoal before it was used, the amount of organic matter in
the medium was expected to be negligible. Therefore, we did not analyze
the medium for organic matter content.
14
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PESTICIDE SOLUBILITY IN THE CULTURE MEDIUM
The maximum solubility of methoxychlor, mirex, and 2,4-D in synthetic
seawater (33-34 ppt salinity) at 20°C was 22.8 ppb, 10.2 ppb, and 220 ppm,
respectively. In these studies, we examined the effect of 2,4-D on
seaweeds at a concentration equivalent to its maximum solubility in the
medium, although it is unlikely that 2,4-D will be present in the marine
environment at such a high concentration. However, lack of effect of
pesticide at a concentration many times that occurring in the environment
establishes the safety of a compound with regard to an organism.
PHYSIOLOGICAL EFFECTS OF METHOXYCHLOR, MIREX, AND 2,4-D
In these studies, the algae were exposed to the pesticides for an
appropriate period after which the photosynthetic activity and concen-
trations of tissue constituents were measured. Enteromorpha and
Rhodymenia were incubated with the pesticide for three weeks. Ulva was
exposed to the pesticide for one week because the tissue disintegrated
if maintained in the laboratory for a longer period. In studies con-
cerning physiological responses of algae to pesticides, it would be
desirable to measure response to a chemical at different intervals
during the incubation period. However, since only a limited amount of
algal tissue was available and a relatively long period was required
to culture it, effects of pesticides were assessed only at the end of
the treatment periods.
In the toxicity studies, the amount of tissue in 100 ml of medium ranged
from 350-450 mg of fresh weight in the case of Ulva and Rhodymenia
and from 450-650 mg for Enteromorpha.
Photosynthesis
In preliminary studies, effect of the pesticides on photosynthesis was
determined by measuring oxygen evolution from algal tissue. The polaro-
graphic method for determining oxygen evolution permitted use of a very
15
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small amount of tissue. Though It was possible to obtain a measurable
rate of oxygen evolution, an inherent error in the weighing of small
quantities of tissue produced variable results. Therefore, we studied
effects of 2,4-D, mirex, and methoxychlor on photosynthesis by measuring
1'*CO_-fixation, which has the following advantages: (1) this method
permits use of larger amounts of plant tissue, thus reducing the magnitude
of error encountered when weighing smaller amounts of tissue used in
polarographic determinations; (2) the variations among replications in
CO -fixation were much smaller than those for 0_ determinations; (3)
the ***C02-fixation method is less time-consuming than that used to make
0« determinations; and (4) the rapid stirring required in measuring ()„
exchanged appears to physically damage algal tissue. The ^CC^-fixation
method requires only gentle agitation to achieve adequate gas exchange.
Mirex, methoxychlor, and 2,4-D failed to inhibit photosynthesis in Ulva,
Enteromorpha, or Rhodymenia when the algae were incubated with saturating
concentrations of the pesticides (Table 1).
Lack of effect of 2,4-D on photosynthesis by the seaweeds can be explained
by the fact that the herbicide does not enter the tissue in sufficient
amounts, as shown by uptake studies (Table 9). In our experiments,
2,4-D in the growth medium (pH 7.8) was present mostly in the ionized
form. Since only the undissociated 2,4-D molecule penetrates the cell
membrane readily, the concentration of 2,4-D in the algae under the
conditions of these experiments is expected to be low. Wedding et al
(1954) and Erickson et al (1955) observed that inhibition of photosynthesis
by 2,4-D in Chlorella was related to concentration of undissociated
2,4-D acid molecules in the bathing medium. With a decrease in pH, the
same concentration of 2,4-D caused a greater inhibition of photosynthesis.
_3
The inhibition by 2 x 10 M 2,4-D (440 ppm) was complete at a pH of 3-4
while at pH values above 6 there was no inhibition. Their results
suggest that the undissociated 2,4-D molecule is the effective agent in
inhibiting photosynthesis.
16
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Table 1. PHOTOSYNTHESIS IN SEAWEEDS TREATED WITH
METHOXYCHLOR, MIREX, AND 2,4-D a
Photosynthetic 11+C02-Fixation
Pesticide
Organism
% Control
Methoxychlor Enteromorpha
Ulva
Rhodymenia
Mirex Enteromorpha
Ulva
Rhodymenia
2 , 4 , -D Enteromorpha
Ulva
Rhodymenia
105.5
111.7
108.0
99.0
102.0
94.0
101.0
98.7
96.0
a. The seaweeds were exposed to pesticide concentrations representing
their maximum solubility in seawater. Treatment times for
Enteromorpha, Rhodymenia, and Ulva were 3, 3 and 1 weeks, respectively.
b. Mean of two experiments.
The effects of mirex, methoxychlor, and 2,4-D on growth and photosynthesis
in phytoplankton have been reported by other workers. However, in most
studies, effects were examined at concentrations exceeding the maximum
solubility of the pesticides in water. Sikka and Rice (1974) observed
that methoxychlor at a concentration of 100 ppb significantly reduced
growth of three marine algae, Skeletonema, Tetraselmis, and Thalassiosira,
but had no effect on Dunaliella and Porphrydium. At 50 ppb, the pesticide
was toxic only to Skeletonema. Butler (1963) reported a slight decrease
in carbon fixation by estuarine phytoplankton following a 4-hr exposure
to 1 ppm of methoxychlor. de la Cruz and Naqvi (1973) examined the
effect of mirex on photosynthesis and respiration in fresh-water phyto-
plankton. Photosynthesis was reduced by 16, 10, 33, and 19% in a
17
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naturally occurring phytoplankton population after 5, 10, 15, and 20 days
exposure to 1 ppb of mirex. Treatment of a pure culture of Chlamydomonas
with 1 ppm of mirex for 7 days reduced photosynthesis by about 55%.
