Table 2. DIFFERENT MORPHOLOGICAL AND/OR PHYSIOLOGICAL STRAINS
REPRESENTED BY THE 36 ISOLATES FROM THE WARRIOR RIVER

Strain Isolate Number
Chlorella sp. 1,5,8,27
Chlorella sp. 2,26
Golinkiniopsis sp, 3
Chlorella sp. 4
Chlorella sp. 6,24,25
Chlorella sp. 7
Chlorella sp. 9,10,13
Monoraphidium sp. 11
Actinastrum sp. 12
Koliella sp. 14,23
Chlorella sp. 15
Chlorella sp. 16
Carteria sp. 17,19
Chlorella sp. 18
Chlorella sp. 20
Scenedesmus sp. 21
Chlorella sp. 22
Scenedesmus sp. 28,29,30
Nitzschia sp. 31,35, 36
Nitzschia sp. 32,34
Nitzschia sp. 33

12


plankton species. Walsh (1972), studying the effect of 2,4—D upon the
growth of four marine phytoplankton species, found that the technical
acid of 2,4-D was more toxic than was the butoxy ethyl ester.

Among the significant results of the current study of interactions
of 2,4-DBE and algae is the discovery that this formulation of 2,4-DBE
shows a relatively low toxicity, compared to the toxicity of the other
pesticides being studied. Another finding of possible significance is
the minimal uptake of this formulation of 2,4-D by Chlorella sp., iso-
late #1, and by Anacystis nidulans. Neither organism appears to have
significant binding capacity, or binding sites, for this ester form of
2,4-D.

DIAZINON

Cultures of Dunaliella euchlora and Platymonas sp. as well as
natural freshwater phytoplankton communities (Butler, 1963) showed re-
duced 18co, uptake in the presence of 1 mg/l diazinon. Estuarine phyto-
plankters are similarly inhibited by 1 mg/1 diazinon (Ware and Roan,
1970). In contrast, in Scenedesmus quadricaudata 1405 uptake is repor-
ted to be stimulated by 0.1 and 1 mg/l. Growth of the nitrogen-fixing
blue-green alga Cylindrospermum sp. in N-free medium is inhibited only
slightly by diazinon up to 200 mg/l. Higher levels of diazinon are
inhibitory to this organism and also to Aulosira fertilissima and to
Plectonema boryanum (Singh, 1973).

The effect of diazinon upon the Warrior River isolates was variable
both in regards to concentrations of diazinon and to different isolates.
However, at concentrations of 10 mg/1 and higher most isolates were sig-
nificantly inhibited. Paris et al. (1975) observed no degradation
of diazinon that could be attributed to the action of mixed populations
of bacteria and fungi. However, diazinon degradation has been reported
in submerged soils and in rice paddies in India (Sethunathan and
Yoshida, 1969; Sethunathan and Pathak, 1972). While it was determined
that the degradation was biological, the microorganisms involved were
not identified. It is not known whether any of the degradation should
be attributed to action by soil algae in the microflora. Our studies
involving small scale cultures of Warrior River isolates indicate that
there is some diazinon degradation by these algae but the extent of
degradation is not great.

33


Plots of the logarithm of malathion concentration in these cul-
tures against time indicated that after a brief lag period (2 to 4 hr)
malathion disappearance approximated that expected from a first order
reaction. Therefore the process was treated as a pseudo-first-order
reaction in these cultures, and in controls, in order to obtain approxi-
mate malathion degradation rates and malathion half-lives in the cul~
tures. Duplicate rate constants and half-lives obtained for Chlorella
and for Anacystis, as well as for degradation of malathion in the
controls, are shown in Table 13.

Samples from the Chlorella culture represented in Table 11 were
extracted, and the extracts were placed on thin-layer chromatographic
plates along with standards of malathion degradation products. Mala-
thion monocarboxylic acid was found first in the 8-hr sample, reached
a maximum concentration in the 12-hr sample and was not found in the
24-hr samples or later, Other degradation products were not identified.

29


Kapoor, I. P., R. L. Metcalf, R. F. Nystron and G. K. Sandha. 1970.
Comparative metabolism of methoxychlor, methiochlor and DDT in
mouse, insects, and in a model ecosystem. J. Agr. Food Chem. 18:
1145-1151.

Kratky, B. A. and G. F. Warren. 1971. A rapid bioassay for photosyn~
thetic and respiratory inhibitors. Weed Res. 19:658-661.

Kruglov, Yu. V. 1970. Algologic method of atrazine determination in
soil. I.Z.V. Akad. Nauk. S.S.S.R. Ser. B. 1:144-147.

Lewin, J. 1966. Boron as a growth requirement for diatoms. J.
Phycol. 2:160-163.

Loeppky, C. and B. G. Tweedy. 1969. Effects of selected herbicides
upon growth of soil algae. Weed Sci. 17:110-113.

Mann, J. E. and J. Myers. 1969. On pigments, growth and photosynthe-
sis of Phaeodactylum tricornutum. J. Phycol. 4:349-55.

Mendel, J. L., A. K. Klein, J. T. Chen and M. S. Walton. 1967.
Metabolism of DDT and some other chlorinated organic compounds by
Aerobacter aerogenes. Journal of the A.O.A.C. 50:897-903.

Menn, J. J., W. R. Erwin and H. T. Gordon. 1957. Color reaction of
2,6-dibromo-N-chloro-p-quinonemimine with thiophosphate insecti-
cides on paper chromatograms. J. Agr. Food Chem. 5:601-602.

Metcalf, R. L., G. K. Sangha and I. P. Kapoor. 1971. Model ecosystem
for the evolution of pesticide biodegradability and ecological
magnification. Environmental Science and Technology 5:709-713.

Moore, R. B. 1967. Algae as biological indicators of pesticides. J.
Phycol. 3(suppl.):4.

Moore, R. B. 1970. Effects of pesticides on growth and survival of
Euglena gracilis Z. Bull. Environ. Contam. Toxicol. 5:226-230.

O'Kelley, J. C. 1966. An apparatus for intermediate~scale continuous
culture of alage. Turtox News 44:70-71.

Palmer, C. M. and T. E. Maloney. 1955. Preliminary screening for
algicides. Ohio J. Sci. 55:1-8.

Paris, D. F., and D. L. Lewis. 1973. Chemical and microbial degra-
dation of ten selected pesticides in aquatic systems. Res. Rev.
45:95-124,

Paris, D. F., D. L. Lewis, J. T. Barnett, Jr., and G. L. Baughman.
1975. Microbial degradation and accumulation of pesticides in
aquatic systems. U.S. Environmental Protection Agency. Washington,
DC. EPA-660/3-75-007. 46p.

38


The pesticide in each 10-ml culture was extracted using a 80:20
v/v mixture of pesticide grade benzene and hexane (Burdick & Jackson,
Inc.). Five milliliters of the solvent mixture was added to each test
tube and then agitated thoroughly on a Vortex-Genie mixer. The layers
were allowed to separate, and with a disposable Pasteur pipette, the
solvent phase was removed. Each culture was extracted twice and the
two solvent portions combined. Sufficient anhydrous sodium sulphate
was added to take up any water present, and the solvent was transferred
to a graduated centrifuge tube.

The extracts were reduced to a working volume (1-10 ml) using a
gentle stream of air and then analyzed for pesticide content using a
Tracor MI-220 Gas-Liquid Chromatograph equipped with a Nickel~63 elec-
tron capture detector.

The column used was a 6 mm OD x 4 mm ID x 1.83 m glass column
containing Chrom W HP 80/100 mesh support coated with 3% OV-1 (Varian
Aerograph).

Analysis conditions for atrazine, diazinon, methoxychlor and 2,4-
DBE were:

Carrier gas flow M,) 120 ml/min
Detector temperaturé 2505C
Inlet temperature 225°C
Column temperature °
Atrazine, diazinon 145°C
Methoxychlor 180°C
2,4-DBE 165°C

Analysis conditions for malathion were:

Carrier gas flow (N,) 120 ml/min
Detector temperature 240°C
Inlet temperature 1955¢
Column temperature 170 ¢

Calculations to quantitate concentration in a sample involved tri-
angulation of the peaks printed out for the pesticides atrazine,
diazinon, methoxychlor and 2,4-DBE. For malathion an Autolab model
6300 Digital Integrator was used.

LARGE SCALE CULTURE CONDITIONS

In order to provide relatively large quantities of algal cells,
four 10-liter continuous cultures of several organisms were maintained
axenically in culture apparatus that has been described (0'Kelley,
1966). Illumination for these cultures was provided by Westinghouse


Dieldrin

Inhibition of the algal strains by dieldrin was similar to that
observed for aldrin (Table 4). Seven strains were inhibited more than
50% by 1 mg/1 dieldrin. A few strains appeared to be slightly stimu-
lated, rather than inhibited, by dieldrin at concentrations not higher
than 1.0 mg/1.

Diazinon

Diazinon, at the lowest concentration tested, 0.01 mg/l, inhibited
the growth of 31 of the isolates, but only one isolate was inhibited
more than 50%; growth was stimulated for one isolate (Table 3). In
contrast, at 0.1 and 1.0 mg/1, diazinon appeared to stimulate growth
of approximately half of the isolates. At 10 mg/l only 1 isolate
appeared to be stimulated, and 29 isolates were measurably inhibited.
At 25 ppm growth of 30 of the 36 isolates was measurably inhibited,
and growth of 21 of these isolates was less than 50% of that of the
controls.

