EFFECT OF PESTICIDES ON BLUE-GREEN ALGAE

AND NITROGEN-FIXATION
EDGARJ. DASILVA+, LARS ERIC H ENRIKSSON++, and ELISABETH ENRIKSSON
Institute o]'Physiological Botany, University of Uppsala, S- 751 21 Uppsala. Sweden.

The effects of the pesticides, amitrol, a derivative of amitrol (viz. 3,5-diamino- 1,2,4-triazole),
diquat, paraquat, linuron, MCPA, malathion, and monuron, were studied on the nitrogen-fixing
algae, Anabaena cylindrica, Aulosira sp., Calothrix elenkenii, Chlorogloeae fritschii, Cylindrospermum
muscicola, Nostoc sp. from Collema tenax, Nostoc muscorum, Tolypothrix tenuis, and Westiellogsis
sp. In general, two types of response were discernible; an initial period of depression succeeded
by an increased activity and an initial period of depression followed by a distinct decrease on
prolonged incubation. The results indicate that some pesticidal compounds can severely limit
the nitrogen-fixing capacities of blue-green algae, thereby affecting the overall nitrogen econo-
my of soils in general.

The ever-increasing use of pesticides in the agricultural economy and in public health
necessitates an investigation into the biological effects of these substances on the soil
microflora. Amongst these forms are the blue-green algae which significantly influence
the nitrogen economy of temperate and tropical soils (Henriksson 1971, Henriksson et al.
1974, Watanabe & Yamamoto 1971). Whereas Batterton et aL (1971) observed that aldrin,
dieldrin and endrin and their metabolites were inhibitory to the growth of Anacystis
nidulans and Agmenellum quadriplicatum, Venkataraman & Rajyalakshmi (1972) and Singh
(1973) showed that some herbicides and pesticides are capable of markedly affecting the
growth of some nitrogen-fixing blue-green algae. Since Lundqvist (1970) and DaSilva et al.
(1973) showed that some pesticides markedly affect the nitrogen-fixing abilities of several
blue-green algae, it was considered worth while to follow up these aspects in the current
investigation.

The present investigation deals with the influences, at varying time intervals, of eight
pesticides on the nitrogen-fixing abilities of eight asymbiotic blue-green algal species and
the blue-green phycobiont of the lichen Collema tenax: The tolerance of one of them
(Chlorogloea fritschii) against six of the pesticides with respect to general growth develop-
m e n t has also been studied for comparative purposes.

Material and methods

Pesticides and algal species. The pesticide,s used in the current investigation were as
follows: Amitrol, 3,5-D (a derivative of amitrol), diquat, paraquat, linuron, MCPA,

+Current address: Centre of Postgraduate Research and Instruction, University of Bombay, Panaji, Goa, India.
++To whom correspondence should be directed.

Archives of Environmental Contamination 193

and Toxicology, Vol. 3, No. 2, 1975
9 1975 by Spdnger-Verlag New York Inc.
194 E . J . DaSilva et al.

malathion and monuron. All pesticides were either of purum or puriss grade, except
malathion which was obtained as a technical product. The chemical designations, grades of
purity, types and sources of the pesticides used are given in Table I.

The algal species used were as follows: Anabaena cylindrica Lemm., Aulosira species,
Calothrix elenkenii Kossinskaja, Chlorogloea fritschii Mitra, Cylindrospermum muscicola Ktitz.,
Nostoc species from Collema tenax (Sw.) Ach., em Degel., Nostoc muscorum Ag., Tolypothrix
tenuis Kiitz., and Westiellopsis species. Table II provides some salient features and the ob-
tention of the various species that were used in this investigation.

Culture m e d i a and culture conditions. All algal species were routinely maintained on
slopes of Go mineral (Holm-Hansen 1964) agar and on slopes of the nitrogen-deficient
medium r e c o m m e n d e d in the International Biological Programme (IBP) (Gorham et aL
1964, supplemented with trace elements according to Clendenning et al. 1956). The
cultures, prior to and under test of the nitrogen fixing activity, were grown in the IBP-
medium, and the cultures in the growth experiments were grown in the Go mineral
medium. The chemicals used were of pure grade.