Moore (1972) reported that mirex was not toxic to marine phytoplankton
populations exposed for 24-48 hrs to concentrations ranging from 0.5 ppb
to 1 ppm. Butler (1963), on the other hand, reported a 42% decrease in
1*tC02~fixation by estuarine phytoplankton following a 4-hr exposure to
1 ppm of mirex. Hollister et al (1975) observed that 0.2 ppb of mirex
did not affect either population growth or oxygen evolution of six species
of marine unicellular algae.
The available information on toxicity of 2,4-D indicates that the pesti-
cide has little or no effect on phytoplankton even at very high concen-
trations. Elder et al (1970) reported that 2,4-D had no effect on the
growth of fresh-water and marine algae tested at a concentration
representing the maximum solubility of the herbicide in water (240 ppm).
In another study, 2,4-D up to a concentration of 400 ppm did not inhibit
growth of the algae Chlorella vulgarls, Chlorococcum sp., and Cylindro-
spermum lichenforme (Arvik et al, 1971). Wedding et al (1954) observed
_3
that 2,4-D at a concentration of 2 x 10 M (440 ppm) in the culture
medium at pH above 6 did not inhibit photosynthesis in Chlorella. Treat-
ment of estuarine phytoplankton with 1 ppm of 2,4-D for 4 hrs did not
reduce photosynthesis (Butler, 1963). However, Walsh (1972) reported that
treatment of four species of marine phytoplankton with 50-75 ppm of the
herbicide for 10 days reduced growth by about 50%.
On the basis of our findings and those of other workers, it appears that
mirex, methoxychlor, and 2,4-D do not adversely affect growth and photo-
synthesis in phytoplankton and seaweeds at concentrations several times
those found in natural waters.
Effect of Mirex, Methoxychlor, and 2,4-D on Tissue Composition
To learn if 2,4-D, mirex, or methoxychlor induce changes in algal composi-
tion, the tissues were analyzed for protein, carbohydrates, total lipids,
18
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pigments, and trace elements following incubation with saturating concen-
trations of the pesticides. As in the photosynthesis studies, both
Enteromorpha and Rhodymenia were exposed to the pesticides for three
weeks, whereas Ulva was treated for one week. Unless otherwise indicated,
the data are the average of two experiments.
Protein - The data in Table 2 show that saturating concentrations of 2,4-D,
mirex, or methoxychlor did not significantly influence the protein content
of Ulva, Enteromorpha, and Rhodymenia.
Table 2. PROTEIN CONTENT OF SEAWEEDS TREATED WITH
METHOXYCHLOR, MIREX, AND 2,4-D
Protein
Pesticide Organism % Control
Methoxychlor Enteromorpha 113.3
Ulva 93.0
Rhodymenia 113.5
Mirex Enteromorpha 87.0
Ulva 95.0
Rhodymenia 110.7
2,4-D Enteromorpha 110.7
Ulva 97.3
Rhodymenia 88.0
Carbohydrates - Treatment with 2,4-D, mirex, or methoxychlor failed to
alter the total carbohydrate content in the three algae (Table 3).
However, mirex and 2,4-D increased the amount of soluble carbohydrates
present in Enteromorpha by 34 and 44%, respectively. The changes in the
concentrations of soluble carbohydrates would seem to be indicative of
19
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either a mobilization of polysaccharides or a reduction in rate of
incorporation of soluble carbohydrates into polysaccharides.
Table 3. TOTAL AND SOLUBLE CARBOHYDRATE CONTENT OF SEAWEEDS
TREATED WITH METHOXYCHLOR, MIREX, AND 2,4-D
Pesticide
Organism
Total Soluble
Carbohydrate Carbohydrate
% Control
Methoxychlor
Mirex
2,4-D
Enteromorpha
Ulva
Rhodymenia
Enteromorpha
Ulva
Rhodymenia
Enteromorpha
Ulva
Rhodymenia
97.5
99.3
93.0
97.5
102.0
89.5
89.0
98.7
84.0
110.5
91.7
99.5
134.5
92.0
112.0
144.5
93.0
104.5
Pigments - The data in Table 4 show that saturating concentrations of
2,4-D, mirex, or methoxychlor did not change chlorophyll concentration
in Ulva, Enteromorpha, or Rhodymenia. The carotenoid content of the
algae was unaffected by mirex or methoxychlor. However, 2,4-D increased
the concentration of carotenoids in Enteromorpha and Rhodymenia but had
little effect on the pigment content in Ulva.
Lipids - The lipid content of the three algae was essentially unaffected
by saturating concentrations of 2,4-D, mirex, or methoxychlor (Table 5).
Sumida and Ueda (1973) reported that treatment of Chlorella elipspidea
with 5 ppm of 2,4-D produced no change in lipid content of the alga.