Endrin

Toxicity of endrin to the algal strains tested appeared to be
somewhat lower than that of aldrin and dieldrin (Table 4). Eight
strains showed some inhibition at an endrin level of 0.01 mg/1. How-
ever, only four strains were inhibited 50% or more by 1.0 mg/1 endrin.
One strain was stimulated by endrin at all three concentrations tested.

Heptachlor

Heptachlor showed limited toxicity to the algal strains isolated
from the Warrior River (Table 4). Fourteen strains showed some inhi-
bition, however, at 0.01 mg/l. While a concentration of 0.1 mg/1
appeared to be no more inhibitory, 1.0 mg/1 was more toxic to two
strains. A few of the strains of algae were stimulated by these con-
centrations of heptachlor.

Malathion

Malathion was sparingly toxic to the algal strains tested at con-
centrations of 1.0 and 5.0 mg/l (Table 4). About half of the strains
were inhibited slightly, nearly half were unaffected, and several were
stimulated. A few more strains (12) were inhibited at 10.0 mg/l. At
25 and 50 mg/1 malathion most or all of the algal strains were inhibi-
ted significantly.

16


ENDRIN

Sensitivity of algae to endrin appears to be variable. Batterton
et al. (1971) found that the growth of Anacystis nidulans and Agmenellum
quadriplicatum was inhibited by 0.0002 to 0.95 mg/1. Vance and Drummond
(1969) reported that the LDj99 for four unicellular algae ranged from
less than 5 to more than 20 mg/1. In other tests three blue-green algae
tolerated concentrations of endrin as high as 600 mg/1, although growth
was less than in the absence of this insecticide (Singh, 1973). In the
Warrior River isolates, several organisms were slightly inhibited at
0.01 and 0.1 mg/1; inhibition was more severe at 1.0 mg/1.

HEPTACHLOR,

Little has been written about the effect of heptachlor on algae.
A concentration of 60 mg/l has been reported inhibitory to Euglena
gracilis (Parrakova and Kremery, 1967). Only a few of the Warrior.
River isolates show inhibition of growth, and only at the highest con-
centration tested, 1 mg/l.

MALATHION

Malathion in low concentration, less than 5 mg/1, appears to have
little effect upon the growth of algae that have been tested so far.
Moore (1970) reported a 48.9% inhibition of the growth of Euglena
gracilis Z by a concentration of 7.25 mg/1. In contrast, Poorman (1973)
reported growth stimulation of Euglena gracilis following a one week
treatment of 10, 50 and 100 mg/l malathion. Malathion at 100 mg/1 had
little effect upon the growth of Chlorella (Christie, 1969). The
effect of malathion upon nitrogen fixation by blue-green algae was
studied by DaSilva et al. (1975) who found that N-fixation was initially
depressed and ultimately stimulated by 100 mg/1. Concentrations of 1,
5 and 10 mg/l malathion had no significant effect upon growth of the
Warrior River isolates, but 25 and 50 mg/l inhibited some of these
organisms.

Degradation of malathion by both bacteria and fungi has been demon-
strated, and degradation rates for the bacteria have been calculated
(Paris et al., 1975). Studies of the loss of malathion in small-scale
cultures of the Warrior River algal isolates suggested that degradation
rates for algae might also be determined. The large-scale culture ex-
periments with Chlorella sp. (isolate #1), and with Anacystis nidulans
showed that malathion is degraded by these algae, and also that the
degradation by Chlorella is strongly dependent upon light. The only
degradation product that has been isolated and identified is the mala-
thion 8-monoacid (from the Chlorella cultures), which disappears from

34


ABSTRACT

In this investigation interactions of 12 pesticides with 37
strains of fresh water algae were studied in an effort to determine
something of the variability in responses of fresh water algae to
the variety of pesticides in use or projected to be used in the future.

Three interactions were investigated. One was the toxicity of
the pesticides to these algae. Another was the sorption of several of
the pesticides by some of the species of algae. The third was the
possibility that some of the pesticides can be degraded by action of
algae.

In general it was found that sensitivity of algae to pesticides
varied greatly with the strains tested.

Sorption of methoxychlor appeared to be mainly physical, since
much of the methoxychlor sorbed was exchangeable. The butoxyethyl
ester of 2,4-D (2,4-DBE) was not sorbed to a significant extent by
two green algae tested, and sorption of carbaryl was very slow.

Malathion can be degraded by algae in the presence of light.
One breakdown product, malathion monoacid (beta form), appeared as
the malathion was being degraded, and later disappeared. Investiga-—
tions of the fate of 2,4-DBE and methoxychlor in algal cultures
suggest that these pesticides may also be degraded by algal activity.

iii


Table 11. @MALATHION CONCENTRATION IN LARGE-SCALE CULTURES OF
FRESH-WATER PALGAE. VALUES ARE PER CENT OF INITIAL
MALATHION CONCENTRATION

Time, Chlorella Nitzschia Anacystis Control
Hours (Isolate #1) (Isolate #35) nidulans (no algae)

0 100.0 100.0 100.0 100

1 97.6 95.1 98.0 --

2 98.9 92.9 97.3 97.3

4 83.9 90.9 94.6 --

8 29.0 72.9 87.2 88.2
12 12.2 63.0 60.0 --
24 0 59.8 32.7 85.2
36 -- 59.8 14.5 “=
48 0 56.5 0) 84.3
60 -- 54.9 - --
72 0 50.1 0 82.0
84 -- 44.3 -- -
96 0 41.8 0 77.2
108 -- 40.1 -- --
120 0 34.7 0 75.5

4 Initial malathion concentration was 1 mg/1.

> Initial algal cell concentration was 4 g/1 medium for Chlorella
and Anacystis; for Nitzschia it was 0.9 g/l.

27


Table 3. NUMBER OF DIFFERENT ISOLATES OF TFE WARRIOR RIVER ALGAE,
IN CULTURES CONTAINING PESTICIDE, THAT GREW TO A CERTAIN
PERCENTAGE RANGE, AS COMPARED TO GROWTH IN CONTROL CULTURES
(NO PESTICIDE)

Pesticide Concentration Growth Range, Per Cent of Controls

mg/1 0-50 51-90 91-110 111-150 151-190
Atrazine 0.001 2 24 10 -- --
0.01 -- 31 5 -- --
0.1 5 16 15 -- --
1.0 32 -- 4 -- --
10.0 36 -- -- -- --
25.0 36 -- -- -- --
Carbaryl 0.01 — 19 12 5 —
0.1 - 14 18 4 --
1.0 -- 18 18 -- --
10.0 4 10 12 10 --
25.0 15 4 5 12 -
2,4-DBE 0.001 -- 8 12 13 3
0.01 -- 5 15 13 3
0.1 -- 5 ll 15 5
1.0 -—- 5 13 14 4
4.0 -- 16 9 7 4
Diazinon 0.01 1 30 4& 1 --
0.1 -- 9 7 12 8
1.0 - 10 10 10 6
10.0 7 22 6 — 1
25.0 21 9 3 2 1
Methoxychlor 0.001 -- 9 12 13 2
0.01 -- 13 il 8 4

13


oxychlor was a Chlorella sp. (isolate #2) and the least active was a
Nitzschia sp. (isolate #34). The values for pesticide lost in cultures
as a result of algal presence ranged in percentage from 23 to 81.

Diazinon

Loss of diazinon in 2-week small scale cultures (Table 5) was
lower than was loss of 2,4-DBE, methoxychlor or malathion. Only
isolate #6, a Chlorella sp., was effective; in cultures of this or-
ganism, pesticide loss was 61% of the pesticide in control tubes
at the end of the 2-week period. Less than 25% was removed by the
other 20 strains.

Atrazine

Atrazine loss (Table 5) did not occur significantly in small
scale algal cultures containing this pesticide. This may have resulted
because of the high toxicity of atrazine to these isolates (see Table
3); algal growth was meager in the presence of atrazine and, perhaps,
for this reason the algae did not degrade the atrazine significantly.

SORPTION OF LABELED PESTICIDES

Methoxychlor

Results of the > G-methoxychlor sorption experiments are seen in
Table 6. Four organisms were used in the study, two green algae
(Chlorella sp., isolate #1; Monoraphidium sp., isolate #11), one
diatom (Nitzschia sp,, isolate #35), and one blue-green alga (Anacystis
nidulans). For each organism, there was a rapid initial sorption of
methoxychlor, indicative of physical adsorption. For Chlorella (iso-
late #1), this rapid sorption involved approximately 7% of the pesti-
cide initially added to the medium; for Monoraphidium (another green
alga), the rapid sorption involved about 13% of the pesticide in the
medium; for Anacystis, it involved about 7% of the pesticide; for
Nitzschia (isolate #35), it involved about 84% of the total pesticide
available for sorption. In Monoraphidium and Chlorella there was a
slower sorption, as represented by increased radioactivity associated
with the cells up to 6 hr after beginning the experiment. For all of
the organisms the label held by the algal cells diminished after 6 hr.
The medium was counted to see if the label was being excreted back
into it, but counts revealed that medium activity did not increase con-
comitant with loss, pf label within the celjs. The loss may represent
conversion of the C in methoxychlor to Co, .

19


Atrazine

Of all pesticides studied, atrazine was by far the most inhibi-
tory to the algal isolates (Table 3). Of the 36 isolates, 26 were
measurably inhibited by 0.001 mg/1 atrazine; two isolates at this con-
centration were inhibited more than 50%. Inhibition was slightly
more severe at 0.01 mg/1 and 0.1 mg/l. At 1 mg/l atrazine all 36
isolates were inhibited and 32 of these grew at a rate less than 50%
of that of the controls. At 10 mg/1 inhibition was even more severe,
and at 25 mg/1 all isolates grew at a rate less than 50% of that in
the control tubes.