Temperature of incubation in the growth-controlled chambers was 30~ for the main-
tenance of the algal cultures and for the growth experiments and 25~ for the experiments
on the nitrogen-fixing activity.

The illumination came from fluorescent lamps (Philips TL/33) and was 3000 lux.

Table I. Chemical designations and class o f pesticides used currently

Common Source and

name Chemical designation Solvent Remarks purity grade
amitrol 3-amino- 1,2,4-triazole water herbicide purum, Gullviks, Sweden
cotton defoliant
-- 3,5-diamino-1,2,4-triazole water derivative purum, Fluka, Switzerland
diquat 6,7-dihydrodipyrido water herbicide puriss 100%, Plant
(1,2-a: 2', 1'-c)pyrazidifi Protection Ltd. (ICI), U.K.
dibromide
linuron 3-(3,4-dichlorophenyl)- 1- methanol: herbicide puriss, recrystallized,E.I.
methoxy.l-methylurea water du Pont de Nemours,
(1:9, v/v) France
malathion O,O-dimethyl water insecticide techn., Plantex AB,
phosphorodithioate of effective Sweden
diethylmercaptosuccinate against spiders
mites and aphids
monuron 3-(4-ch Iorophen yl)-1,I- methanol herbicide puriss, recrystallized, E.I.
dimethylurea water du Pont de Nemours,
(1:9, v/v) France
MCPA 4-chloro-2-methylphenoxy- water herbicide puriss, Bayer, Germany
acetic acid
paraquat 1,1 '-dimethyl-4,4'- water herbicide purum > 96%, Plant
bipyridylium dichloride Protection, Ltd. (ICI), U.K.
 Effect of Pesticides on Blue-Green Algae 195

Table II. Source and characteristics o f the algal species used in the investigation

Species State Source Remarks

Anabaena cylindrica
A ulosira sp.
Calothrix elenkenii
Chlorogloeafritschii
Cylindrospermum muscicola
Nostoc sp.
Nostoc muscorum
Tolypothrix tenuis
Westiellopsis sp.
aRepurified by DaSilva, E.J. during the current investigation.
Axenic Culture Collection of Algae No. 1403/2A
and Protozoa, Cambridge,
U.K.
Axe nic Pattnaik, H., Ravenshaw
College, Orissa, India.
Axenic Singh, R.N., Banaras Hindu
Univ., Varanasi, India.
Axenic Fogg, G.E., Marine Science
Laboratories Anglesey, U.K.
Axenic lchimura, T., Institute No.M--32
Applied Microbiology,
Univ. of Tokyo, Japan.
Axenic a Henriksson, E., Institute of phycobiont of the
Physiological Botany, lichen Collema tenax
Univ. of Uppsala, Sweden. (Henriksson, 1951).
Axenic Cult. Coll. Cambridge
Axenic lchimura, T. Tokyo No. M--29
Axenic Singh, R.N. Varanasi

The sand used was obtained commercially (washed sea sand, Kebo, Stockholm, Sweden)
and analyzed by the National Swedish Laboratory for Agricultural Chemistry, Uppsala.
The methods of analysis are confirmed by Royal Proclamation of the National (Swedish)
Board of Agriculture (1965). Table III shows the analyses of the sand.

Nitrogen-fixing analyses. The activity of the nitrogen-fixation was measured by the

acetylene reduction technique of Stewart et al. (1967, 1968, 1971). Contrary to the original
method, however, the same samples and controls were used during the whole experimen-
tal period (Henriksson et al. 1972). The analyses were carried out with a gas chromatograph
(Perkin-Elmer Model 880) fitted with a Porapak T column (50-80 mesh). Uninoculated con-
trois ("zeros") for each of the pesticides were also tested in similar manner.

Growth Measurements. The cultures under test were grown in 20-ml aliquots in 125 ml
Erlenmeyer flasks equipped with a side-arm. Growth was followed turbimetrically at 610
nm using a photometer (Kipp-Zonen H 34, modified for the sidearms) during 14 days.