20
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Table 4. PIGMENT CONTENT OF SEAWEEDS TREATED WITH
METHOXYCHLOR, MIREX, AND 2,4-D
Pesticide
Methoxychlor
Mirex
2,4-D
Organism
Enteromorpha
Ulva
Rhodymenia
Enteromorpha
Ulva
Rhodymenia
Enteromorpha
Ulva
Rhodymenia
Chlorophylls
%
118.7
106.3
99.0
106.3
96.0
93.5
89.3
88.3
106.5
Carotenoids
Control
115.0
91.0
95.0
108.7
89.5
109.5
129.0
91.3
124.0
Table 5. TOTAL LIPID CONTENT OF SEAWEEDS TREATED WITH
METHOXYCHLOR, MIREX, AND 2,4-D
Pesticide
Organism
Lipids
Control
Methoxychlor
Mirex
2,4-D
Enteromorpha
Ulva
Enteromorpha
Ulva
Enteromorpha
Ulva
98.0
104.0
104.0
99.0
100.0
117.0
21
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Trace Metals Content - Table 6 shows the Mg, Cu, Mn, Fe, and Zn content
of Enteromorpha and Ulva treated with methoxychlor, mirex, and 2,4-D.
Methoxychlor at saturating levels did not affect the trace metal content
of Enteromorpha or Ulva. Treatment with mirex increased the concentrations
of Fe and Mn in Ulva and of Cu in Enteromorpha, but did not change those
of the other metals in the two algae. Incubation with 2,4-D had no effect
on the concentration of trace metals in Ulva. However, in Enteromorpha,
saturating concentrations of this herbicide decreased the Mg and Zn content
and slightly increased the Cu content. The pesticide showed no effect on
content of Fe and Mn. The findings suggest that the three pesticides have
no marked effect on the concentration of the above-mentioned trace metals
in Ulva and Enteromorpha.
Table 6. TRACE METAL CONTENT OF SEAWEEDS TREATED WITH
METHOXYCHLOR, MIREX, AND 2,4-D
Pesticide
Methoxychlor
Mirex
2,4-D
Organism
Enteromorpha
Ulva
Enteromorpha
Ulva
Enteromorpha
Mg
96.5
99.0
102.0
113.0
67.0
Cu
a
95.5
109.0
139.0
86.0
129.0
Mn
I Control
103.0
100.0
92.0
164.0
98.0
Fe
94.5
91.0
85.5
134.0
103.5
Zn
105.0
109.0
86.5
81.0
61.0
Effects of the pesticides on the trace-metal content of Rhodymenia was
not examined since it could not be grown in sufficient quantities. The
amount of boron and cobalt in Ulva and Enteromorpha could not be determined
since their concentrations in the algae were below the limit of detection.
22
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UPTAKE OF METHOXYCHLOR, MIREX, AND 2,4-D
Methoxychlor
The uptake (absorption and/or adsorption) of ll*C-methoxychlor by all three
species was rapid. More than 50% of the maximum methoxychlor uptake by
a given species occurred within three hrs of treatment. In the cases
of Ulva and Enteromorpha, uptake of pesticide reached its maximum 24 hrs
after treatment, whereas the maximum uptake was observed 48 hrs after
treatment of Rhodymenia (Table 7). In all three organisms, there was a
gradual decline in concentration of radioactivity once maximum accumula-
tion had been reached. The three algae varied in their ability to
accumulate methoxychlor from the medium, and they accumulated the
pesticide in the following order: Enteromorpha > Ulva > Rhodymenia.
Table 7. UPTAKE OF 14C-METHOXYCHLOR BY SEAWEEDS3
Organism Concentration of ^C-Residue in the Organism-ppm
(expressed as Methoxychlor equivalent ")
Hours After Treatment
Enteromorpha
Ulva
Rhodymenia
3
106
25.7
3.9
6
117
29.2
7.2
24
165
36.1
8.7
48
120
21.3
9.0
72
102
24.8
8.0
Concentration of llfC-methoxychlor in the medium at the time of
treatment was 25 ppb.
^Calculated from the specific activity of ll+C-methoxychlor.
Mirex
The three species readily removed 14C mirex from the medium. However,
uptake was relatively slow as compared to that of methoxychlor. The
algae continued to remove mirex up to 144 hrs after treatment, when the
experiment was terminated (Table 8), whereas the concentration of
23
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methoxychlor in the algae reached its maximum within 24-48 hrs (Table 7)
Enteromorpha was more effective in removing mirex from the medium than
Plva or Rhodymenia. which did not differ greatly in their ability to
accumulate the insecticide.
Table 8. UPTAKE OF C-MIREX BY SEAWEEDS
Organism
Mirex Concentration in the Organism (ppm)
Hours After Treatment
Enteromorpha
Ulva
Rhodymenia
3
1.0
1.0
0.9
6
1.0
1.5
1.6
24
1.6
2.6
3.0
48
4.3
3.1
4.3
72
8.8
4.3
4.9
144
15.7
4.8
6.3
Concentration of
was 15 ppb.
C-mirex in the medium at the time of treatment
Although mirex is more lipid-soluble than methoxychlor, its uptake by the
seaweeds was less than that of methoxychlor. It is possible that uptake
of mirex was limited by its molecular weight (546) which is higher than
that of methoxychlor (345).
2.4-D
Although maximum solubility of 2,4-D in the growth medium used was about
200 ppm, uptake of the pesticide was studied at a concentration of 25 ppb.
We chose this concentration because it approximates the concentration of
2,4-D in water in the estuarine environment. Since this is close to the
concentration at which uptake of mirex and methoxychlor was determined,
the results would also permit us to compare the relative uptake of the
three pesticides by the algae. Maximum uptake of 2,4-D by the algae
occurred within 24 hrs of treatment, after which the concentration of
radioactivity in the tissue did not change (Table 9). Uptake of 2,4-D
24
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was low, the total amount of 2,4-D absorbed varying from 0.01 to 0.03%
of the pesticide added to the culture medium. Rhodymenia and Ulva appeared
to remove somewhat greater amounts of 2,4-D from the medium than
Enteromorpha.
llf a
Table 9. UPTAKE OF C-2.4-D by SEAWEEDS
Organism
2,4-D Concentration in the Organism (ppb)
Hours After Treatment
Enteromorpha
Ulva
Rhodymenia
6
0.01
0.13
0.11
24
0.01
0.44
0.28
48
0.01
0.42
0.20
72
0.01
0.34
0.19
Concentration of C-2,4-D in the medium at the time of treatment
was 25 ppb.