Captan

Captan inhibited several strains (of the 21 tested) slightly at a
concentration of 0.01 mg/1 (Table 4). Inhibition increased progres-
sively as the captan concentration was increased, up to at least 8.0
mg/l. At a concentration of 5.0 and 8.0 mg/l approximately half of
the strains were inhibited more than 50%, compared to controls with no
captan.

Carbaryl

Overall, the growth of the 36 isolates was about as good in 0.01
and 0.1 mg/1 carbaryl as was observed in the controls (Table 3). Some
organisms appeared to be stimulated slightly while others were slightly
inhibited. However, at 1 mg/l carbaryl no isolates were stimulated and
18 were inhibited. At 10 mg/l carbaryl 4 isolates were severely inhi-
bited (less than 50% of control growth), but the growth of 10 isolates
was stimulated by this pesticide. At 25 ppm 15 isolates were inhibi-
ted such that their growth was 50% (or less) of growth in control cul-
tures; again the response of other isolates was quite different in that
12 isolates were measurably stimulated by this level of carbaryl.

2,4-DBE

At 0.001, 0.01, 0.1 and 1.0 mg/1 2,4-DBE approximately as many
isolates were stimulated by the herbicide as were inhibited by it
(Table 3). At 4 mg/l, the highest level tested, more isolates were
inhibited (16 isolates) than stimulated (11 isolates). The isolates
inhibited at this level of 2,4-DBE grew at a rate more than 50% of
that of the controls, and the 4 isolates most stimulated had grown
significantly more than did the controls (151-190% of growth in the
controls).

15


Table 12. COMPARISON OF ®MALATHION CONCENTRATION IN LARGE-SCALE CUL-
TURES CONTAINING ILLUMINATED CHLORELLA (ISOLATE #1) CELLS WITH
CONCENTRATION IN DARKENED CHLORELLA CULTURE, AND IN "AGED"
MEDIUM CENTRIFUGED TO REMOVE CELLS AND ILLUMINATED

Time, Chlorella Chlorella Chlorella Medium
Hours (Illuminated) (Dark) (Illuminated)
0 100.0 100.0 100.0
1 97.6 -- --

2 98.9 96.7 85.0
4 83.9 -- --
8 29.0 74.1 71.1
12 12.2 -- 75.5
24 0 65.8 70.0
36 -- -- boi
48 0 65.8 --
60 ~- 61.2 --
72 0 60.3 -
84 -- 57.1 --
96 0 51.1 -
108 -- 46.5 --
120 0 46.0 -
132 -- 43.3 --
144 0 38.2 --

@ Initial malathion concentration 1 mg/l.

b Culture had become green from the production of new Chlorella
cells from the small number remaining after centrifugation.

28


SECTION IV

RESULTS

ISOLATION OF ALGAE USED IN THE STUDY

A number of algae were isolated from the Warrior River water
samples and 36 isolates were obtained in unialgal axenic culture
(Table 1). Subsequent study of the isolates revealed that some were
replicates of the same morphological and physiological strain. These
replications are indicated in Table 2, which also shows that the 36
isolates represent 21 different strains of algae.

TOLERANCE TO PESTICIDES BY ALCGAL ISOLATES

Interpretation of Tabulated Results

Tables 3 and 4 show the response of the Warrior River algae (not
specifically identified) to varying levels of pesticides in the culture
medium. The vertical columns of each table indicate algal growth as a
range of the percent of growth of the same organism with no pesticide
present; the horizontal rows in each table indicate pesticide concen-
tration. The figures in the body of each table show the number of iso-
lates that grew to a certain percentage range, relative to the growth
of the same isolate without any pesticide. For example, in Table 3,
in 0.01 mg/l carbaryl, five isolates were stimulated to grow to a level
111-150% of the control growth; 12 isolates grew approximately as well
in this concentration of pesticide as they did in control tubes (91-
110%), and the growth of 19 isolates was inhibited (51-90% of controls)
in medium containing 0.01 mg/1 carbaryl.

Aldrin

Aldrin effects were measured on the 21 strains of Warrior River
algae (Table 4). A majority of the strains were inhibited by concen-
trations of 0.01 and 0.1 mg/l. Seven of the strains were inhibited
more than 50% by a concentration of 1.0 mg/l; in contrast to this, two
strains appeared to be slightly stimulated by this concentration of
aldrin.

10


SECTION IT

CONCLUSIONS AND RECOMMENDATIONS

While the toxicity of the different pesticides used in this study
varies significantly according to the pesticide involved, each pesti-
cide inhibited growth in one or several of the Warrior River isolates
at concentrations of a few parts per million or less. The sensitivity
to a particular pesticide varied significantly with different isolates.
For this reason, it is concluded that estimates of environmental damage
to the flora of streams and other bodies of fresh water are prone to
error if they are based only upon studies of the effects of pesticides
on a few laboratory strains of algae. It is recommended that such
estimates be based in the future upon studies that utilize algae repre-
sentative of those native to a particular aquatic environment.

Of the pesticides tested in this study atrazine was by far the
most toxic to the Warrior River algae. Some of these organisms, which
are much more sensitive to atrazine than has been reported previously,
can be inhibited by atrazine levels as low as 10-3 mg/l. Thus
it is recommended that more extensive studies of atrazine in the aqua-
tic environment be carried out.

Sorption of methoxychlor, 2,4-DBE, and carbaryl varied signifi-
cantly with different strains and species of fresh water algae. The
14¢-methoxychlor rapidly taken up by these organisms was also rapidly
exchanged with unlabeled methoxychlor. Thus, it is concluded that much
of the uptake was physical adsorption to cell surfaces. Some strains
of fresh water algae lack binding sites for 2,4-DBE and consequently do
not take it up in detectable quantities. A long lag in the uptake of
14c-carbaryl by some fresh water algae suggests that these organisms
only take up breakdown products of this pesticide and not the parent
compound. These limited sorption studies lead to the conclusion that
generalizations about uptake of pesticides by algae may lead to errors
in estimations of environmental effects of such uptake.

A biological breakdown of malathion mediated by fresh water algae
can occur in the presence of light. It also appears that some fresh
water algae can degrade methoxychlor and 2,4-DBE even though the lat-
ter may not be absorbed. It is recommended that additional studies of
these phenomena be conducted in order to obtain better estimates of
the fates of these pesticides in the aquatic environment.


The sorption of 2,4-DBE was negligible for all organisms tested
except for Nitzschia, isolate #35, Thus the only exchange experiments
using 2,4-DBE involved this isolate. Table 10 shows that the labeled
pesticide taken up by this organism in 5 min was rapidly exchanged with
unlabeled pesticide.

The peculiar pattern of 14c-carbaryl sorption by the three algal
strains tested, namely the long delay in uptake of the 14¢ label, was
such that exchange experiments did not appear to be appropriate for
any of the algal strains being studied.

MALATHION DEGRADATION

Large scale algal cultures were utilized to provide a large
enough sample of algal cells to permit a sequential study in the lab-
oratory of malathion breakdown caused by the action of algal cells.
Three organisms, two of them indicated to be active in breaking down
malathion in test tube cultures, were selected; they were Chlorella
sp. (isolate #1), Nitzschia sp. (isolate #35) and Anacystis nidulans.

Two of these organisms, under illumination, were very active in
breaking down malathion (see Table 11); one was the Chlorella isolate
and the other was Anacystis nidulans; malathion breakdown was very
rapid compared to the no-cell controls. In contrast, the Nitzschia
isolate showed lower activity. Each experiment had a no-cell control
consisting of medium with equivalent initial malathion concentration
and subjected to similar physical conditions (temperature, light,
aeration) to the extent possible. Malathion breakdown without algae
varied somewhat in different experiments, but in all of these controls
the malathion half-life was calculate to be 110 hr or longer.

Since the Chlorella isolate appeared the most active in breaking
down malathion, its activity was studied in more detail. In one experi-
ment both the Chlorella culture and its no-alga control were placed in
darkness, rather than in light. While there was measurable breakdown
of malathion by Chlorella in the dark, as compared to malathion break-
down in the dark control, the dark rate was very much lower than the
malathion breakdown rate in illuminated Chlorella cultures (see Table
12). In another experiment, a Chlorella culture was grown to the cell
density desired and the medium was separated from the cells by refrig-
erated continuous flow centrifugation. Then malathion was added to
illuminated supernatant, and its fate was followed. Malathion break~
down was negligible in the illuminated supernatant for 24 hr. At 36
hr the experiment was terminated since the culture was becoming green
from the renewed production of Chlorella cells from a few that had not
been removed by centrifugation.

25


Table 7. SORPTION OF !"C-2,4-DBE BY FRESH-WATER ALGAL SPECIES

Time of 4amount of !4¢-2,4-DBE taken up by indicated organism,

Sorption ug per g fresh weight
Chlorella Nitzschia Anacystis
(Isolate #1) (Isolate #35) nidulans
1 min 2.6 193.6 10)
5 min - 195.2 0
10 min 0.4 - 0
20 min 0 174.8 0
1 hr 0) 127.9 0
3 hr 0 81.7 0
6 hr 0 18.0 0
12 hr 0 7.7 0
24 hr 0 4.8 1)

4 The initial concentration of 2,4-DBE was 1 mg/l. The values
reported are the means from duplicate experiments involving
cells from two different cultures of the same organism. The
initial cell density of all organisms except Nitzschia was 4
g wet weight per liter of medium; for Nitzschia it was 0.9 g/l.