Experiments
The effect of pesticides on the activity of nitrogen-fixation. In determining the effect of
pesticides with respect to different time intervals, the pesticides mentioned in "Material
and methods" were employed. The application rates used were according to Alexander
(1969) as far as possible. These, however, exceed the recommended rates and were as
follows: Amitrol, (3,5-D), diquat, paraquat, 20 ppm; linuron, 10 ppm; MCPA, 25 ppm;
malathion, 100 ppm; and monuron, 10 ppm. The algal species employed in these experi-
ments were all those which are mentioned in "Material and methods".
196 E. J. DaSilva et al.

Table Ill. Sand analyses

mg/lO0 g
Constituen~a air dried sand
Phosphorus, P-AL 0.1
P-HC1 1.
Potassium, K-AL 0.7
K-HC1 5.
Calcium, Ca-AL 4.0
Ca-HC1 5.
Sodium, Na-AL 3.6
Na-HC1 9.
Magnesium, Mg-AL 1.4
Mg-HC1 35.
Nitrogen (Kjeldahl) < 1.
Iron, Fe-AI 1.0
Manganese, Mn 2.0.10 -3
Cupper, Cu-HC1 0
Boron, B 7.9.10 -3
Spec. conductance, 20"C 30.0.10 .-6
pH 6.1
aThe easily soluble constituents, marked -AL (e.g. P-AL, K-
AL) are extracted by standard AL-solution (0.10 M NH4-1ac-
tat, 0.40 m acetic acid). Values from ~.0 M HCl-extractions
(e.g. P-HC1, K-HC1) also include stored constituents.

One-ml aliquots of the pesticides were incorporated in the reactions flasks containing
one g o f sand resulting in final pesticide concentrations as shown above, and in Figure 1
and Table IV. Since all the pesticide solutions were incorporated immediately after pre-
paration into the reaction mixture without prior sterilization, it is assumed that bacterial
contamination, if any did not affect the experimental investigations. Following a 30-min
interval, one ml of exponentially growing cultures of the nitrogen-fixing algae under test
was introduced into the pesticide-sand mixture. Nitrogen-fixation activities were assayed
at the end of 1 hour and I-, 8-, and 17-day incubation periods, and in some cases also after
28 days.

The effect of pesticides on growth. In these experiments, amitrol, 3,5-D,diquat, paraquat

and malathion were tested at concentrations of 25 and 200 ppm on the growth of
Chlorogloeafritschii, This alga was found to be the most fitting test representative species
for the growth measurements. Some comparative experiments were carried out on the
growth of Nostoc muscorum and the Nostoc species o f the lichen Collema.

R e s u l t s and d i s c u s s i o n

A varied pattern of response, with respect to time, in the nitrogen-fixing ability of nine
algal species was observed with different pesticides (Table IV., Fig. 1). The effects of the
pesticides on the nitrogen-fixing activity are recorded in relation to the controls. In the
controls, as a function of time, a slight decrease in nitrogen-fixing activity occurred after
 Table IV. The effect on the nitrogen-fixing ability o f diquat and paraquat on different nitrogen-fixing species

Nitrogen-fixing activity in % of controls a

Effect of diquat 20 ppm after Effect of paraquat 20 ppm after
Species 1 hour 1 day 8 days 1"/days 1 hour I day 8 days 17 days ~
Anabaena cylindrica 10.3 0.7 0 0 41.1 0 0 0 t~
A ulosira sp. 17.7 0 0 0 0 0 0 0
Calothrix elenkenii 34.1 2.2 1.3 0 0 0 0 0 o
Chlorogloea fritschli 74.8 71.4 89.0 0 100.0 28.6 10.5 0
Cylindrospermum muscicola 149.9 39.0 5.5 0 100.0 27.9 0.6 0 E"
Nostoc sp. / Collema -- 38.9 6.2 0 -- 69.4 75.7 0 ?
Nostoc must orum 28.6 6.9 2.8 0.5 17.8 9.2 6.1 0 ~3
Tolypothrix tenuis 41.7 5.9 6.1 0 262.5 9.3 7.8 0 =
Westiellopsis sp. 108.7 18.8 18.4 0 35.9 4.7 0 0

aAn initial inhibitory effect is followed by a continued depression and final ceasing. For the exceptions see text!