Our results are similar to those reported by Valentine and Bingham (1974)
lit
on uptake of C-2,4-D by the fresh water algae Chlorella pyrenoidosa,
Scenedesmus quadricauda, Chlamydomonas reinhartii, and Euglena gracilis.
Only S. quadricauda removed a measurable amount of 2,4-D from the medium
but only at a pH below 6. A greater uptake at low pH was presumably caused
by a relatively high concentration of the undissociated 2,4-D molecule
which may enter the cell more readily than the ionized form. In the
marine environment, where the average pH is 8.3, 2,4-D would be expected
to be present in an ionized form (pK of about 3) and consequently its
uptake by the cells may not be high, possibly because of an interaction
between charged groups on the cell surface and the ionized carboxyl
group of 2,4-D.
A separate experiment was conducted to study uptake of 2,4-D by Ulva
as a function of pesticide concentration in the medium. Since uptake of
25
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2,4-D by the alga was extremely low, the blomass of Ulva in the medium was
increased to 2.75 g/100 ml to give a greater amount of 2,4-D in the tissue.
Uptake increased linearly with increase in concentration of the pesticide
from 2 to 200 ppm. When Ulva was incubated in a medium containing 2,
10, and 200 ppm of 2,4-D, the concentration of pesticide in the tissue
was 12.4, 58.9, and 1150 ppb, respectively, 96 hrs after treatment.
BIOACCUMULATION OF METHOXYCHLOR, MIREX, AND 2,4-D
All three algae accumulated both mirex and methoxychlor from the medium
(Table 10). In the case of Enteromorpha treated with methoxychlor, a
llf 1U
small percentage of the total C in the tissue was present as C-
Table 10. BIOACCUMULATION OF METHOXYCHLOR, MIREX,
AND 2,4-D BY SEAWEEDS
a
Pesticide
Methoxychlor
Mirex
2,4-D
Organism Bioaccumulation Factor
Enteromorpha
Ulva
Rhodymenia
Enteromorpha
Ulva
Rhodymenia
Enteromorpha
Ulva
Rhodymenia
5375
1174
289
1112
332
419
.001
.001
.003
TT
a C-Methoxychlor, mirex and 2,4-D were added at initial concentration
of 25, 10, and 25 ppb in the medium, respectively.
"Concentration of pesticide in the tissue/concentration of pesticide in
the medium; calculated at the time of maximum pesticide uptake by the
tissue.
26
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metabolites. Enteromorpha accumulated considerably more mirex and
methoxychlor than Ulva or Rhodymenia. Although the algae accumulated
both methoxychlor and mirex from the medium, bioaccumulation of methoxy-
chlor was greater than that of mirex except in Rhodymenia. The con-
lit
centration of C-methoxychlor and its metabolites in the algae three
days after treatment ranged from about 300 to 5400 times the concentra-
14
tion in the medium. In the case of algae treated with C-mirex, the
bioaccumulation factor ranged from about 350 to 1100.
Like other chlorinated hydrocarbon pesticides, mirex and methoxychlor have
been reported to undergo bioaccumulation in various organisms. Sikka
and Rice (1974) observed that five species of marine phytoplankton
accumulated methoxychlor from a medium containing 20 ppb of the pesti-
cide. Concentrations of methoxychlor in the algal species were 710 to
about 8200 times greater than that in the medium. In a fresh-water
model ecosystem, methoxychlor was found in fish as a concentration
1500 times that of the water (Kapoor et al, 1970). Bioaccumulation of
mirex by various species of aquatic organisms has been reported (Butler,
1969; Cooley et al, 1972; Borthwick et al, 1973; Wolfe and Norment, 1973;
Hollister et al, 1975). In a fresh-water model ecosystem, mirex was
concentrated 214- and 1165-fold in fish and snails, respectively
(Metcalf et al, 1973).
Although both mirex and methoxychlor appear to have no adverse effects
on the seaweeds at the concentrations tested, their bioaccumulation is
of significance. Pesticide accumulated by algae may be transferred to
the higher trophic levels and thereby may have an impact on the estuarine
ecosystem. In contrast to mirex and methoxychlor, 2,4-D did not
accumulate in the seaweeds. Wojtalik et al (1971) reported no harmful
effects or accumulation in zooplankton, phytoplankton, or microinverte-
brates in water treated at 20 or 50 Ib of 2,4-D/acre. Hence, unlike the
chlorinated hydrocarbon insecticides, there is little danger of bio-
magnification of 2,4-D.
27
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METABOLISM OF METHOXYCHLOR, MIREX, AND 2,4-D
These studies were done using Ulva and Enteromorpha. Metabolism of the
pesticides by Rhodymenia was not investigated because the algal tissue
was not available in sufficient quantities. To learn if the pesticides
were metabolized, tissues and the culture media were analyzed for the
parent compounds and their possible metabolites after the organisms had
absorbed the maximum amounts of pesticides as determined from the results
of the uptake studies.