22


Number

1

2

1o

1L

12

13

TABLES

List of algae isolated from the Warrior River

Different morphological and/or physiological strains
represented by the 36 isolates from the Warrior River

Number of different isolates of the Warrior River
algae, in cultures containing pesticide, that grew
to a certain percentage range, as compared to growth
in control cultures (no pesticide)

Number of different strains of the Warrior River
isolates, in cultures containing pesticide, that
grew to a certain percentage range, as compared
to growth in control cultures (no pesticide)

Pesticides recovered from algal cultures after 2-
week growth period, as percentage of pesticide
in controls

Sorption of !*c-methoxychlor by fresh-water algal
species

Sorption of 14¢ 2,4-DBE by fresh-water algal species
Sorption of 14¢_carbaryl by fresh-water algal species

Exchange of 14¢_methoxychlor from algal cells to
medium containing unlabeled methoxychlor

Exchange of 14¢_1abeled-2,4-DBE using Nitzschia
sp. (isolate #35)

Malathion concentration in large-scale cultures of
fresh water algae. Values are per cent of initial
malathion concentration

Comparison of malathion concentration in large-
scale cultures containing illuminated Chlorella
(isolate #1) cells with concentration in darkened
Chlorella culture, and in "aged" medium centrifuged
to remove cells and illuminated

Malathion degradation rate constants and pesticide
half-lives for illuminated cultures (calculated as
a pseudo-first-order process)

13

14

18

20

22

23

24

26

27

28

30


CONTENTS

Abstract

List of Tables

Acknowledgments

I Introduction

II Conclusions and Recommendations
III Materials and Methods

IV Results

v Discussion

VI References

VIL Publications

iv

10
31
37

4L


Table 1.

LIST OF ALGAE ISOLATED
FROM THE WARRIOR RIVER

12.
13.
14,
15.
16.
17.

18.

Chlorella

Chlorella

Golinkiniopsis sp.

Chlorella
Chlorella
Chlorella
Chlorella
Chlorella
Chlorella

Chlorella

sp.

sp.

sp.

sp.

sp.

sp.

sp.

sp.

sp.

Monoraphidium

sp.

Actinastrum sp.

Chlorella

sp.

Koliella sp.

Chlorella

Chlorella

sp.

sp.

Carteria sp.

Chlorella

sp.

19.
20.
2l.
22.
23.
24,
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.

36.

Carteria sp.
Chlorella sp.
Scenedesmus sp.
Chlorella sp.
Koliella sp.
Chlorella sp.
Chlorella sp.
Chlorella sp.
Chlorella sp.
Scenedesmus sp.
Scenedesmus sp.
Scenedesmus sp.
Nitzschia sp.
Nitzschia sp.
Nitzschia sp.
Nitzschia sp.
Nitzschia sp.

Nitzschia sp.

11


SECTION I

INTRODUCTION

Most prior studies of the interactions of pesticides and fresh
water algae have dealt with a specific pesticide or a few, and have
involved one or only a few species or strains of algae. Generally
the algae employed have been common laboratory strains rather than
organisms recently isolated from streams, lakes or ponds. The fresh-
water algae, as a group, are very heterogeneous, physiologically as
well as morphologically. Because of this, predictions of pesticide
interaction with algae in streams based upon such laboratory studies
may be subject to significant error.

In addition, most of the studies carried out so far have been
concerned only with the toxicity of pesticidesto algal species or
strain. Only a few exceptions have considered other aspects of pesti-
cide-alga interaction.

The present study is an attempt to broaden the base upon which
predictions can be made about the interactions of fresh water algae
and pesticides in nature. It also considers, in addition to sensitiv-
ity of algae to pesticides, sorption of pesticides and algal activi~
ties leading to pesticide breakdown. It involved 36 strains of algae
isolated from the Warrior River near Tuscaloosa, Alabama and one
additional laboratory strain, Anacystis nidulans.

The pesticides investigated included: aldrin, atrazine, captan,
2,4-DBE (the butoxy ethyl ester formulation), diazinon, dieldrin,
endrin, heptachlor, malathion, methoxychlor and toxaphene.


Methoxychlor

Since methoxychlor is sparingly soluble in aqueous solutions, it
was added to algal cultures only at levels of 0.001 and 0.01 mg/1
(Table 3). At these levels, methoxychlor inhibited the growth of about
one-third of the 36 isolates, about one-third had their growth unaf-
fected by methoxychlor, and approximately one third of the isolates
were stimulated to grow by methoxychlor.

Toxaphene

The pattern of response of twenty algal strains to toxaphene was
as follows: at 0.001 mg/l 12 strains were inhibited, but growth was
more than 50% of that of the controls (Table 4). At 0.01 mg/1 toxa-
phene a number of the algal strains were stimulated. Slightly more
inhibition was observed at 0.1 mg/l. At a concentration of 1.0 mg/1
eight of the strains were inhibited more than 50%; one strain did
not grow in this concentration of toxaphene.

PESTICIDE LOSS IN SMALL-SCALE ALGAL CULTURES
2,4-DBE

Loss of this formulation of 2,4-D in small scale algal cultures
was high in all organisms tested (Table 5). Isolate #18, a Chlorella
sp., retained the highest quantity of 2,4-DBE relative to the control
tubes, 53%, while isolate #17, a Carteria sp., removed all but 13% of
the 2,4-D relative to control tubes.

Methoxychlor

These culture experiments indicate that algae will remove methoxy-
chlor from the environment (Table 5). The most active organism in
this respect was a Nitzschia species (isolate #32), and of the green
algal species tested, Chlorella (isolate #1) was most active. The
values of methoxychlor in culture tubes, expressed as the percentage
of methoxychlor in control tubes at the end of the two week growth
period, ranged from 29 to 79.

Malathion
As in the case of methoxychlor and 2,4-D, malathion was lost from

all of the small scale cultures more rapidly than from control tubes
lacking algae (Table 5). The most active organism for removing meth-

17


ENVIRONMENTAL
PROTECTION
AGENCY

EPA-600,'5.76-622 DALLAS, TEXAS
Esotegical Recs

March 1976 . eh By

DEGRADATION OF PESTICIDES BY ALGAE

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Environmental Research Laboratory
Office of Research and Development

U.S. Environmental Protection Agency
Athens, Geergia 30601


Walsh, G. E. 1972. Effects of herbicides on photosynthesis and growth
of marine unicellular algae. Hyacinth Control J. 10:45-48.

Ware, G. W. and C. C. Roan. 1970. Interaction of pesticides with
aquatic microorganisms and plankton. Residue Rev. 33:15-45.

Wells, J. S. and W. E. Chappel. 1965. The effects of certain herbi-
cides on the growth of Chlorella pyrenoidosa 7-11-05. Proc. 19th
Northeast Weed Control Conf. 1958:449-450.

Wheeler, W. B. 1970. Experimental absorption of dieldrin by Chlorella.
J. Agr. Food Chem. 18:416-419.

Wiedeman, V. E., P. L. Walne, and F. R. Trainor. 1964. A new tech-
nique for obtaining axenic cultures of algae. Can. J. Bot. 42:
958-959.

Zweig, G., I. Tamas and E. Greenberg. 1963. The effect of photosynthe-

sis inhibitors on oxygen evolution and fluorescence of illuminated
Chlorella. Biochim. Biophys. Acta. 66:196-205.

40


dimethyldithiophosphate, potassium dimethylthiophosphate and potassium-
0-desmethyl malathion.

ALGAL ISOLATION AND MAINTENANCE OF STOCK CULTURES

The algae utilized in this study (except for one culture strain)
were isolated from water samples collected from the Black Warrior River
at Bennett's Marina boat landing just above the Hugh Thomas Bridge at
Tuscaloosa, Alabama. Water samples were collected in sterile 1 liter
bottles and returned to the laboratory where the algae were isolated.
Some of the samples were used for isolations on the day of collection
and some samples were enriched by mixing with equal volumes of double
strength medium. Three media were used for enrichment: 1) Bristol's
Inorganic Mineral Solution (Deason and Bold, 1960), 2) Diatom Medium
FW-1 (Lewin, 1966), and 3) Blue-green Algal Medium BG-11 (Stanier et
al., 1971). The enriched samples were incubated 1-2 weeks to allow
the algal populations to increase in number. Following the incubation
period the algae were isolated.

Isolations were made from the original samples and enriched
cultures by plating and spraying (Pringsheim 1946; Wiedeman et al.,
1964). Axenic cultures were obtained by successive plating and spray-
ing of the unialgal cultures. All cultures were examined periodically
for contamination by inoculating from stock cultures into microbiolo-
gical media (nutrient broth, nutrient broth + 1.0% w/v glucose) and
by direct microscopic observations.

The isolated algae were maintained on slants of FW-1 medium
(Lewin, 1966) in 18 x 150 mm test tubes capped with plastic caps.
FW-1 was also used as the culture medium following the initial iso-
lations. All stock cultures were maintained in a walk-in growth
chamber on a 16-8 hr light-dark cycle at 20 + 1°C. Illumination of
3500 lux was provided by Westinghouse cool-white fluorescent tubes.

Since the isolates did not include a blue-green alga several
large-scale experiments utilized a laboratory strain of Anacystis
nidulans, strain B 625 from the Indiana University Culture Collection
of Algae (Starr, 1964), which was maintained in stock as a liquid
culture in the BG-11 medium of Stanier et al. (1971). These stocks
were maintained with the isolate stocks and under the same environmen-
tal conditions.

Media used for maintaining stock cultures, and all other media
used, were sterilized by autoclaving for 15 min, at 121°C and 105.5
g/cm2.