-.-4
198 E. J. DaSilva et al.

500

~> 400
".~ Monuron, 10 ppm

c~
e-
300

c~ 200
o
Z 100

An.cyl. Aul. Cal. Chlor. Cyl. N.

, JJ N.
fL~
T. West.sp.
sp. elen. fr. musc. Coll. rnusc, ten.

500

Linuron, 10 ppm
400

~300
.E
X
~7
200
g
100

,J
An.cyl. Aul. Cal. Chlor. Cyl. N. N. T..West.sp.
sp. elen. fr. musc. Coll. musc. ten.

500

MCPA, 20 ppm
.~ 400
.>

o= 300

c 200
o

z 100

An.cyl. Aul. Cal. Chlor. Cyl. N. N. T. West.sp.

sp. elen. fr. musc. Coll. musc. ten.

Fig. 1. The effect on the nitrogen-fixing ability of monuron, linuron, MCPA, malathion,
amitrol, and 3,5D on the nitrogen-fixing species Anabaena cylindrica, Aulosira sp.,
Calothrix elenkenii, Chlorogloea fritschii, Cylindrospermum muscicola, Nostoc sp. from
 Effect of Pesticides on Blue-Green Algae 199

5OO
Malathion, 100 ppm
4(70

~ == 300
"5

z 100

An.cyl. Aul. Cal. Chlor. Cyl. N. N T. West.sp.

sp, elen. fr. musc. Coll. musc. ten.

500
Amitrol, 20 ppm

4OO

:~ 300
e=

~ 200

z 100

An. Aul. Cal. Chlor. CyI. N. N. T. West.sp.

Cyl. Sp. elen. fr. musc. Coll. muse. ten.

500 [] 1 h o u r
FI 1 day
3,5-D, 20 ppm 8 days
> 400
fl 17 days
I 28 days
300 Controls = 100.

200
o
.~_
Z
100

A n cyl. Aul. Cal. Chlor. CyI. N. N. T. West. Sp.

sp. elen. ft. musc. Coll. Musc. ten.

Collema, Noatoc muscorum, Tolypothrix tenuis, and Westiellopsis sp. An initial inhibitory
effect is more or less followed by gradual recovery. In some cases a stimulation may occur.
 200 E.J. DaSilva et al.

the first day of experimentation. The algae under test, however were sometimes able, after
a period of low activity, to recover and exhibit a strong response in the form of a large in-
crease in the nitrogen-fixing activity as compared with that of the controls. In addition to
this "recovering ability" of the algae the pesticides may also have stimulating effects as is
observed with amitrol (Fig. 1).

With the phycobiont, a Nostoc species, of the lichen Collema tenax, and for Calothrix
elenkenii erratic responses were encountered after a one-hr interval; nevertheless on the
basis of succeeding time intervals, general conclusions could be arrived at, which are dis-
cussed with respect to each pesticide.

In the case of the bipyridilium salts, diquat and paraquat (Table IV), apart from a possi-
ble initial stimulatory effect on Tolypothrix tenuis (paraquat) and Cylindrospermum muscicola
(diqua0, both herbicides exhibited a marked inhibitory action on nitrogen-fixation. After
eight days the effect of inhibition was striking, and by the end of the 17th day all the
cultures were completely inhibited with respect to their nitrogen-fixing activities. No at-
tempt was made to determine whether inhibition of nitrogen-fixation occurred as a result
of the disruption of the nitrogen-fixing enzyme system, or whether it was due to the reduc-
tion of these herbicides to toxic radicals which interfere with photosynthesis (Baldwin
1969, Van Rensen 1971).

