United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
Research and Development
EPA-600/S3-83-065 Nov. 1983
Project Summary
The Potential for Biological
Controls of Cladophora
glomerata
Ruth Patrick, Charles F. Rhynme, R.William Richardson, III, Richard A. Larson,
Thomas L. Bott, and Kurt Rogenmuser
The purpose of this research program
was to determine whether or not natu-
ral biological controls of Cladophora
glomerata could be developed. Two
avenues of research were pursued. One
was to study macroscopic organisms
that were known to occur in the Great
Lakes area to see if any of them would
prefer C. glomerata as a food source
and under what conditions the
desirability of C. glomerata could be
increased. The second approach was to
study microorganisms, particularly
fungi, that commonly occurred in
aquatic ecosystems to determine if they
might function as contols for C.
glomerata.
The results of the first avenue of
research show that C. glomerata was a
poor food for Physa heterostropha,
Orconectes propinquus, Rana pipiens,
Ictalurus punctatus, and Pimephales
promelas. Not only were these species
averse to eating the food, but when they
did, it appeared to have a deleterious
effect. In the studies with snails, egg
production was greatly curtailed on the
C. glomerata diet.
The unialgal C. glomerata was less
preferred than the C. glomerata plus
epiphytes. The main effect on
organisms other than Physa
heterostropha was loss in weight. The
crayfish did not molt even when the
eyestalks were removed, and the frog
tadpoles did not show any signs of
metamorphosing. All organisms
steadily lost weight during the
experiments.
An examination of the chemical
constituents of cells of C. glomerata
and of diatoms showed that they
differed considerably from diatom-
dominated periphyton, which was the
preferred diet in all experiments. These
differences were mainly that the
diatoms had larger amounts of amino
acids, such as serine, aspartic acid, and
glutamic acid. Also the free and
combined fatty acids of diatoms were
different from those of Cladophora. The
diatoms were dominated by C16 and C20
combined fatty acids, whereas the
unsaturated C18 fatty acid, particularly
linolenic acid, was common in C.
glomerata. Of the free fatty acids, the
diatom-dominated periphyton
contained mainly C14 and C16 isomers,
whereas C. glomerata contained mostly
C18. Of the short-chain fatty acids Cjj
acids, particularly lauric acid, was
present in C. glomerata and absent
from the periphyton. It is known that
lauric acid is toxic to several organisms.
The second avenue was to study the
effect of fungi as parasites on C.
glomerata. One fungus, Acremonium
kiliense (Fungi Imperfect!) was found to
have an antagonistic effect on C.
glomerata. The effect varied in various
experiments, those carried out in the
summer months showing the greatest
antagonistic effect. It may be that the
lesser effect in the fall was due to
temperature and other environmental
conditions or to the fact that A. kiliense
had reduced virulence. A toxin was
present in the supernatant derived from
cultures of C. glomerata that had been
damaged by this pathogen. It is water
soluble and can withstand heat up to
80°C.
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This Project Summary was developed
by EPA's Environmental Research Lab-
oratory. Duluth. MN. to announce key
findings of the research project that is
fully documented in a separate report of
the same title (see Project Report order-
ing information at back).
Introduction
In this research, two approaches were
pursued in trying to identify a biological
control of Cladophora glomerata. One of
these was to study macroorganisms
common to the Great Lakes region that
select algae as a considerable portion, if
not the main portion, of their diets. The
other avenue of research was to find a
microorganism which, by parasitism,
would destroy C. glomerata. It was
realized that this organism must be
specific for C. glomerata, and, if present
in fairly large amounts, would not destroy
other algae that were desirable food
sources.
The organisms selected for me
macroorganism studies were the pond
snail, Physa heterostropha; the crayfish,
Orconectes propinquus; the tadpole Rana
pipiens; the channel catfish, Ictalurus
punctatus; and the fathead minnow,
Pimephales promelas. All of these
organisms feed on algae. Some of these,
such as Tilapia aurea, are known to be
prodigious algal feeders. In these
experiments most of the work was done
on Physa heterostropha, Orconectes
propinquus, and Pimephales promelas.
Three diets were tested on eacn
species: Unialgal Cladophora,
Cladophora plus epiphytes, and diatom-
dominated periphyton. All experiments
were run in the natural soft waters of
White Clay Creek, Pennsylvania. Condi-
tions under which tests were run are
detailed in the full report. For all tests
oxygen was maintained above 7 ppm, pH
between 7 and 8.2 and ammonia less
than 40 /ug/l. Growth and respiration
were measured for all test species, and
fecundity was determined for snails.
