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
80C.

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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 80C. 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
                                                                      I.II..II.,..M..H....I..l.l.JJ

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