The phenylureas, monuron and linuron, exhibited somewhat dissimilar effects (Fig. 1).
Monuron has shown promise as an algicide (Fitzgerald 1957), inhibitory effects on growth
being evident at 0.5 and 1.0 ppm. Independent serial liquid culture tests (DaSilva, un-
published) have shown that in the growth of the two nostocacean species, Nostoc species
from the lichen Collema and N. muscorum, a partial inhibitory effect occurs at five ppm,
which observation is in good agreement with that of Pillay & Tchan (1972). In all cultures
with monuron (Fig. 1) a marked inhibition occurred after an hour, but with an increase in
time the cells of Nostoc muscorum, Nostoc from Collema and Tolypothrix tenuis were able to
recover. Nevertheless, in comparison with the control culture, a 10 to 20% inhibition was
still present after 17 days. The reason for the behaviour of these species cannot be easily
explained, although monuron is known to interfere with photosynthesis (Van Overbeck
1964).

With linuron (Fig. 1) in nearly all cultures, a strong initial inhibition was obtained in
nitrogen-fixation activity. As in the case of Tolypothrix tenuis and Nostoc species in the
pesticide test, all the cultures exhibited a gradual recovery and in some cases increased ac-
tivity as compared with the controls.

The phenoxyacetic acid, MCPA, initially strongly retarded nitrogen-fixation with gra-
dually increased nitrogen-fixing activity as a function of time (Fig. 1). Nevertheless an 80
to 90% inhibitory effect was evident in Calothrix elenkeniL Aulosira species, Cylindrosper-
mum muscicola and the phycobiont Nostoc of Collema tenax.

In the case of the insecticide malathion (Fig. 1) the phycobiont species was markedly
depressed in its ability to fix nitrogen. In most species, following an initial retardation in
 Effect of Pesticides on Blue-Green Algae 201

nitrogen-fixation activities, a resurgence of their ability to do so was observed as a function

of time, with marked increases beyond those of the control cultures. Although the organo-
phosphorus compounds have been extensively studied, there are relatively few studies
concerning algal and other microbial forms. Alexander (1969) in reviewing the modes of
action of different pesticides noted that large quantities of insecticides were required to
have an inhibition of any great consequence.

With amitrol (Fig. 1) and 3,5-D (Fig. 1) similar patterns of response with respect to par-
tial inhibition followed by recovery were noted in all species with the sole exception of the
A ulosira species. In addition, 3,5-D had a similar marked effect on the Westiellopsis species
and Chlorogloea fritschii. Aaronson (1960) has shown that amitrol inhibits multiplication
and chlorophyll synthesis in Ochromonas danica and several other photosynthetic protists,
the mode of action of the herbicide being attributed to its ability to chelate iron. Whether
amitrol functions in a similar manner with the blue-green algae is not known and no at-
tempt was made in this investigation to establish its mode of action with the species used
in the analyses.

The growth of Chlorogloea fritschii is markedly affected by high concentrations of

different pesticides (Fig. 2). Paraquat and diquat were lethal at 25 ppm respectively. Single
experiments with Nostoc muscorum, and the Nostocspecies of the lichen Collema provide a
similar response to these pesticides.

In determining the tolerance of blue-green and green algae to diquat and paraquat using
a soil Plate paper-disc technique (DeSilva, unpublished), it was found that most filamen-
tous non-nitrogen-fixing blue-green algae could resist the action of the herbicides at 500
ppm. Chlorococcum humicolum and Scenedesmus quadricauda could tolerate 2500 ppm.
Tolerance of these two species to paraquat is ascribed to the multiple uncontrolled factors
such as pH, organic and mineral content, etc. Levels of 100 ppm inhibited both Anacystics
nidulans and Chroococcus cohaerens. The soil-plate paper disc-technique possesses certain
inherent inaccuracies in the absence of controlled conditions and factors. Nevertheless, it
does offer the advantage of a rapid visual assessment of the response of different algal
species to an herbicide in any desired concentration in a natural growth medium in more
simulated in situ conditions. In addition the technique offers itself for use in the screening
of soils towards different test species.

However, it is felt that, in order to obtain a perspective view of the effects of pesticides
on the soil algal flora, a supplementing correlation of its situ determinations with respect to
laboratory pure culture studies is necessary, since it has proved possible to obtain some
organisms that utilise these compounds through the technique of elective culture (Cripps
1971).

In conclusion, this investigation reveals that two patterns of response occurred while
studying the effects of different pesticides on eight asymbiotic cyanophycean species and
the blue-green phycobiont of the lichen entity, that are capable of fixing nitrogen. The
response to the pesticides was as follows:
202 E. J. DaSilva et al.

