WATER POLLUTION CONTROL RESEARCH SERIES •  16010DOU06/70
     PHARMACOLOGICAL TESTING
         OF BLUE-GREEN ALGAE
           FOR CONSTITUENTS
     HAVING THERAPEUTIC VALUE
ENVIRONMENTAL PROTECTION AGENCY • WATER QUALITY OFFICE

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     WATERvPOLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control Research Series describes
the results and progress in the control and abatement
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Reports should be directed to the Head, Project  Reports
System, Office of Research and Development, Water Quality
Office, Environmental Protection Agency, Room 1108,
Washington, D.  C.  20242.

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PHARMACOLOGICAL TESTING OF BLUE-GREEN ALGAE
   FOR CONSTITUENTS HAVING THERAPEUTIC VALUE
                                by
                 World  Life Research Institute
                    Colton,  California  92324
                              for  the

                      WATER QUALITY OFFICE

               ENVIRONMENTAL PROTECTION AGENCY
                       Project #16010 DOU
                      Contract #  14-12-535
                            June,  1970
 For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price 30 cents
                           Stock Number 5501-0123

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            EPA Review Notice
This report has been reviewed by the Water
Quality Office, EPA, and approved for publication.
Approval does not signify that the contents
necessarily reflect the views and policies of
the Environmental Protection Agency, nor does
mention of trade names or commercial products
constitute endorsement or recommendation for
use.
                        -11-

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                         ABSTRACT
The freshwater alga Aphanizomenon flos-aquae (Linnaeus) Ralfs,
commonly referred to as waterbloom and pondscum, is an economic
pest infesting many freshwater ponds and lakes of North America.
This alga has been incriminated in fatalities in fish,  fowls,  and cattle.

Samples of the alga were collected at Upper Klamath Lake,  Oregon,
during the peak of its primary bloom on 12  June  1969.   The samples
were taken in Upper Klamath Lake near Bare Island, along the south
side of  Buck Island, and at Pelican Marina  where the bloom was most
luxuriant.

Acetone and  ethanolic extracts were prepared  for pharmacological and
microbiological testing.  The extracts were tested at 50 and 500 mg/kg
i.p.  in  the rat pharmacological screen of the Atlas Chemical Industries,
Inc. ,  at Wilmington,  Delaware.   Antimicrobial tests were performed
on Escherichia coli,  Beta streptococcus, Pseudomonas aeruginosa,
Mycobacterium fortuitum,  Staphylococcus aureus,  and Candida albicans.
The results of these tests showed that there was low potency,  no fractional
localization of activity, no significant neurological, cardiovascular,  or
outstanding biological activity of  any type.  No antimicrobial activity
was observed.  However,  there was evidence that the alga could be
classified  as "toxic"  if sufficient quantities  of  it were ingested.

Algal extracts were tested for antitumor activity in Leukemia  L-1210
and P388 in mice.  The tests showed inactivity and low toxicity.

The tests conducted in these studies fail to  show evidence of pharmacol-
ogical properties having commercial pharmaceutical potential.
This report was submitted in fulfillment of project 16010 DOU 06/70
(contract #14-12-535) under the sponsorship of the Federal Water
Quality Administration.
                             -ill-

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                            CONTENTS
Section                                                     Page
   I            Conclusions                                  j_




   II            Introduction                                  _




   III           Purpose of the Project                        3




   IV           Description of Work                          t




   V            Acknowledgments                            12




   VI           References                                  13
                             -IV-

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                         SECTION I

                        CONCLUSIONS
1.  In general the fractions exhibited little acute overt symptomatology
and thus probably do not contain products that exert pharmacological
effects  similar to the type seen by drugs which ordinarily directly
produce psychomotor, neurological,  or cardiovascular effects.  The
possibility of opposing drug activities cancelling each other out exists,
but it is unlikely that such an effect would be  evident in all fractions
or over the whole observational period.  Fraction 1R was the most
active,  but at 500 mg/kg its effect was not outstanding.