All studies showed that C. glomerata
was not a satisfactory diet. Diatom-
dominated periphyton was a more
satisfactoy diet, as the organisms had a
better rate of growth on that diet than on
Cladophora plus epiphytes. The
Cladophora plus epiphytes tended to be a
better food source than unialgal
Cladophora. In the experiments using P.
heterostropha, some snails were starved.
Their decline in weight and their
respiration rates tended to be greater
when starved than when fed unialgal C.
glomerata.
The results of the experiments with P.
heterostropha showed that the relative
value of the three diets presented were
diatom-dominated periphyton > Clado-
phora plus epiphytes > unialgal Clado-
phora. The respiration rates of snails fed
Cladophora or Cladophora plus epiphytes
were less than those on the periphyton
diet. There was not much difference
between diets of Cladopohora plus epi-
phytes and unialgal Cladophora.
The reproductive success was much
greater on the diatom-dominated
periphyton diet and was very poor on the
unialgal Cladophora diet. Egg-laying
greatly decreased in a relatively few days
(11-15 days) after the experiments
started. However, the eggs produced
from all three diets had similar caloric
content. These experiments were
repeated at various seasons of the year
and showed similar results.
The experiment with Orconectes
propinquus showed that none of the diets
was very satisfactory, but that the
diatom-dominated periphyton was much
better than the other two diets. The
crayfish molted for only a short time after
the experiment started. This may be due
to the normal life cycle, as crayfish tend to
hibernate in the cooler months. However,
the temperature at which the
experiments were run was similar to
summer temperatures. Molting was forced
by removing the eyestalks and associated X
organ-sinus gland. Following this opera-
tion, the females on the periphyton diet
all molted. The respiration rates on all
three diets declined during the
experiments and the respiration rates of
the females tended to be greater than
those of the males.
The experiments with the tadpoles of
Rana pipiens resulted in increases in
size, and there were signs of
metamorphosis on the diatom-dominated
periphyton diet and on the Cladophora
plus epiphyte diet. Those fed on unialgal
Cladophora grew very little and showed
no signs of metamorphosis. The
respiration rates for tadpoles were
similar on the periphyton and Cladophora
plus epiphyte diets and were much lower
for tadpoles on the Cladophora diet.
These tadpoles were very small and
weighed very little.
The experiments with the channel
catfish (Ictalurus punctatus) showed a
similar pattern . The diatom-dominated
diet was more satisfactory than the
unialgal Cladophora diet. However, none
of the diets was as satisfactory as the
prepared fish food diets.
The fathead minnow (Pimephales
promelas) ingested and assimilate
much more of the diatom-dominate
periphyton than the Cladophora pk
epiphytes or the unialgal Cladophora. Tt1
fish fed the last two diets decreased i
weight, with those on unialg<
Cladophora showing the greates
decrease. The respiration rates wei
variable for fish on the periphyton diet.
was highest for those fed on Cladophoi
plus epiphytes. On both the Cladophoi
diets, the cal/mg/hr consumed wei
higher than on the periphyton diet.
These results led to an examination i
the relative food value of Cladophoi
glomerata and diatom-dominated per
phyton. Calories/mg were similar. A
analysis of the chemical composition i
diatoms and Cladophora showed sorr
interesting differences. The amino acic
present in larger amounts in the diatorr
were serine, aspartic acid, glutamic aci
and a small amount of cystine, which ws
absent from C. glomerata.
The combined fatty acids in diatorr
were dominated by C16 and C2o- Th
amount of unsaturated Ci8 fatty acid:
particularly linolenic acid, was muc
smaller in diatoms. The composition of (
glomerata was more like that of oth(
green plants, having C18 unsaturate
fatty acids, particularly linolenic acid, ar
low concentrations of C16 and C,2 acid:
Periphyton contained more free fat
acids than C. glomerata, with the major!
being Cu and C16 isomers, whereas thos
present in Cladophora were mostly Ci
Of the short chain free fatty acids C
(lauric acid) was present in Cladophoi
and absent from periphyton, and C
(capric acid) was in periphyton but not i
Cladophora. Lauric acid has been show
from many reports to be toxi
Experiments with yeasts (Saccharomyct
cervisiae) showed that the extract
Cladophora containing this acid was tox
to yeast. This suggests that the preseru
of this acid may be in part the reason wl
C. glomerata was an unsatisfactory foo
C. glomerata contained 4.5% solub
carbohydrates, whereas the periphytc
contained 21.6%. There was also mo
phosphorus in the periphyton.