SECTION III

MATERIALS AND METHODS
PESTICIDES AND PESTICIDE DERIVATIVES

The unlabeled pesticides used were: 1) aldrin (1,2,3,4,10,10-
hexachloro-1,4,4a,5,8,8a-hexahydro-1, 4~endo-exo-5 , 8-dimethanonaphtha-
lene) (analytical gtandard, 99.5%) supplied by Shell Chemical Company;
2) atrazine (2-chloro-4-ethylamino-—6-isopropylamino-s-triazine) (99.0%)
supplied by Aldrich Chemical Co., Inc.; 3) captan (N-(trichloromethyl
thio)-4-cyclohexene-1,2-dicarboximide) (MP 173-174°C) supplied by
Matheson, Coleman and Bell; 4) carbaryl (l-naphthyl N-methylcarbamate)
(99.7%) supplied by Union Carbide Corporation; 5) 2,4-DBE (2,4-dichlo-
rophenoxyacetic acid, butoxy ethyl ester) (98.2%) supplied by Amchem
Products, Inc.; 6) dieldrin (1,2,3,4,10, 10~hexachloro-exo-6, 7-epoxy-
1,4,4a,5,6,7,8,8a-octahydro-1,4-endo, exo-5,8 dimethanonaphthalene)
(analytical standard, 99.5%) supplied by Shell Chemical Company; 7)
diazinon (0,0- -diethy1- ~0-(2-isopropyl-4-methy1-6-pyrimidinyl) phospho-
rothioate) (99.9%) supplied by CIBA-GEIGY Corporation; 8) endrin (1,2,3,
4,10, 10-hexachloro-6, 7-epoxy-1,4,4a,5,6,7,8,8a-octahydro-1,4-endo,
endo-5,8 dimethanonaphthalene) (analytical standard, 99.5%) supplied by
Shell Chemical Company; 9) heptachlor (1,4,5,6,7,8,8-heptachloro-3a,
4,7,7a-tetrahydro-4, 7-methanoindene) (purity 99.4% by labile chlorine)
supplied by Velsicol Chemical Corporation; 10) malathion (0,0-dimethyl
S-(1,2-dicarbethoxy ethyl phosphorodithioate) (purity 99.3%) supplied
by American Cyanimid Company; 11) methoxychlor (2,2-bis (p-methoxy-
phenyl)-1,1,1-trichloroethane) (88.0% p-methoxyphenyl isomer, 12.0%
other isomers) supplied by E. I. DuPont; 12) toxaphene (chlorinated
camphene compounds of uncertain identity, combined chlorine 67-69%)
supplied by Hercules, Inc.

The labeled pesticides used included l4c-carbaryl (rin ~labeled),
American Radiochemical Corporation; methoxychlor- (ring-uL-! C), Mall-
inckrodt; and 2,4-dichlorophenoxyacetic acid- 1-14¢- (butoxy ethyl ester
formulation), New England Nuclear.

Analytical standards for gas chromatography were provided by J. F.
Thompson, Chief, Quality Assurance Section, Chemistry Branch, EPA,
Pesticides and Toxic Substances Effects Laboratory, National Environ-
mental Research Center, Research Triangle Park, N. C., 27711.

The following derivatives of malathion were provided by M. H.
Woolford, Jr. and R. C. Blinn, American Cyanimid: malaoxon, malathion
dicarboxylic acid, malathion monocarboxylic acid (beta-form), potassium


to partial inhibition of growth. Stadnyk et al. (1971), working with
Scenedesmus quadricaudata, found 0.1 mg/l toxaphene had no effect, but

1 mg/1 caused a 19% decrease in cell number. The Warrior River isolates
were not inhibited by 0.001 or 0.01 mg/1 toxaphene; however, a signifi-
cant number were inhibited by 0.1 and 1.0 mg/1. Toxaphene sorption

has been demonstrated in several microorganisms, including Chlorella
pyrenoidosa strain 395, and a field sample containing Scenedesmus sp.
and Chlorella sp. (Paris et al., 1975).

36


Table 9. EXCHANGE OF !*C-METHOXYCHLOR FROM ALGAL CELLS TO MEDIUM
CONTAINING UNLABELED METHOXYCHLOR
Isolate Organism Uptake Exchange Per Cent Per Cent
# Time Time Exchanged Not-exchanged
1 Chlorella sp. 5 min 5 min 75 25
(Isolate #1) l hr 89 ll
6 hr 88 12
6 hr 5 min 72 28
1 hr 93 7
6 hr 92 8
Anacystis 5 min 5 min 87 13
nidulans l hr 93 7
6 hr 95 5
6 hr 5 min 87 13
1 hr 88 12
6 hr 92 8
i Monoraphidium sp. 5 min 5 min 83 7
(Isolate #11) 1 hr 88 12
6 br 88 12
6 hr 5 min 43 57
1 hr 54 46
6 hr 58 42
35 Nitzschia sp. 5 min 5 min 33 67
(Isolate #35) 1 hr 42 58
6 hr 40 60
6 hr 5 min 25 75
1 hr 35 65
6 hr 34 66

24


SECTION VII

PUBLICATIONS

Aldridge, E. F., G. L. Blume, J. C. O'Kelley and T. R. Deason. Degra-
dation of malathion by planktonic algae. In preparation.

Butler, G. L. 1974. Effects of five pesticides on twenty-one fresh-
water algae. Ph.D. Dissertation, University of Alabama, 1975.
University, AL 35486.

Butler, G. L., T. R. Deason and J. C. O'Kelley. 1975. Loss of five
pesticides from cultures of twenty-one planktonic algae. Bull.
Environ. Contam. Toxicol. 13:149-152.

Butler, G. L., T. R. Deason and J. C. O'Kelley. The effect of atra-
zine, 2,4-D, methoxychlor, carbaryl and diazinon on the growth of
planktonic algae. In preparation.

Butler, G. L., T. R. Deason and J. C. O'Kelley. The effect of endrin,
heptachlor, aldrin, dieldrin, captan, toxaphene and malathion on
the growth of planktonic algae. In preparation.

Moss, S. W., G. L. Blume, J. C. O'Kelley and T. R. Deason. Sorption

and degradation of methoxychlor by planktonic algae. In prepara-
tion.

41


——————S

Table 8. SORPTION OF !4C-CARBARYL BY FRESH-WATER ALGAL SPECIES

Time of Aamount of 14c-carbaryl taken up by indicated organism,
Sorption vg per ml fresh weight
Chlorella Nitzschia Anacystis.
(Isolate #1) (Isolate #35) nidulans
1 min 0 - 0
5 min - 0.4 -
10 min 0 1.2 0
20 min 0 2.1 0
Ll hr 0 3.4 0
3 hr 0.3 7.8 0
6 hr 0.3 8.2 -
12 hr 6.4 7.3 -
24 hr 2.4 6.8 14.9

4 The initial concentration of carbaryl was 1 mg/1. The values
reported are the means from duplicate experiments involving
cells from two different cultures of the same organism. The
initial cell density for all organisms except Nitzschia was 4
g wet weight/of medium; for Nitzchia it was 0.9 g/l.

23


ACKNOWLEDGMENTS

Appreciation is expressed to the companies donating the pesticides
used in these studies and to J. F. Thompson, National Environmental
Research Center, Research Triangle Park, North Carolina, for analyt-
ical standards. Special thanks are due M. H. Woolford and R. C. Blinn,
American Cynamid Company, for standards of malathion products.

Appreciation is expressed to Professor John L. Mego who assisted
in some of the work with the butoxyethyl ester of 2,4-D.

Work carried out by graduate students Gary L. Butler and Sam W. Moss
is included in this report.

The technical assistance of Deborah L. Clayton, Edward F. Aldridge,
and Gary L. Blume is gratefully acknowledged.

The authors wish, particularly, to thank Mrs. Doris F. Paris and
Mr. George Baughman, Environmental Research Laboratory, Athens, Georgia,
for helpful discussions during this investigation.


Table 4, NUMBER OF DIFFERENT STRAINS OF THE WARRIOR RIVER ISOLATES,
IN CULTURES CONTAINING PESTICIDE, THAT GREW TO A CERTAIN PERCENTAGE
RANGE, AS COMPARED TO GROWIH IN CONTROL CULTURES (NO PESTICIDE).

Pesticide Concentration Growth Range, Per Cent of Controls
mg/1 0-50 51-90 91-110 111-150 151-190
Aldrin 0.01 3 14 4 -- --
0.1 2 12 5 2 --
1.0 7 8 4 2 --
Captan 0.01 -- 8 12 1 --
0.1 -- 6 10 5 =
1.0 _ 15 6 -- --
5.0 3 8 8 2 --
8.0 4 13 3 1 --
Dieldrin 0.01 3 13 3 2 --
0.1 2 12 6 1 --
1.0 7 9 4 1 --
Endrin? 0.01 -- 8 11 1 --
0.1 2 14 3 1 --
1.0 4 14 1 1 --
Heptachlor@ 0.01 -- 14 5 1 --
0.1 -- 10 7 2 --
1.0 2 il 4 1 1
Malathion 1.0 -- 9 8 4 -—
5.0 -- 7 9 4 1
10.0 -- 12 4 4 1
25.0 12 6 2 1 --
50.0 17 4 -- -- --
Toxaphene 0.001 -- 12 6 3 --
0.01 -- 2 13 5 1
0.1 1 7 8 5 --
1.0 8 7 6 -- -

2 One strain of Nitzschia, of the 21 strains, was not tested.

14


SECTION V

DISCUSSION

ALDRIN AND DIELDRIN

Dieldrin is toxic to the alga Agmenellum quadruplicatum at concen-
trations of 0.475 and 0.95 mg/l while aldrin is not; these concentra-
tions of dieldrin are non-toxic to this species and also to Anacystis
nidulans (Batterton et al., 1971). Poorman (1973) reported that there
was stimulation, rather than inhibition of growth of Euglena gracilis
by 10, 50 and 100 mg/1 of aldrin. Growth of the diatom Navicula semin-
ulum was reduced 10% by 1.8 mg/1 dieldrin and was stopped by 32 mg/1
(Cairns, 1968). Stadnyk et al. (1971) found a 10% decrease in the cell
number of Scenedesmus quadricaudata at 0.1 and 1 mg/l dieldrin after
10 days of exposure. In contrast to the tolerance of some of these
organisms to aldrin and dieldrin, growth of a number of the Warrior
River isolates was inhibited by these insecticides at 0.01, 0.1 and
1.0 mg/l. In-so-far as accumulation is concerned, Wheeler (1970) found
that Chlorella pyrenoidosa accumulated dieldrin, and Rice and Sikka
(1973) reported that marine diatoms could remove dieldrin from culture
media.