2.  Generally the activity of the fractions had a delayed onset (e. g. ,
approximately 24 hours).  This type of activity,  resulting in depression
of function, alertness, and appetite and decreased body weight, is
characteristic  of an  antimetabolite action and is common to many
naturally-occurring  substances.  Many fractionated mushroom species,
for instance, produced similar effects, and this activity has been shown
to be related to their antimetabolic ability.   The necropsy results
indicated that there was some general inflammation and delayed toxic
activity.

3.  In summary, there was low potency, no fractional localization of
activity, no significant neurological,  cardiovascular, or outstanding
biological activity of any type.  No antimicrobial activity was observed.
However,  there was  evidence that the alga could be classified as "toxic"
if sufficient quantities of it were  ingested.  The tests conducted to date
fail to  show evidence of pharmacological properties having commercial
pharmaceutical potential.

4.  On the basis  of the studies, further investigation as to the commercial
pharmaceutical potential of the phytochemical constituents of
Aphanizomenon flos -aquae  does not appear to be warranted.
                             -1-

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                         SECTION II

                        INTRODUCTION
The freshwater alga Aphanizomenon flos-aquae  (Linnaeus) Ralfs,
commonly referred to as waterbloom and pondscum, is an economic
pest infesting many freshwater ponds and lakes  of North America.
A. flos-aquae has been incriminated in fatalities in cattle which have
been watered in ponds infested by this  alga (Nelson, 1904).   It has
also been involved  in fish mortalities in lakes in Iowa {Prescott,  1933).
Durrell and Deem (1940) found this  alga to be toxic to rabbits, guinea
pigs,  and chickens  when they were fed A_. flos -aquae or injected with
extracts from this  alga.  This alga  has also been reported to cause
deaths in wild birds and domestic ducks in a  water reservoir in north-
ern Colorado.  Over  the years various reports have appeared from
time to time concerning the toxicity of this alga to fish,  fowl, and
cattle.

The studies of Fitch et al. (1934),  Ingram and Prescott (-1954),  Olson
(1951),  Schwimmer and Schwimmer (1955), and others have  shown that
toxic waterblooms  involving A. flos -aquae are sporadic,  and the  symptoms
produced and survival times of poisoned animals vary considerably.  It
was later determined by Gorham (1964) that a fast-death factor was
present in this species of alga, but  the chemical nature of the poison was
not defined.  In their studies  on A_.  flos-aquae in Burton Lake, Saskatche-
wan, Canada, Gorham et al.  (1964) found that there were both toxic and
nontoxic strains of  this species.

Studies conducted under the Eutrophication Program by the Pacific
Northwest Water Laboratory,  Federal Water Quality Administration,
Corvallis, Oregon, revealed  that the alga A_.  flos -aquae was  a pest
present in Upper Klamath  Lake, Oregon.  It  was therefore considered
advisable to try and find an economic application for the use of the alga
involved.  In view of  the biological activity of A. flos-aquae  it was
suggested that perhaps the alga contained phytochemical constituents
that might ultimately have potential value as  pharmaceutical agents.
                                -2-

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                         SECTION III

                  PURPOSE  OF THE PROJECT
The objectives of this contract were to determine if the phytochemical
constituents of Aphanizomenon flos-aquae from Upper Klamath Lake,
Oregon,  contained pharmacological properties that might have potential
pharmaceutical value as a therapeutic agent.
                              -3-

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                         SECTION IV

                   DESCRIPTION  OF WORK
A description of the scope of each of the major technical areas follows:

             A.  Collection of algal samples:  After consultation with
Dr.  L. P. Seyb, Deputy Director; Pacific Northwest Water Laboratory,
FWQA, it was decided that the ideal time  to collect the alga at Upper
Klamath Lake, Oregon, was during the peak of its primary bloom in
the middle of June.  On 12 June 1969 Mr.  and Mrs. Richard Marin of
the World Life Research Institute accompanied Mr. Sanville of the
Pacific Northwest Water Laboratory to Upper Klamath Lake to collect
the algae.  A small skiff from the Eutrophication Research Program of
FWQA was -used to collect the algae.   All  algal samples were collected
with the use  of dip nets.  Samples of the algae were squeezed to remove
the excess water, and the  samples were immediately deposited in plastic
iced chests.   This material was  later frozen.  In addition samples
were placed  in concentrated isopropyl alcohol. All samples were taken
in Upper Klamath Lake near Bare Island,  along the south side of Buck
Island,  and at Pelican Marina.  These were the areas  where the  algal
growth  was most profuse.  Other areas of the lake were examined, but
the growth was insufficient to warrant collecting.  Approximately 8
gallons of algae were collected.  The  iced material was promptly frozen.
A small sample  of the algae was air  dried, but it was  found that the
material broke down rapidly,  and it was finally decided that this  process
was  not suitable for the studies to be undertaken.

             B.  Pharmacological  screening  procedures:  In view of
the fact that  the pharmacological evaluation of this alga was aimed at
trying to develop a commercial use of this pest it was  suggested  that
the pharmacological screening be done by a reputable pharmaceutical
company.  Samples  of the alga were therefore submitted to Atlas
Chemical Industries, Inc., at Wilmington, Delaware.   This phase of
the work was under  the supervision of Dr. K. K. Kimura, Director of
Medical Research, and Dr. D.H. McCurdy. Head of Pharmacology.
The  alga was then submitted through  their routine natural products
screening program according to the following  scheme:
                 1.  Chemical Extraction Procedure
Two liters of the ethanolic suspension of Aphanizomenon flos-aquae was
processed for pharmacological screening  by preparing extracts for test-
ing according to the following schematic procedure:
                             -4-

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                    EthanolSuspension
                (25 gms. wet wt. of algae)
                                 Centrifuged at 3000 rpm
                                 (International-Hn) for 15 mins ,
       Ethanol Supernatant
   r
Slimy ppt
                          ppt
                 Add 2 vols.
                 acetone,  vacuum
                 filter
	1
 Filtrate
                              Triturate with
                              acetone,  800 ml
     Wash off filter
     paper with
     & lyophilize
Labeled 2R
wt. =3.15 gms.
       Flash
       evaporate
       in vacuo to
       aq.  phase
       and lyophilize
                   Labeled 2AR
                   wt. = 6.7 gms.
Cell Residue     Acetone
  Labeled IR_    Extract
  wt. = 20.6 gms.
                      Flash
                      evap-
                      orate
                      to  aq.
                      phase
                      in  vacuo
                      and lyoph'
                      ilize
                                                      Labeled IAR
                                                      wt.  = 0. 88 gms.
                                                         (oily)
                             -5-

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                 2.  Pharmacological Tests Performed
The fractions obtained from the chemical extractions were passed
through a screening procedure which was designed by the pharma-
cology staff of Atlas Chemical Industries to elicit a broad spectrum
of psychomotor, neurological, and cardiovascular  responses.   These
techniques employ a rat screen in which the various fractions (2R,
2AR,  1R, and 1AR) were injected intraperitoneally into Laboratory
rats which were carefully observed over a period of 48 hours.

The signs observed and recorded included the following:
             a.  change in motor activity
             b.  ataxia
             c.  analgesia
             d.  loss of righting reflexes
             e.  loss of corneal reflexes
             f.  loss of pinna reflexes
             g.  paralysis of hind legs
             h.  screen grip hind leg loss
             i.  screen grip front  leg loss
             j.  tremors
             k.  fasciculation
             L.  tonic or  clonic convulsions.

Respiratory Changes:
             a.  change in rate of respiration.
             a.  enophthalmus
             b.  exophthalmus
             c.  palpebral ptosis
             d.  changes in pupillary size
             e.  nystagmus
             f.  lacrimation
             g.  chromodacryorrhea.

Ears and Mucosa:
             a.  blanching
             b.  hyperemia
             c«  cyanosis.