Methoxychlor
Enteromorpha - To obtain relatively large amounts of the methoxychlor
metabolites produced by Enteromorpha, 50 culture dishes, each containing
approximately 500 mg of germling tissue, were incubated with 500 ppb of
^C-methoxychlor. After 7 days, both the tissue and medium were
extracted separately as described in the Methods section, and the
extracts were concentrated. To remove pigments and other extraneous
material coextracted from the tissue and the medium, the concentrated
extract was applied in a narrow band on several 1-mm preparative thin-
layer silica gel plates, which were developed in chloroform. This
procedure separated essentially all the ^C-compounds from the pigments,
which were found between 5 and 6 cm from the origin. The area of silica
gel containing the radioactive material was scraped off the plate and
extracted twice with chloroform; the extract was then filtered to remove
the silica gel. The extract was concentrated by flash evaporation, and
about 96% of the 14C material was recovered. Aliquots of the extracts
were spotted on thin-layer silica gel plates, which were then developed
in the following solvent systems.
I. Petroleum ether:ethyl ether (9:1)
II. Hexane:acetone (8:2)
III. Petroleum ether: chloroform:methanol (3:2:1)
IV. Benzene:acetic acid (9:1)
28
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The chromatograms were scanned for radioactivity on a Nuclear Chicago
14
Actigraph. The C-containing areas were scraped off the plate into
liquid scintillation vials and counted for radioactivity.
14
C-Analysis of tissue - Two successive extractions with acetone and 80%
acetone removed more than 95% of the radioactivity from the tissue.
Thin-layer chromatography of the purified extract in solvent systems I and
II revealed the presence of methoxychlor and a minor metabolite
which co-chromatographed with authentic 2,2-bis (£-methoxyphenyl)-l,l-
dichloroethylene (MPDE). Solvent systems III and IV did not effectively
separate the metabolite from methoxychlor. The relative amounts of
14
C-methoxychlor and the metabolite in the tissue 7 days after treatment
are shown in Table 11 along with their respective Rf values.
14
Table 11. DISTRIBUTION OF C-METHOXYCHLOR AND
1JtC-MPDEa IN ENTEROMORPHA
„ - 14 Rf Value in Solvent
% of C xn Acetone System
Compound
Methoxychlor
MPDE
Extract
96.8
3.2
I
0.51
0.60
II
0.62
0.68
a2,2-bis (p_-methoxyphenyl)-l,l, 1-dichloroethylene
C-Analysis of culture medium - Extraction of the culture medium with
ether following acidification removed more than 90% of the total radio-
activity in the medium. Thin-layer chromatography of the ether extract
in the solvent systems revealed that radioactivity in the extract was
14
present in the form of C-methoxychlor and six minor metabolites. Two
of the latter were identified as 2,2-bis (p_-methoxyphenyl)-l, 1-dichloro-
ethylene and 2,2-bis (£-hydroxyphenyl)-l,l,l-trichloroethane by
29
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co-chromatography with authentic standards. System IV gave the best
resolution of the various C-compounds, but it did not effectively
separate methoxychlor and MPDE. Systems I and II separated the two
m
compounds. The relative amounts of C-methoxychlor and various meta
bolites in the ether extract of the medium are given in Table 12.
Because only extremely small amounts of the metabolites were present,
they could not be identified by spectral methods.
Table 12. DISTRIBUTION OF C-METHOXYCHLOR AND
ITS METABOLITES IN THE MEDIUM
Compound
Methoxychlor
MPDEa
HPTEb
Unknown 1
it 2
3
4
% of ll*C in
Ether Extract
84.0
4.6
1.0
0.7
2.1
4.3
3.3
Rf Value in Solvent Systems
I II IV
0.51 0.62 0.90
0.60 0.68 0.90
0.29
0.15
0.40
0.52
0.64
a2,2-bis (p_-methoxyphenyl)-l,l-dichloroethylene
b 2,2-bis (p_-hydroxypheny1)-1,1,1-trichloroethane
Enteromorpha metabolized methoxychlor although only to a limited extent.
The presence of 2,2-bis (p_-methoxyphenyl)-l,l-dichloroethylene and
2,2-bis (pj-hydroxyphenyl)-1,1,1-trichloroethane suggests that methoxy-
chlor undergoes dehydrochlorination and 0-demethylation in cultures of
Enteromorpha, Another chlorinated hydrocarbon pesticide, DDT, is also
dehydrochlorinated by marine algae to produce DDE (Rice and Sikka, 1973),
0-demethylation of methoxychlor has also been reported in other
30
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biological systems. Kapoor et al (1970) observed that methoxychlor was
converted by 0-d erne thy lat ion into 2,2-bis (p_-hydroxyphenyl)-!,!, 1-trichloro-
ethane in mice. However, they found no evidence of dehydrochlorination
of the pesticide.
Since the Enteromorpha plants were not axenic, metabolism of the pesti-
cide may have resulted from the action of the algae or microorganisms
present in the medium, or a combination of both. To ascertain the role
of the microorganisms and non-biological factors in methoxychlor
metabolism, the transformation of the pesticide was studied in the
following systems: (1) ^C-methoxychlor was incubated in the culture
medium after 3-week old germlings had been removed (hereafter referred
to as old medium); the purpose was to determine if microorganisms in
the medium- were capable of metabolizing the pesticide; (2) the pesticide
was incubated in the old medium without algae, but supplemented with
glucose and nutrient broth to enhance the growth of microorganisms;
(3) methoxychlor was incubated in a sterile fresh medium to account for
any non-biological conversion. In each treatment the pesticide was
incubated for 7 days, after which the algae, microorganisms, and the
medium were extracted separately and analyzed, for 1^C-methoxychlor and
its possible metabolites by chromatography on thin-layer silica gel
plates and radiochromatographic scanning.