ATRAZINE

The effect of atrazine on several laboratory strains of algae has
been studied previously. Ashton et al. (1966) reduced the growth of
Chlorella vulgaris 85% by treating with 70 mg/1 for 72 hours. Atkins
and Chan (1967) also reported inhibition to a Chlorella sp., but found
that the inhibition was overcome by the organism and growth equalled
that of the controls after 12 days. Zweig et al. (1963) reported a 50%
inhibition in 02 evolution by Chlorella pyrenoidosa using 6.2 mg/1
atrazine. Wells and Chappel (1965) found that a concentration of 0.1
mg/1 atrazine was only slightly inhibitory to a thermophilic strain,
Chlorella pyrenoidosa 7-11-05. Both Wells and Chappell (1965) and
Kratky and Warren (1971) found that concentrations of 1 mg/l and higher
were severely inhibitory. Arvik et al. (1971) found that 0.73 mg/1
atrazine reduced growth of an unidentified soil alga by about 50%.
Gramlich and Frans (1964) observed a 70% reduction of growth of Chlor-
ella pyrenoidosa by 1.25 x 10° M atrazine. Less than 2.5 mg/1 reduced
growth of a Chlorella-like alga by 95% (Kruglov, 1970). Loeppky and
Tweedy (1969) found that the effect of atrazine varied depending upon
the alga involved, some species being inhibited completely by 0.5 mg/l.
Walsh (1972) obtained similar results with four unicellular marine
algae. Our studies with the Warrior River isolates demonstrate that
some aquatic algae are much more sensitive to atrazine than has been

31


reported previously and that organisms can be inhibited by atrazine
levels as low as 1073 mg/1.

The Warrior River algae were severely inhibited by atrazine at 1
mg/1; there was no atrazine loss in cultures that could be attributed
to action by these algal species.

CAPTAN

Soeder et al. (1973) found that the amount of growth inhibition
caused by captan was dependent upon the algal strain being studied;
while 37 strains of Chlorella were strongly inhibited by 2.5 to 5 mg/l
captan, three strains of Chlorella and two strains of Scenedesmus were
tolerant of 50 mg/l. Moore (1967) found that the blue-green alga
Nostoc muscorum was not inhibited by 1 mg/l. Similarly, the Warrior
River isolates were not inhibited by 1 mg/l captan, However, at 5 and
8 mg/1 captan growth was inhibited in about one-quarter of these
isolates.

CARBARYL

Prior studies of interactions of carbaryl and algae have been
concerned mainly with toxicity of carbaryl. Carbaryl at 0.1 and 1.0
mg/1 stimulates growth of some algae and inhibits others (Ukeles, 1962,
Stadnyk et al. 1971). Higher concentrations (100 mg/1) kill some or-
ganisms (Ukeles, 1962) while others are reported to be inhibited only
slightly (Butler, 1963; Christie, 1969). Carbaryl inhibited growth of
the Warrior River isolates at concentrations of 0.01, 0.1 and 1.0 mg/l.
As the concentration was increased to 10 and 25 mg/1, growth of several
isolates was stimulated. Because the non-biological breakdown of car-
baryl is rapid, this stimulation may have been from a breakdown product
or products. Carbaryl appears to be taken up readily by the Nitzschia
sp. (isolate #35); however, neither the Chlorella sp. (isolate #1) nor
Anacystis nidulans take up carbaryl readily. In the latter algae there
is no uptake of label for several hours; the label ultimately taken up
may represent a breakdown product (or products) of carbaryl rather than
the parent compound.

2,4-DBE

While the effects of some formulations of 2,4-D on laboratory
strains of algae have been studied extensively, only a few such studies
have involved 2,4-DBE. Ware and Roan (1960) found that this ester of
2,4-D, at 1 mg/l, reduced !4CO, fixation about 15% in several phyto-

32


Table 6. SORPTION OF !¥C-METHOXYCHLOR BY FRESH~WATER ALGAL SPECIES

Time of amount of 14¢_Methoxychlor taken up by indicated organism,

Sorption ng per g fresh weight
Chlorella Monoraphidium Nitzschia Anacystis
(Isolate #1) (Isolate #11) (Isolate #35) nidulans
1 min 214 799 5205 399
5 min 426 762 -- 424
10 min 397 731 -- 415
15 min = -- 5642 --
20 min 378 764 -- 390
30 min -- -- 5558 --
1 hr 367 836 5528 383
3 hr 278 -- 5687 --
6 hr 259 1063 5704 428
12 hr 155 -- -- 391
18 hr -- -- 5097 -—
24 br 22 645 -- 316

2 the initial concentration of methoxychlor was 0.01 mg/l. The
values reported are the means from duplicate experiments invol-
ving cells from two different cultures of the same organism.

The initial cell density for all organisms except Nitzschia was
4 g wet weight per liter of medium; for Nitzschia it was 0.9 g/1
Volume of all cultures was 500 ml.

20


DISCLAIMER

This report has been reviewed by the Environmental Research
Laboratory, 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
constitute endorsement or recommendation for use.

di


Paris, D. F., D. L. Lewis, and N. L. Wolfe. 1975. Rates of degrada~
tion of malathion by bacteria isolated from an aquatic system.
Environ. Sci. and Tech. 9:135-138.

Parrakova, E. and V. Kremery. 1967. Effects of insectides and their
combinations on the growth of Euglena gracilis. Cesk. Hyg. 12:
473-476,

Poorman, A. E. 1973. Effects of pesticides on Euglena gracilis. I.
Growth studies. Bull. Environ. Contam. Toxicol. 10:25-28.

Pringsheim, E. G. 1946. Pure Cultures of Algae. London, Cambridge
University Press, 119 pp.

Rice, C. and H. C. Sikka. 1973. Fate of dieldrin in selected species
of marine algae. Bull. Environ. Contam. Toxicol. 9:116-123.

Sethunathan, N. and M. D. Pathak. 1972. Increased biological hydroly-
sis of diazinon after repeated application in rice paddies. J.
Agr. Food Chem. 20:586-589.

Sethunathan, N. and T. Yoshida. 1969. Fate of diazinon in submerged
soil. Accumulation of hydrolysis product. J. Agr. Food Chem.
(17:1192-1195.

Singh, P. K. 1973. Effect of pesticides on blue-green algae. Arch.
Microbiol. 89:317~-320.

Soeder, C. J., R. Liersch and U. Triiltzsch. 1973. Differential action
of captan on the growth of some strains of Chlorella and Scenedesmus.
Arch. Microbiol. 67:166-172.

Stadnyk, L., R. S. Campbell, and B. T. Johnson. 1971. Pesticide effect
on growth and !4C assimilation in a freshwater alga. Bull. Environ.
Contam. Toxicol. 6:1-8.

Stanier, R. Y., R. Kunisawa, M. Mandel, and G. Cohen-Bazire. 1971.
Purification and properties of unicellular blue-green algae (order
Chroococcales). Bact. Rev. 35:171-205.

Starr, R. C. 1964. The culture collection of algae at Indiana Uni-
versity. Amer. J. Bot. 51:1013-1044.

Ukeles, R. 1962. Growth of pure cultures of marine phytoplankton in
the presence of toxicants. Appl. Microbiol. 10:532-537.

Vance, B. D. and W. Drummond. 1969. Biological concentration of pesti-
cides by algae. J. Amer. Water Works Assoc, 61:361-362.

39


SECTION VI

REFERENCES

Arvik, J. H., D. L. Wilson, and L. C. Darlington. 1971. Response of
soil algae to picloram--2,4-D mixtures. Weed Sci, 19:276-278.

Ashton, F. F., T. Bisalputra and E. B. Risely. 1966. Effect of atra-
zine on Chlorella vulgaris. Amer. J. Bot. 53:212-216.

Atkins, C. A., and Y. T. Tchan. 1967. Study of soil algae. VI. Bio-
assay of atrazine and the prediction of its toxicity in soils using
an algal growth method. Plant Soil 27:432-442.

Batterton, J. C., G. M. Boush, and F. Matsumura. 1971. Growth re-
sponse of blue-green algae to aldrin, dieldrin, endrin and their
metabolites. Bull. Environ. Contam. Toxicol. 6:589-594.

Butler, P. A. 1963. Commercial fisheries investigation. Fish and
Wildlife Circ. No. 167:10-20.

Cairns, J., Jr. The effects of dieldrin on diatoms. 1968. Mosquito
News 28:177-179.