The second approach was to determir
whether fungi commonly occurring intf
aquatic environment would have £
antagonistic effect on C. glomerata. Or
isolate, Acremonium kiliense (Fun
imperfect!) was found to have this effec
This fungus was isolated from the stoi
culture of Cladophora where it wj
present in small amounts. This fungus
flask cultures deleteriously affected tf
cell condition of C. glomerata and caus<
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a reduction in chlorophyll a when the
stock cultures of Cladophora were used.
Ctadophora isolated from Lititz Creek was
more susceptible to the fungus than
Cladophora from stock cultures. In the
Lititz Creek community experiments, the
correlation of loss of chlorophyll a with
da mage to the cells was not as good as that
from stock cultures. This may be due to
the fact that small amounts of algae were
present that were not damaged by A.
kiliense and thus would obscure the loss
of chlorophyll a. Therefore, it was decided
that direct microscopic examination of
the cells was the most reliable way to
determine the damage of this fungus.
Supernatant culture medium from the
flasks containing damaged and
undamaged Cladophora cells also had
deleterious effects on the algae. This
antagonistic substance from A. kiliense is
water soluble and does not seem to be
damaged by heating to 80°C. Cultures of
A. kiliense were raised on Czapek-Dox
broth, and Cladophora was inoculated
with conidia, but these did not produce
damage.
Microcosm experiments were then set
up in which the C. 0/o/nerafa-dominated
communities were exposed to varying
concentrations of A. kiliense.
In exposures carried out in the summer
months, there was a striking effect of this
fungus on the Cladophora, although
diatoms and Ulothrix living in the
microcosms were unaffected. In fact, the
Ulothrix divided rapidly and occupied the
habitats formerly occupied by C.
glomerata. Scenedesmus and Spirogyra
were also present, as were protozoans
and rotifers. They did not seem to be
affected by this fungus. Static
microcosms produced similar results.
However, experiments repeated in the
fall showed far less effect.
Conclusions
Experiments in which a number of
different consumer organisms were fed
C, glomerata showed that the alga was
either not eaten or caused deleterious
physiological changes.
C. glomerata is lower in overall nutritive
value (less protein, lipid and phosphorus
per unit dry weight) than many other
readily eaten algae. It contains low
concentrations of steroids and free
polyunsaturated fatty acids. Although
low in sulfur-containing amino acids,
Cladophora is not unique in this respect,
and it appears to contain most of the other
essential amino acids in adequate
quantities.
C. glomerata contains substances that
are toxic, based on yeast growth
inhibition. One of these substances is
free lauric acid, which is present in the
organism at a concentration of about 170
ppm.
The microbial pathogen, Acremonium
kiliense, may provide a useful tool for
controlling extensive growths of C.
glomerata in nature. The results of these
experiments, though striking in some
instances, have been variable, and more
study is needed. Nevertheless,
Cladophora from both stock cultures and
field sites were adversely affected in
laboratory experiments.
The results of these experiments
showed that unialgal Cladophora and
Cladophora plus epiphytes were unsatis-
factory diets for the flathead minnow, P.
promelas. These data, plus those on
snails, showed the importance of chemi-
cal analysis of the food value of C.
glomerata.
Cladophora plus epiphytes were unsati-
factory diets for the fathead minnow, P.
promelas. These data, plus those on
snails, showed the importance of
chemical analysis of the food value of C.
glomerata.
The fungus Acremonium kiliense may
be a potential biological control. However,
a good deal more experimentation has to
be done to understand the conditions
under which it develops and what the
toxin is.
The results from the experiments with
megaorganisms as biological controls of
C. glomerata produced negative results.
However, these experiments did turn up
some very interesting information as to
the nutritional value of C. glomerata. This
alga has a very different assortment of
free and combined fatty acids, as well as
different amino acid proportions than
diatom-dominated periphyton, which
was obviously the preferred diet.
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Ruth Patrick, Charles F. Rhynme, R. William Richardson. Ill, Richard A. Larson,
Thomas L Bott, and Kurt Rogenmuser are with the Academy of Natural
Sciences of Philadelphia, Philadelphia, PA 19103.
Nelson Thomas is the EPA Project Officer (see below).
The complete report, entitled "The Potential for Biological Controls o/Cladophora
glomerata," (Order No. PB83-251298; Cost: $17.50. subject to change) will be
available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Research Laboratory
U.S. Environmental Protection Agency
6201 Congdon Blvd.
Duluth. MN 55804
if U.S. GOVERNMENT PRINTING OFFICE. 1983-659-017/7214
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
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