No transformation of ^C-methoxychlor was observed in the old medium
with or without an exogenous source of carbon. In these treatments, all
of the 1!tC was present in the form of a single compound which co-chroma -
tographed with authentic methoxychlor. These findings suggest that
microorganisms in the culture medium did not metabolize methoxhchlor.
No transformation of pesticide was observed in fresh, sterilized growth
medium. These findings demonstrate that the metabolism of methoxychlor•
in the cultures of Enteromorpha resulted primarily from the action of
algae, although the role of microorganisms associated with the plant
surface cannot be ruled out.
31
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Ulva - The tissue and the incubation medium were analyzed after 7 days
of incubation with 14C-methoxychlor. Thin-layer chromatographic analysis
of extracts of the tissue and medium showed that all radioactivity in
these extracts was present as unchanged methoxychlor. Our findings show
that Ulva and Enteromorpha differ in their ability to metabolize
methoxychlor.
Mirex
Thin-layer chromatographic analysis of the extracts of Ulva, Rhodymenia,
and Enteromorpha incubated with ^(Mnirex for 7 days revealed the presence
of one compound which co-chromatographed with authentic mirex. When the
hexane extract of the culture medium was chromatographed, only one spot
with an Rf value of 0.93 in hexane:acetone (8:2) and 0.79 in heptane
could be detected. This compound co-chromatographed with authentic mirex
in both solvent systems. The results show that neither organism was
able to metabolize mirex. Our findings support the observations of
other workers that mirex is resistant to transformation by plants and
microorganisms (Mehendle et al, 1972; Jones and Hodges, 1974).
2,4-D
Extremely low uptake of 2,4-D by the seaweeds made it necessary to use
a relatively large amount of algal tissue in the metabolism studies so
that the pesticide or its metabolites might be extracted in quantities
sufficient for their characterization by conventional methods. To study
the metabolism of 2,4-D by Ulva and Enteromorpha, the algae were incubated
with 2 ppm of l^02,4-0 for 4 days. Both the tissues and medium were
then analyzed separately for 2,4-D and its metabolites.
All of the llfC in the methanol extract of Ulva and Enteromorpha incubated
with 2,4-D for 4 days was present in the form of a single compound which
co-chromatographed with 2,4-D in the following solvent systems:
(1) butanol:benzene:water (1:9:1), Rf 0.04; and (2) chloroform, Rf 0.11.
32
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Thin-layer chromatography of the ether extract of the medium did not
reveal the presence of any 14C-compound other than 2,4-D. These findings
indicate that neither Ulva nor Enteromorpha is able to transform 2,4-D.
It may be pointed out that because of extremely low uptake of 2,4-D by
the algae, it would be difficult to detect herbicide metabolites which
may have been formed in small amounts by these organisms.
33
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SECTION IV
CONCLUSIONS
1. Methoxychlor, mirex, and 2,4-D at concentrations corresponding to
their maximum solubility in water do not adversely affect photosynthesis
and composition of Ulva sp., Enteromorpha sp., and Rhodymenia sp.
2. The seaweeds accumulate mirex and methoxychlor but not 2,4-D,
which is taken up by the organisms in extremely small amounts.
3. Although the seaweeds accumulate mirex and methoxychlor, they vary
in their ability to concentrate the pesticides.
4. Ulva and Enteromorpha do not metabolize mirex and 2,4-D. Enteromorpha-,
however, can transform methoxychlor to a minor extent.
5. We conclude that methoxychlor, mirex, and 2,4-D, at concentrations
approaching their maximum solubility in seawtater, do not adversely
affect the seaweeds. However, the ability of the algae to accumulate
mirex and methoxychlor suggests that these organisms may act as physical
agents in transporting the pesticides in an estuarine ecosystem.
34
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SECTION V
RECOMMENDATIONS
1. In the present studies, mirex, methoxychlor, and 2,4-D were used
in highly purified forms. It is recommended that for purposes of com-
parison, effects of the pesticides in the form of commercial formula-
tions be examined. It is possible that the pesticide formulation may
contain by-products and other impurities which may alter toxicity of
the pesticide to algae.
2. It is likely that in an estuarine environment, algae will be
exposed to more than one pollutant at a time and that problems arising
from the interaction of a pesticide with other toxicants may result.
Therefore, it is recommended that the effects and fate of mirex,
methoxychlor, and 2,4-D in algae be studied in the presence of other
pesticides and environmental contaminants.
3. The pesticides may be converted into other products as a result of
biological and/or non-biological transformation in an estuarine
environment. To fully assess the effects of mirex, methoxychlor, and
2,4-D on estuarine algae, we suggest that effects of known metabolites
of these pesticides be examined.
4. Algae growing in estuaries are subjected to changes in environ-
mental factors, such as temperature, salinity, and nutrients. Since an
algal species may become more susceptible to an outside stress under
environmental conditions which are not optimal for that species, it is
recommended that the effects and metabolism of mirex, methoxychlor,
and 2,4-D be examined in algae growing under varying environmental
»
conditions.
35
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SECTION VI
REFERENCES
Alley, E. G. The Use of Mirex in Control of the Imported Fire Ant.
J. Environ. Quality, 12:52-54, 1973.
Arvik, J. H., D. L. Willson and L. C. Darlington. Response of Soil
Algae to Picloram-2,4-D Mixtures. Weed Sci. 19_: 276-278, 1971.