General:
             a.  salivation
             b.  tail erection
                                -6-

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             c.   pilomotor erection
             d.   urination
             e.   diarrhea
             f.   Robichaud test - skin fold reaction
             g.   circling motions
             h.   tail-lashing
             i.   writhing
             j.   rectal temperature
             k.   body weight
             1.   body tone
             m.  head tap reaction
             n.   body touch reaction
             o.   catalepsy
             p.   excessive curiosity.

The  responses of the test animal are plotted against an arbitrary
standard which has been developed over the years in the Atlas
Pharmacology Laboratory as  a result of testing compounds having
known pharmacological activity.   These compounds include a broad
range  of psychomotor, neurological, and cardiovascular drugs.  The
degree of the response is assigned a number value.  For example a
good central nervous system  depressant might have a Central Nervous
System Depressant rating of 30 with  a theoretical maximum rating of
100.  Unless a response rating of a minimum  of 10 is obtained the
results are generally not considered as very significant.

Table  I summarizes the results in terms of the pharmacological
responses obtained from the algal extracts. It will be noted that
the pharmacological responses were very low  in every aspect,
even when the extracts were administered in very high dosages
(500 mg).  The headings for the various general categories reflect
the results affecting the various systems psycho-neurological
(ANS= Autonomic nervous  system; CNSS= Central nervous  system
stimulation;  CNSD= Central nervous  system depressant; Misc. =
general signs not included within the other  categories).  Any evidence
of cardiovascular activity would have been  included in this category
although there may be some overlap  in the  autonomic responses.

             Comments on the results:

Fraction 2R

      50 mg/kg:     There was very little activity.  A slight increase in
                    rectal temperature was noted at 30 min.  post inject-

                                 -7-

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      500 mg/kg:
ion,  which slowly returned to normal by the
24-hour reading.  At the experimental necropsy,
48-hour post injection, the intestines were
immotile, the heart was not beating, the liver
was  engorged with blood and there was rhexis
hemorrhage in the lungs.

There was very little activity.  There was a
decrease in body weight recorded from  1-48
hours post injection. Necropsy, after comple-
tion  of the experiment, 48 hours post injection
showed that the heart was not beating, the
liver was  engorged, and that there was rhexis
in the lungs.
Fraction 2AR
      50 mg/kg:
      500 mg/kg:
There was very little activity.  Some slight
fluctuation in pupil size occurred throughout
the observation period.  There was a decrease
in body weight noted from 0. 5 to 48 hours post
injection.  At 24 and 48 hours post injection
the animal had  lost an apparent 12  gms.  The
experimental necropsy, 48 hours post inject-
ion, revealed no gross abnormalities.

There was very little activity.  There was  some
pupillary size fluctuation throughout the observa-
tion period.  Body weight was decreased and by
24-48 hours post injection the animal had lost
10-11 gms.  The experimental necropsy, 48
hours post injection,  revealed no outstanding
gross abnormalities.
Fraction 1R
      50 mg/kg:
There was a slightly decreased pupil size from
1-48 hours post injection.  The symptomatology
seemed to be delayed in onset.  Slight piloerection;
slight rectal temperature decrease, and  slight
                            -8-

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                    decreased body tone were noted at approximately
                    24-48 hours post injection.   The animal lost
                    approximately  15 gms.  in body weight by 24-48
                    hours post injection.  At the experimental necropsy,
                    48 hours post injection, the right kidney was enlarged,
                    whereas the left kidney was normal. There was
                    petechial hemorrhaging in the  liver and lungs.  The
                    intestines, although motile, were empty.