OD at 610 nm,
Kipp-Zonen Photometer
0,5

0.3

0.1 1
~---0~ 0 .... O - - O -

.__ I . I I ,_ I , . L. , ._I
2 6 10 14 Days

. Un,rea, 2Sp0m _I- Pa q,,atl.

[3 A m i t r o l 25 ppm 9 200 ppm O 1 Diquat J 25 &ppm200
l
[] 200 ppm 0 Malathion 25 ppm LMalathion 200 ppm

Fig. 2. Growth response o f Chlorogloea[ritschii in presence of 25 and 200 ppm of diaquat,

paraquat, amitrol, malathion, and the derivative of amitrol.

a) In some cases, an initial period of depression was succeeded by an increased activity

in the nitrogen-fixing ability. The ability of pesticides to stimulate microbial numbers
(Gawad et al. 1972), ammonification, nitrification, CO-/evolution, bacteria, fungi and ac-
tinomycetes has been reviewed (Audus 1964, Alexander 1969). Bartha et al. (1967) found
that pesticides of a particular type stimulated nitrification, whereas others, notably
monuron, were inhibitory in action.

b) In other cases, an initial decrease was observed with a subsequent decrease in

nitrogen-fixation as a function of time.

Finally, the blue-green algae are of significance, because it is well established that apart
from their functions in conserving and preventing soil erosion, they are vitally implicated
 Effect of Pesticides on Blue-Green Algae 203

in the natural process of increasing the nitrogen content of soil Hence, the effect of the
different pesticides on the nitrogen-fixing capacities of blue-green algae is considered to be
significant when these substances are employed to control pests and insects. The results of
this investigation on the nitrogen-fixing abilities of the blue-green algae show that the in-
hibition of their capacity to fix elemental nitrogen in the presence of certain pesticides may
well affect the overall nitrogen economy of either tropical or temperate soils.

Acknowledgements

The authors acknowledge the facilities extended towards this investigation by Prof. Nils
Fries, Head, Institute of Physiological Botany, University of Uppsala, and Prof. Bengt Kihl-
man, Department of Genetics and Plant Breeding, University of Uppsala, Sweden.

This investigation was undertaken as part of the International Biological Programme on

Nitrogen-Fixation. One of us (E.J. DS.)gratefully acknowledges the award of an UNESCO
Fellowship.

References

Aaronson, S.: Mode of action of 3-amino-1,2,4-triazole on photosynthetic microorganisms.

J. Protozool, 7, 289 (1960).
Alexander, M.: Microbial degradation and biological effects of pesticides in soil. In Soil
Biology--Reviews of Research, pgs. 209-236. Publ. Unesco (1969).
Audus, L.J.: Herbicide behaviour in the soil. In The Physiology and Biochemistry of the
Herbicides. ed. Audus, L. J., pgs. 163-206. Acad. Press. N. York (1964).
Baldwin, B. C.: On the mode of action of bipyridylium herbicides in photosynthesis. In
Progress in Photosynthesis Res. ed. Metzner, H., Tubingen, Vol. 3, pgs. 1737-1741
(1969).
Bartha, R., R. P. Lanzilotta, and D. Pramer: Stability and effects of some pesticides in soil.
Appl. Microbiol. 15, 67 (t967).
Batterton, J. C., G. M. Bonsh, and F. Matsumura: Growth response of blue-green algae to
aldrin, dieldrin, endrin and their metabolites. Bull. Environ. Contamin. Toxicol. 6,
589 (1971).
Clendenning, K. A., T. E. Brown, and H. C. Eyster: Comparative studies of photosynthesis
in Nostoc muscorum and Chtorella pyrenoidea. Canad. J. Bot. 34, 943 (1956).
Cripps, R. E.: The microbial breakdown of pesticides. In Microbial Aspects of Pollution.
eds. Sykes, G. and Skinner, F. A. Soc. Appl. Bact. Symp. No. i. pgs. 255-266 (1971).
DaSilva, E. J., L. E. Henriksson, and E. Henriksson: Effects of pesticides on asymbiotic
and symbiotic nitrogen-fixing blue-green algae. Abstr. No. 14, p. 3, 4th Int. Conf.
Global Impacts Appl. Microbiot., Sao Paulo (1973).
Fitzgerald, G. P.: The control of the growth of algae with CMU. Wisconsin Acad. Sci. Arts.
Letters. 46, 281 (1957).
Gawaad, A. A. A., M. Hammad, and F. H. EI-Gayan: Studies on soil insecticides XI. Effect
204 E.J. DaSilva et al.