Ashwell, G. Colorimetric Analysis of Sugars. In; Methods of
Enzymology, Colowick, S. P. and N. 0. Kaplan (ed.). New York,
Academic Press, 1957. p. 73-105.
Borthwick, P. W., T. W. Duke, A. J. Wilson, J. I. Lowe, J. M. Patrick,
and J. C. Oberheu. Accumulation and Movement of Mirex in Selected
Estuaries of South Carolina. Pestic. Monit. J. 2.'.6-lbt 1973.
Burdick, G. E., H. J. Dean, E. J. Harris, J. Skea, C. Frissa and
C. Sweeney. Methoxychlor as a Black Fly Larvicide, Persistence
of its Residues in Fish and its Effect on Stream arthropods.
J. N. Y. Fish Game, 15:121-142, 1968.
Butler, P. A. Commercial Fisheries Investigations. In; Pesticide-
Wildlife Studies, Fish and Wildlife Service, Department of the
Interior Cir. 167, Washington, D.C., 1963. p. 11-25.
Butler, P. A. Monitoring Pesticide Pollution. Bioscience, 19:889-891,
1969.
Cooley, N. R., J. M. Keltner and J. Forester. Mirex and Archlor 1254 -
Effect on and Accumulation by Tetrahymena pyroformis. J. Protozool.
19:636-638, 1972.
de la Cruz, A. A. and S. M. Naqvi. Mirex Incorporation in the Environment:
Uptake in Aquatic Organisms and Effects on the Rates of Photosynthesis
and Respiration. Arch. Environ. Contam. Toxicol. !_:255-265, 1973.
Davies, B. H. Analysis of Carotenoid Pigments. In: Chemistry and
Biochemistry of Plant Pigments, Goodwin, T. W. (ed.). New York,
Academic Press, 1965. p. 489-532.
Elder, J. H., C. A. Lembi and D. J. Morre. Toxicity of 2,4-D and Picloram
to Fresh Water Algae. National Tech. Inform. Service, Report
No. PB 199114, 1970, 13 p.
36
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Erickson, L.C., R.T. Wedding and B.L. Brannaman. Influence of pH on
2,4-dichlorophenoxyacetic Acid and Acetic Acid Activity in Chlorella.
Plant Physiol. 30:69-74, 1955.
F^yn, B. Lebenzyklus, Cytologie und Sexualitat der Chlorophycee
Cladophora suhriana. Kutz. Arch. Protistenk. jJ3:l-56, 1934.
Guillard, R.R.L. and J.H. Ryther. Studies of Marine Planktonic Diatoms.
Can. J. Microbiol. j8:229-239, 1962.
Hamilton, R.H., J. Hurter, J.K. Hall and C.D. Ercegovich. Metabolism
of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic
Acid by Bean Plants. J. Agr. Food Chem. 19_: 480-483, 1971.
Hollister, T.A., G.E. Walsh and J. Forester. Mirex and Marine Uni-
cellular Algae: Accumulation, Population Growth and Oxygen
Evolution. Bull. Environ. Contain. Toxicol. 14:753-759, 1975.
Jones, A.S. and C.S. Hodges. Persistence of Mirex and its Effects on
.Soil Microorganisms. J. Agr. Food Chem. 22:435-439, 1974.
Kirk, J.T.O. Studies of the Dependence of Chlorophyll Synthesis on
Protein Synthesis in Euglena gracilis, Together with a Nomogram for
Determination of Chlorophyll Concentration. Planta (Berlin)
2^:200-207, 1968.
Kirk, J.T.O. and R.L. Allen. Dependence of Chloroplast Pigment
Synthesis on Protein Synthesis: Effect of Actidione. Biochem. Biophys.
Res. Comm. 21:523-530, 1965.
/•
Kapoor, I.P., R.L. Metcalf, R.F. Nystrom and G.K. Sangha. Comparative
Metabolism of Methoxychlor, Methiochlor, and DDT in Mouse, Insects
and in a Model Ecosystem. J. Agr. Food Chem. 1.8:1145-1152, 1970.
Lawrence, J.M. Aquatic Herbicide Data. USDA Agric. Handbook No. 231. 1962.
McLachlan, J. Some Considerations of the Growth of Marine Algae in
Artificial Media. Canad. J. Microbiol. 10:769-782, 1964.
McLachlan, J. Growth Media-Marine. In; Phycological Methods (Janet R.
Stein, ed.) Cambridge University Press, 1973, p. 25-51.
Mehendle, H M., L. Fishbein, M. Fields and H.B. Matthews. Fate of
Mirex- C in the Rat and Plants. Bull. Environ. Contain. Toxicol.
1:200-207, 1972.
Metcalf, R.L., I.P. Kapoor, P.Y. Lu, C.K. Schuth and P. Sherman.
Model Ecosystem Studies of the Environmental Fate of Six Organo-
chlorine Pesticides. Environ. Health Perspect. 4^35-44, 1973.
37
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Moore, S. A. Some Effects of Selected Organochlorine Compounds on Radio-
carbon Uptake by Marine Phytoplankton Populations in St. George
Sound, Florida. Abstracts, 35th Annual Meeting Amer. Soc. Limnol.
and Oceanogr., Tallahassee, Fla., 1972.
Ott, F.D.. Personal Communication, 1973.
Pimental, D. Ecological Effects of Pesticides on Non-target Species.
U.S. Govt. Printing Office, Washington, B.C., 1971.
Provasoli, L. Growing Marine Seaweeds. In; Proceedings 4th International
Seaweed Symposium. 1963. p. 9-17.