      500 mg/kg:    Symptomatology was  delayed in onset.  At 4-24
                    hours post injection,  the animal was slightly to
                    moderately depressed with a slight loss of
                    screen grip and a marked loss in its ability to
                    function on the  rotorod.  There was slight to
                    marked enophthalmus, slight decreased pupil
                    size,  slight piloerection,  slight decreased body
                    weight,  and slight decrease in  body tone.  The
                    animal was found dead at 48 hours post injection.
                    A necropsy was not performed.
Fraction 1AR
      50 mg/kg:
      500 mg/kg:
There was very little activity.  A slight decrease
in rectal temperature was noted from 10 minutes
to four hours post injection.  At the post experi-
ment necropsy, 48 hours post injection,  the heart
was  not beating,  the liver was engorged with blood,,
there was rhexis hemorrhaging in the lungs and
kidney.  Some  particles of injected material were
found in the peritoneal  cavity.

There was only slight activity which  seemed to be
delayed in onset.  At 24-48 hours post injection,
a marked Robichaud test,  12  gms. loss in body
weight, and slight decrease in body tone  were noted.
At the experimental necropsy, 48 hours post inject-
ion,  the peritoneal wall was irritated.  There was
rhexis hemorrhage in the lungs and kidney. Particles
of injected material were found in the abdominal cavity,
                              -9-

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          3.  Antimicrobial Tests Performed
The various fractions utilized in the pharmacological screen were also
assayed for antimicrobial activity.   These fractions were tested against
Escherichia coli,  Beta streptococcus,  Pseudomonas aeruginosa,  Myco-
bacterium fortuitum, Staphylococcus aureus,  and Candida albicans.
The extracts were tested in concentrations of 1.0,  0. 1,  and 0.01 percent
and incubated at 37°C for 48 hours.  There was no evidence of inhibition
observed  at any dose level, and in fact, may have promoted bacterial
growth.  In view of the complete lack of activity, no further testing was
done for antiviral  activity.

          4.  Antitumor Tests Performed
A sample  of the alga preserved in concentrated isopropyl alcohol was
submitted to the Natural Products Section,  Development Branch,  Cancer
Chemotherapy National Service Center, National Institutes  of Health,
Bethesda, Maryland, for evaluation for antitumor activity.   The sample
was then placed through their routine screen and tested in Leukemia
L-1210 and  Leukemia P388 in mice. It was inactive in all and was low
in toxicity.  It is noteworthy that  the CCNSC had  previously tested four
other samples of this same alga species which had been cultured in the
laboratory.  These samples were tested in Leukemia L-1210,  Leukemia
P388,  and in Walker 256 carcinosarcoma in rats, but none  in KB cell
culture.   In none of  the tests were the samples found to have any signifi-
cant antitumor activity.
                                -10-

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                            TABLE  I
                           Weighted Score Expressed as the Percent
                           of the Maximum Possible Score Per Category

Fraction   Dose (mg/kg)   ANS      CNSS     CNSD       Misc^

   2R
   2AR
   1R
   1AR
50
500
50
500
50
500
50
500
0
0
1.89
1. 57
4. 09
4. 51
0
0
0.30
0
0
0
0.41
0
0
0
0
0
2.30
0. 15
1. 15
2. 84
0.76
1. 92
2. 06
5. 50
0
6.42
4. 81
1.37
0. 91
4. 12
                              -11-

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                            SECTION V

                     A C KN O W LE DG MEN TS
We are indebted to Doctors K. K. Kimura, D.H. McCurdy, R.C.
Landis, and their associates  at Atlas Chemical Industries, and
Dr. J.L.  Hartwell of the National Cancer Institute for his assist-
ance in testing the algal extracts.  We wish to acknowledge the
cooperation of Dr. L. P.  Seyb and his associates of the Pacific
Northwest Water Laboratory,  Federal Water Quality Administra-
tion, for their assistance in collecting the algal samples.
                                -12-

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                            SECTION VI

                           REFERENCES
Durrell, L. W. , andA.W. Deem
   1940         Toxic algae in Colorado.   J.  Colo. -Wyo. Acad.
                Sci.  2(6): 18.

Fitch, C.P., Bishop, JL. M. , Boyd,  W. L. ,  Gortner,  R.A.,  Rogers,
C.F., and I.E. Tilden
   1934         "Water bloom" as a cause of poisoning in domestic
                animals.  Cornell Vet.  24(1): 30-39.