Christie, A. E. 1969. Effects of insecticides on algae. Water Sewage
Works 116:172-176.

DaSilva, E. J., L. E. Henriksson, and E. Henriksson. 1975. Effect of
pesticides on blue-green algae and nitrogen-fixation. Arch. Envi-
ron. Contam. Toxicol. 3:193-204.

Deason, T. R. and H. C. Bold. 1960. Phycological Studies I. Explora-
tory studies of Texas soil algae. University of Texas Publication
No. 6022, Austin, Texas.

Gramlich, J. R. and R. E. Frans. 1964. Kinetics of Chlorella inhibi-
tion by herbicides. Weeds 12:184-189.

Johnson, B. T. and J. O. Kennedy. 1973. Biomagnification of p, p-DDT
and Methoxychlor by bacteria. Applied Microbiology 26:66-71.

Kadoum, A. M. 1970. Thin-layer chromatography sorption and colori-

metric detection of malathion and some of its metabolites from
stored grains. J. Agr. Food Chem. 18:542-543.

37


cool-white fluorescent tubes at a culture surface intensity of 8.7 x

10" ergs/cm*-sec as determined with a YSI-Kettering radiometer. Cul-
tures were stirred continuously with magnetic stirring bars, and 1%

CO, in air was bubbled through the cultures, except for Anacystis nidu-
lans cultures, which were aerated only with air. Cultures were continu-
ously maintained in a logarithmic stage of growth by periodically
draining the culture and adding fresh, sterile medium.

The culture medium for green algal isolates was Bristol's Inor-
ganic Mineral Solution (Deason and Bold, 1960); for isolates of the
diatom Nitzschia,it was medium ASP-2, modified (Mann and Meyers, 1968);
and for Anacystis nidulans,it was medium BG~11 (Stanier et al., 1971).

PESTICIDE UPTAKE AND EXCHANGE BY ALGAE

The pesticides used in these studies included both !4c-labeled
and unlabeled forms of methoxychlor, carbaryl and 2,4-DBE described
previously. The initial concentration of pesticide in these experi-
ments was 0.01 mg/l methoxychlor, or 1 mg/1 2,4-DBE, or 1 mg/l carbaryl.
Organisms studied include Chlorella sp. (isolate #1), Nitzschia sp.
(isolate #35), Monoraphidium sp. (isolate #11) and Anacystis nidulans.

In order to study uptake of one of these pesticides by algal
cells, 350 ml of medium containing the proper concentration of labeled
pesticide was prepared. In the case of methoxychlor this was stirred
for 24 hours to insure dissolution. A 350 ml aliquot of liquid algal
culture was then removed from a 10 liter continuous culture, and a
turbidimetric reading was made using a Klett~Summerson colorimeter
equipped with a red filter. Except in the case of Nitzschia cultures,
the cells from the 350 ml were then collected by centrifugation at
9750 x g in a Sorvall RC2-B refrigerated centrifuge, resuspended in 2.0
ml of medium and added to the 350 ml of stirring medium containing
labeled pesticide. Cells from the diatom cultures were collected on
5.0 » pore size Millipore filters (Millipore Corporation), and then
scraped off the filters. Diatom cells were then resuspended in 2.0 ml
of growth medium and added to the 350 ml of stirring medium containing
labeled pesticide. Medium and cells were stirred, aerated and iilumi-
nated under the same conditions as for the 10 liter continuous cultures.
Three 10 ml samples were then taken from the stirring culture at pre-
scribed times periods following the addition of cells. Samples were
collected on glass fiber filters (Reeve Angel 934AH, Arthur Thomas Com-
pany) to collect the cells (for Nitzschia and green algae) or ultra
fine glass fiber filters (Reeve Angel 984H, Arthur Thomas Company) for
Anacystis. Filters with cells were dried at room temperature for eight
hours and cut up into small pieces in polyethylene scintillation vials
(Primavials, Nuclear Associates) containing 10.0 ml of a scintillation
cocktail. The cocktail was prepared by mixing 500 ml of Triton X-100


point malathion, dissolved in acetone, was added axenically to give an
initial malathion concentration in the culture of 1 mg/l. Culture
conditions remained the same as before the malathion addition except
that aeration for all organisms was by air, rather than by 1% C02 in
air. A control was set up that was treated similarly, but included
medium and malathion and no algal cells. After addition of the mala-
thion, samples were taken immediately (time zero), after 1 hr, 4 hr,

8 hr, 12 hr, and then at 12 hr intervals until 144 hr. Ten-ml samples
were taken in triplicate and extracted as described earlier for quan-
titative determination of malathion by gas chromatography. Fifty-ml
samples were extracted by the following procedure: The 50 ml sample
of algal culture containing pesticide was placed in a 259 ml separa-
tory funnel and extracted twice with 100 ml chloroform (technical grade,
Fisher). Following the extractions sufficient anhydrous sodium sulfate
was added to remove water. After separating and washing the sodium
sulfate with an additional 10 ml of chloroform the chloroform extract
was evaporated to | ml using vacuum or a slow stream of air. The 1 ml
samples were then used to spot thin-layer chromatographic plates.

The extracts condensed to 1 ml were then co-chromatographed on
thin layer plates with the following standards: malathion, malaoxon,
malathion dicarboxylic acid, malathion monocarboxylic acid (beta-form),
potassium dimethyl dithiophosphate, potassium dimethylthiophosphate
and potassium-O0-desmethyl malathion. Silica gel 60 (E. Merck) plates
were equilibrated in a glass chamber and developed using hexane:acetic
acid:ether (75:15:10) as the solvent system (Kadoum, 1970). Following
development the plates were air-dried and then sprayed with 2,6-dibro-
mo-N-chloro-p-quinoneimine (Menn. et al., 1957). Following the spray~
ing, the plates were heated 10 min at 110°C and examined.

In several experiments using the Chlorella strain, at the time
malathion was added, the culture was placed in darkness where it was
kept until all samples were taken. In one experiment when the
Chlorella cell density reached 4 g/l the medium was separated from the
cells by continuous flow centrifugation and the experiment was con-
ducted under illumination but using only the supernatant, without
algal cells.


EPA-600/3-76-022
March 1976

DEGRADATION OF PESTICIDES BY ALGAE

by

Joseph C. O'Kelley and Temd R. Deason
Department of Biology
The University of Alabama
University (Tuscaloosa), Alabama 35486

Grant No. R800371

Project Officer

Doris F, Paris
Environmental Processes Branch
Environmental Research Laboratory
Athens, Georgia 30601

U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
ATHENS, GEORGIA 30601


Table 13, MALATHION DEGRADATION RATE CONSTANTS AND PESTICIDE
HALF-LIVES FOR ILLUMINATED CULTURES
(CALCULATED AS A PSEUDO-FIRST-ORDER PROCESS)

Organism Replicate k Ts, hr
aChiorella 1 0.454 1.54
(Isolate #1)

2 0.241 2.89
@anacystis 1 0.044 15.9
nidulans

2 0.064 10.8
Control 1 0.006 116.4
(no algae)

2 0.004 240.2

@ Cell concentration 4 g& wet weight/1 of cylture medium.

30


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18


RESEARCH REPORTING SERIES

Researcr ©
Protection
categories w

wersoment US Environmental
Wve series Tnese five broad
development and application of
environmenta' technology Eliminator. of treaitiona, grouping was consciously
planned tc foster tecnnolagy transter and a maximum interface i related fields
The five series are

Environmentai Health Effects Sesearch
Environmental Protection
togical Research

Environmental Monitoring

ne wR

Snaenaranamic Environmental S!

This report has Deen assianed to the ECOLOGICAL RESEARCH series. This series
describes researcn on the effects or pollution or numans, plant ane afitial

species a 1 Prob r fong- and short-term
influ nd pathway stud.es to
dote: tants and thor affante This mark nrowides the tachnical

pasis for setting sianaards to miunumze ot
in the aquatic. terrestrial and atmospheric environments

JS in tiving organisms

Tnis document is avaiable to the public through the Natonal Technical Informa-
tion Service. Springfield Virginia 22163


TECHNICAL REPORT DATA 5
(Please read Instructions on the reverse before completing)

7 REPORTING, 2.
EPA~600/3-76-022

3. RECIPIENT'S ACCESSION NO.

4, TITLE AND SUBTITLE

DEGRADATION OF PESTICIDES BY ALGAE

Is. REPORT DATE
March 1976 (Issuing Date)
6. PERFORMING ORGANIZATION CODE

7. AUTHORS) *
Joseph C. O'Kelley and Temd R. Deason

8. PERFORMING ORGANIZATION REPORT NO.

9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Biology
University of Alabama
University (Tuscaloosa), AL 35486

70, PROGRAM ELEMENT NO.

1BA023

71, CONTRACT/GRANT NO.
R800371

12, SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Athens, GA 30601

13, TYPE OF REPORT AND PERIOD COVERED
Final Report

14. SPONSORING AGENCY CODE

EPA-ORD

15, SUPPLEMENTARY NOTES

76, ABSTRACT

In this investigation interactions of 12 pesticides with 37 strains of
fresh water algae were studied in an effort to determine something of the
variability in responses of fresh water algae to the variety of pesticides in use
or projected to be used in the future.