Provasoli, L., J. A. Mclaughlin and M. R. Droop. The Development of
Artificial Media for Marine Algae. Arch. Microbiol. 25^392-428, 1957.
Radin, N. S. Preparation of Lipid Extracts. In; Methods in Enzymology.
J. M. Lowenstein (ed.) 14:245-254, 1969.
Rice, C. P. and H. C. Sikka. Uptake and Metabolism of DDT by Six Species
of Marine Algae. J. Agr. Food Chem. 21:148-152, 1973.
Sikka, H. C. and C. P. Rice. Effects, Uptake and Metabolism of Methoxy-
chlor in Marine Algae. In; Interaction of Selected Pesticides With
Marine Microorganisms. Research Report to the Office of Naval
Research. 1974. p. 33-47.
Strain, H. H., B. T. Cope and W. A. Suec. Analytical Procedures for the
Isolation, Identification, Estimation and Investigation of the
Chlorophylls. In; Methods in Enzymology, San Pietro, A. (ed.).
14:452-476, 1971.
Strickland, J. D. H. and T. R. Parsons. A Manual of Sea Water Analysis
(2nd ed.). Fisheries Research Board of Canada Bulletin, No. 125.
Ottawa, 1965. 203 p.
Sumida, S. and M. Ueda. Studies of Pesticide Effects on Chlorella
Metabolism. I. Effect of Herbicides on Complex Lipid Biosynthesis.
Plant. Cell Physiol. 14:781-785, 1973.
Valentine, J. P. and S. W. Bingham, Influence of Several Algae on
2,4-D Residues in Water. Weed Sci. 22_:358-363, 1974.
Walsh, G. Effects of Herbicides on Photosynthesis and Growth of Marine
Unicellular Algae. Hyacinth Control J. 10:45-48, 1972.
38
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Wedding, R. T., L. C. Erickson and B. L. Brannaman. Effect of
2,4-Dichlorophenoxyacetic Acid on Photosynthesis and Respiration.
Plant Physiol. 2,9:64-69, 1954.
Wojtalik, T. A., T. F. Hall and L. L. Hill. Monitoring Ecological
Conditions Associated with Wide-scale Applications of DMA-2,4-D
to Aquatic Environments. Pestic. Monit. J. 4^:184-203, 1971.
Wolfe, J. L. and B. R. Norment. 1973. Accumulation of Mirex Residues
in Selected Organisms After an Aerial Treatment, Mississippi
1971-1972. Pestic. Monit. J. 7:112-116, 1973.
39
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TECHNICAL REPORT DATA
(Please read laUructiuns on the reverse before completing)
1. REPORT NO.
EPA-600/ 3-76-048
2.
4. TITLE AND SUBTITLE
Effects, Uptake, and Metabolism of Methoxychlor ,
Mirex, and 2,4-D in Seaweeds
7. AUTHOR(S)
Harish C. Sikka, Gary L. Butler and Clifford P. Rice
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Life Sciences Division
Syracuse University Research Corporation
Syracuse, New York 13210
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
3. RECIPIENT'S ACCESSION-NO.
5. REPORT DATE
June 1976 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO
10. PROGRAM ELEMENT NO.
1EA077
11. CONTRACT/GRANT NO.
Contract No. 68-03-0271
13. TYPE OF REPORT AND PERIOD COVERED
Final (5/73-10/75) ?
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report presents tt
metabolism of mirex, methoxj
and Rhodymenia sp. None of
maximum solubility in seawat
carbohydrate, lip id, chlorop
All three algae removed subs
but uptake of 2,4-D was extr
considerably greater than th
mirex and methoxychlor than
metabolize either mirex or 2
extent. After 7 days of inc
of the label in the tissue a
amount of radioactive metabo
detected in both the tissue
(p-hydroxyphenyl) -1,1, 1-tric
metabolites. Unlike Enterom
17.
•' DESCRIPTORS
le results of a study concerning effects, i
rchlor, and 2,4-D in the seaweeds Ulva sp.
the pesticides, at concentrations corresp<
er, had any significant effect on photosyr
hyll, carotenoid or trace metal content oi
tantial amounts of mirex and methoxychlor
emely low. The rate of uptake of methoxyc
at of mirex. Enteromorpha accumulated con
Ulva or Rhodymenia. Both Ulva and Enteron
,4-D. Enteromorpha metabolis
ubation with carbon-labelled
nd medium was present in uncl
lite, 2,2-bis (p_-methoxyphenj
and medium. In addition, mec
hloroethane and four unident:
orpha, Ulva did not metaboliz
iptake, and
, Enteromorpha sp.
mding to their
ithesis, protein,
: the algae.
from the medium,
.hlor was
tsiderably more
rorpha failed to
:ed methoxychlor to a limited
methoxychlor, a major portion
langed methoxychlor. A small
fl)-l,l-dichloroethylene, was
lium contained 2,2-bis
.fied minor radioactive
se methoxychlor. j
i
KEY WORDS AND DOCUMENT ANALYSIS
b.lDENTIFIERS/OPEN ENDED TERMS
Algae, insecticides, herbicides
18. DISTRIBUTION STATEMENT
Release unlimited
19. SECURITY CLASS (Tttis Report}
None
20. SECURITY CLASS (This page)
None
c. COSATI l-icld/(iroup
6F
21. NO. OF PAf.tS
48
22. PRICE
EPA Form 2220-1 (9-73)
40
U.S. GOVHNMENT PRINTING OFFICE: 1976-657-695/5M5 Region No. 5-11
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