Gorham, P.R.
   1964         Toxic algae, p.  307-336,  9  figs.  InD.F. Jackson,
                     ,  Algae and man.  Plenum Press,  New York.
Gorham,  P.R., McLachlan, J. , Hammer, U.T., and W. K. Kim
   1964         Isolation and culture of toxic strains of Anabaena
                f los -aquae (Lyngb. ) de  Breb.   Verb..  Internat.
                Verein.  Limnol.  15: 796-804, 1 fig.

Ingram,  W. M. , andG.W. Prescott
   1954         Toxic fresh-water algae. Am. Midi.  Nat.  52(1):
                75-87, 14 figs.

Nelson, N.P.
   1904         Observations upon some algae which cause  "water
                bloom."  Minn.  Bot. Stud. 3(1): 51-57, 1 pi.

Olson, T.A.
   1951         Toxic plankton,  p.  86-95.  In Proceedings of
                Inservice Training Course in Water Works  Problems,
                February 15-16,  1951.   University of  Michigan School
                of Public Health, Ann Arbor.

Prescott, G.W.
   1933         Some effects of the blue -green algae,  Aphanizomenon
                f los -aquae,  on lake fish.  Collect.  Net 8(4): 77-80.

Schwimmer, M. ,  and D.  Schwimmer
   1955         The role of algae and plankton in medicine.  Grune
                and Stratton, New York.  86 p.
                              -13-

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1

5
Accession Number
2

Subject Field & Group
04A
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Organization
         World Life Research Institute,  Colton
     Title
         PHARMACOLOGICAL, TESTING OF BLUE-GREEN ALGAE FOR
         CONSTITUENTS HAVING THERAPEUTIC VALUE,
1 Q Authors)
Bruce W. Halstead, M. D.
16

21
Project Designation
Project No: 16010
FWQA Contract # 14-12-535
DOU
Note
  22
      Citation
  23
     Descriptors (Starred First)

        Nuisance algae, Algal toxins,  Cyanophyta,  Eutrophication
  25
     Identifiers (Starred First)

       Pharmaceuticals, Pharmacology,  Chemistry,  Toxicology, Poisonous plants
  27
     Abstract
             Samples of the freshwater alga Aphanizomenon flos-aquae (Linnaeus) Ralfs
were collected at Klamath Lake, Oregon, during the peak of its primary bloom on 12
June 1969.   This alga infests many freshwater ponds and lakes of North America and has
been incriminated in fatalities in fish, fowl,  and cattle.
             Acetone and ethanolic  extracts were prepared for pharmacological and
microbiological  testing.  The extracts were tested at 50 and 500 mg/kg i. p. in the rat
pharmacological screen of the Atlas Chemical Industries, Inc. , at Wilmington, Delaware.
Antimicrobial tests were performed on Escherichia coli, Beta streptococcus, Pseudomonas
aeruginosa,  Mycobacterium fortuitum,  Staphylococcus aureus,  and Candida albicans.
The results  of these tests showed that there was low potency, no fractional localization
of activity,  no significant neurological, cardiovascular, or  outstanding biological activity
of any type.   No antimicrobial activity was observed.  However, there was evidence
that the alga could be classified as  "toxic" if sufficient quantities  of it were ingested.

Algal extracts were tested for antitumor activity in Leukemia L-1210 and P388 in mice.
The tests showed inactivity and low toxicity. The tests conducted in these studies fail to
show evidence of pharmacological properties having commercial pharmaceutical potential.
                                                                      (Halatcad,  WLRI)
                                       World Life Research Institute,  Colton,  Calif.
Abstractor
       Bruce W.  Halstead
                             Institution
  WR:102 (REV. JULY 1969)
  WRSI C
                                             SEND TO: WATER RESOURCES SCIENTIFIC INFORMATION CENTER
                                                    U.S. DEPARTMENT OF THE INTERIOR
                                                    WASHINGTON, D. C 20240
                                                                             * GPO: 1969-359-339

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