of some soil insecticides on the nitrogen transformation in treated soils. Zentralblatt

f~ir Bakt. Parasit. lnfekt, und Hyg. 127, 297 (1972).
Gorham, P. R., R. MacLachlan, U. Hammer, and W. D. Kim: Isolation and culture of toxic
strains of Anabaenaflos-aquae (Lyngb) de Br~b. Verh. int. Ver. Limnol. i5, 796 (1964).
Henriksson, E.: Nitrogen-fixation by a bacteria-free, symbiotic Nostocstrain from Collema.
Physiol. Plant. 4, 542 (1951).
Henriksson E.: Algal nitrogen-fixation in temperate regions. Plant and Soil. Special
Volume, 415 (1971).
Henriksson, E., L. E. Henriksson, and E. J. DaSilva: A comparison of nitrogen fixation by
algae of temperate and tropical soils. In Nitrogen Fixation in the Biosphere. eds.
Stewart, W. D. P. and Nutman, P. S. Cambr. Univ. Press. Cambridge (1975).
Henriksson, L. E., P. H. Enckell, and E. Henriksson: Determination of the nitrogen-fixing
capacity of algae in soil. Oikos. 23, 420 (1972).
Holm-Hansen, O.: Isolation and culture of terrestrial and freshwater algae. Phycologia. 4,
43 (1964).
Lundqvist, I.: Effect of two herbicides on nitrogen-fixation by blue-green algae. Svensk
Bot. Tidskr. 64, 460 (1970).
National Swedish Board of Agriculture.: Royal proclamation regarding the stipulations for
soil analyses and methods. (In Swedish). Proclam. No. 1 with suppl. (1965).
Pillay, A. R., and Y. T. Tchan: Study of soil algae. VII. Adsorption of herbicides in soil and
prediction of their rate of application by algal methods. Plant and Soil. 36, 571 (1972).
Singh, P. K.: Effect of pesticides on blue-green algae. Arch. Mikrobiol. 89, 317 (1973).
Stewart, W. D., G. P. Fitzgerald, and R. H. Burris: In situ studies on N2-fixation using the
acetylene reduction technique. Proc. Nat. Acad. Sci. Wash. 58, 2071 (1967).
Stewart, W. D., G. P. Fitzgerald, and R. H. Burris: Acetylene-reduction by nitrogen-fixing
blue-green algae. Arch. Mikrobiol. 62, 336 (1968).
Stewart, W. D., T. Mague, G. P. Fitzgerald, and R. H. Burris: Nitrogenase activity in
Wisconsin lakes of differing degrees of eutrophication. New Phytol. 70, 479 (1971).
Van Overbeck, J.: Survey of mechanisms of herbicide action. In The Physiology and
Biochemistry of Herbicides. ed. Audus, L. J. pgs. 387-400. Acad. Press. N. York
(1964).
Van Rensen, J. J. S.: Action of some herbicides in photosynthesis of Scenedesmus as
studied by their effects on oxygen evolution and cyclic phosphorylation. Mededlingen
Landbouwhoge-schooL, Wageningen, Neder. 71-9, pgs. 80 (1971).
Venkataraman, G. S., and B. Rajyalakshmi.: Relative tolerance of nitrogen-fixing blue-
green algae to pesticides. Indian J. Agric. Sci. 42, 119 (1972).
Watanabe, A., and Y. Yamamoto: Algal nitrogen-fixation in the Tropics. Plant and Soil.
Special Volume, 403 (1971).

Manuscript received January 12, 1974; accepted July 21, 1974