Three interactions were investigated. One was the toxicity of the pesticides
to these algae. Another was the sorption of several of the pesticides by some of
the species of algae, The third was the possibility that some of the pesticides
can be degraded by action of algae,

In general it was found that sensitivity of algae to pesticides varied
greatly with the strains tested,

Sorption of methoxychlor appeared to be mainly physical, since much of the
methoxychlor sorbed was exchangeable, The butoxyethyl ester of 2,4-D (2,4-DBE)
was not sorbed to a significant extent by two green algae tested, and sorption
of carbaryl was very slow,

Malathion can be degraded by algae in the presence of light. One breaddown
proudct, malathion monoacid (beta form), appeared as the malathion was being
degraded, and later disappeared. Investigations of the fate of 2,4-DBE and
methoxychlor in algal cultures suggest that the fate of 2,4-DBE and methoxychlor

in algal cultures suggest that these pesticides may also be degraded: thy

n7. KEY WORDS AND DOCUMENT ANALYSIS .

ja. DESCRIPTORS b.IDENTIFIERS/OPEN ENDED TERMS |{c. COSATI Field/Group
Algae, Pesticides, Toxicity, Pesticide exchange O6F
Adsorption, Metabolism, Pesticide toxicity O6P
Biodeterioration Organic pesticides 06M
Biodegradation O6T

Hg. DISTRIBUTION STATEMENT 19. SECURITY CLASS (This Report) 21. NO. OF PAGES

Unclassified 48
Release to public 20. SECURITY CLASS (This page) 22. PRICE
Unclassified

EPA Form 2220-1 (9-73)
42 ‘zr U. S. GOVERNMENT PRINTING OFFICE: 1976-657-695/5384 Region No. 5-11


Table 10. EXCHANGE OF !4C-LABELED-2,4-DBE USING NITZSCHIA SP.
(ISOLATE #35)

h he
Uptake Time Exchange Time Exchanged Not exchanged

5 min 5 min 16 84
1 hr 4 96
6 hr 5 95
6 hr 5 min 450 450
1 hr 450 450
6 hr 450 450

4 The quantity of label present on cells after 6 hr expo-
sure to labeled pesticide was only 2 per cent of that
present after 5 min exposure; 50% values represent only
1% of that initially taken up by the Nitzschia cells.

26


the culture soon after the malathion has broken down. Degradation
rates, in the light, appear to be, on a per cell basis, roughly equiva-
lent to those reported for bacteria studied by Paris et al. (1975).

METHOXYCHLOR

There have been only limited prior studies of interactions of
algae and methoxychlor. Poorman (1973) reported that methoxychlor con-
centrations of 50 and 100 mg/1 reduced growth of Euglena gracilis, but
that concentrations of 1, 5 and 10 mg/1 were stimulatory. However,
fixation of 140, in other phytoplanktonic algae was reported to be
inhibited by 1 mg/1 methoxychlor (Butler, 1963; Ware and Roan, 1970).
Because the solubility of methoxychlor in water is considerably lower
than these values, experiments with the Warrior River isolates were
confined to those involving concentrations within the solubility range,
specifically 0.001 and 0.01 mg/1 methoxychlor. Methoxychlor in these
concentrations stimulates growth of some isolates and inhibits growth
of others.

Methoxychlor is reported to be biodegraded more rapidly than DDT
(Kapoor et al., 1970; Metcalf et al. 1971). Methoxychlor can also be
accumulated by bacteria from the medium to a concentration about 300
times the medium concentration (Johnson and Kennedy, 1973). Paris
et al.(1975) reported that Flavobacterium harrisonii degrades methoxy-
chlor to 2,2-bis (p-methoxypheny1)~1,1-dichloroethylene, or methoxychlor-
DDE. Mendel et al. (1967) report the same derivative when methoxychlor
is degraded by Aerobacter aerogenes. Pesticide-loss studies suggested
that most of the Warrior River isolates have some capacity to degrade
methoxychlor. Studies with labeled methoxychlor also indicated that
several isolates degrade methoxychlor, but the specific degradation
products have not been identified.

Methoxychlor sorption by Chlorella pyrenoidosa has previously been
reported (Paris and Lewis, 1973); under the conditions of the study,
equilibrium was reached within 30 min. The Warrior River isolates
tested (Chlorella sp., isolate #1 and Nitzschia sp., isolate #35), as
well as Anacystis nidulans, took up significant quantities of labeled
methoxychlor within 1 to 5 min, indicating physical adsorption. Fur-
thermore, most of this labeled methoxychlor (about 90%) taken up by
Chlorella sp. and by Anacystis nidulans was readily exchanged with non-
labeled methoxychlor, but only about half of the labeled methoxychlor
taken up by the Nitzschia isolate was exchangeable.

TOXAPHENE

Palmer and Maloney (1955) found that the effect of toxaphene, at a
concentration of 2 mg/l, upon 6 unicellular algae ranged from no effect

35


2,4-DBE

Results of the !4*C-2,4-DBE sorption experiments are shown in Table
7. Organisms utilized were Chlorella sp. (isolate #1), Nitzschia sp.
(isolate #35) and Anacystis n Of the three organisms tested
only the diatom, Nitzschia, took up appreciable quantities of this
formulation of 2,4-D. The rapid nature of this sorption indicated
that it was physical adsorption. The label was rapidly lost from the
cells after its initial sorption. The other two organisms possess
few or no binding sites for this form of 2,4-D; a very small, but detec-
table quantity was taken up by the Chlorella isolate and this was soon
lost. There was no detectable sorption of 2,4-DBE by Anacystis nidu-
lans.

Carbaryl

In contrast to !“C-methoxychlor and !*c-2,4-DBE, !"*C-carbaryl did
not appear to be taken up rapidly by any of the three organisms studied
(Chlorella sp., isolate #1; Nitzschia sp., isolate #35; Anacystis
nidulans) (Table 8). Sorption of the label by Nitzschia was approxi-
mately linear for the first three hours, after which time the quantity
of label held by the cells remained essentially unchanged for 24 hr.
In contrast, for Chlorella, there was no detectable sorption of label
for 3 hr and the maximum sorption rate was seen between 3 and 6 hr
after the !4c-carbaryl was introduced. With Anacystis there was no
detectable sorption at 12 hr after exposure of the cells to !4c-
carbaryl, but the cells accumulated some of the label after 12 hr.

EXCHANGE OF LABELED PESTICIDES

Results of the methoxychlor exchange experiments are shown in
Table 9. In Chlorella sp. (isolate #1) and in Anacystis nidulans after
5 minutes of methoxychlor sorption, about 90% will exchange with un-
labeled methoxychlor, indicating that 90% is adsorbed physically to the
cell surface; after 6 hr of methoxychlor sorption 90% can still be
exchanged. This indicates that little methoxychlor has been taken up
metabolically by the protoplasts of the cells. In the case of Mono-
raphidium (isolate #11) after 5 min. sorption, 80% will exchange
with non-radioactive methoxychlor. However, after 6 hr sorption only
55% will exchange; this indicates that 45% has been taken up by the
protoplasm or has been degraded. The uptake behavior of Nitzschia,
isolate #35, is more difficult to interpret. Even after a short sorp-
tion period of 5 min. much of the methoxychlor is not exchangeable.
Specifically, after 5 min. sorption, only 40% will exchange with non-
labeled pesticide offered the cells. The value after 6 hr uptake
decreases slightly, with only 35% exchanging.

21


ALGAL TOLERANCE OF PESTICIDES

All pesticide tolerance (algal growth in the presence of pesti-
cide) experiments were conducted using 18 x 150 mm test tubes capped
with plastic caps. The pesticides tested were aldrin, atrazine, cap-
tan, carbaryl, 2,4-DBE, dieldrin, diazinon, endrin, heptachlor, mala-
thion, methoxychlor and toxaphene.

A quantity of each pesticide was weighed out and dissolved either
in pesticide grade acetone or hexane (Burdick & Jackson, Inc.). Vary-
ing amounts of the pesticide/solvent mixtures were added to liter
quantities of sterilized FW-1 medium to give the desired concentration.
Since preliminary tests showed that the concentrated pesticides and
solvents did not contain bacterial contaminants, the pesticide/solvent
mixtures were not sterilized.

Following the addition of the pesticide to the medium, it was
stirred overnight to provide sufficient time for the pesticide to
dissolve and the solvent to evaporate. The concentration of the sol-
vent used was 0.5% or less, a concentration which preliminary experi-
ments showed did not affect algal growth. Using aseptic technique,
10 ml of pesticide-containing medium was pipetted into sterile test
tubes.

The algae to be used for inocula were transferred from 4-week-old
agar slants to 50 ml FW-1 medium in 125-ml Erlenmeyer flasks and the
flasks were then placed on a shaker in the growth chamber. Liquid
cultures 4-6 days old were used for inocula and all such cultures were
in the logarithmic phase of growth. Each test tube was inoculated
with a standardized inoculum and then placed in the growth chamber.
Cultures were neither aerated nor shaken. At the end of a 2-week
growth period the cultures were removed, agitated on a Vortex-Genie
mixer, and growth was measured turbidimetrically using a Klett-Summer-
son colorimeter equipped with a red filter.

PESTICIDE LOSS IN SMALL ALGAL ‘CULTURES

The cultures for the pesticide loss studies were prepared using
the procedures described for the pesticide tolerance experiments. The
initial concentration of pesticide in the cultures was 1 mg/1 for
atrazine, diazinon and malathion, and 0.01 mg/l for methoxychlor and
2,4-DBE.

At the end of the 2-week growth period the cultures were extrac—
ted along with control tubes containing pesticide and medium but no
algae and the extracts were analyzed for loss of pesticide.


(New England Nuclear), 917 ml of toluene (Eastman Chemicals) and 83

ml of Liquifluor (New England Nuclear). A standard quantity (0.4 g)

of Cabosil thixotropic gel (Beckman Instruments) was added to each vial
to suspend pieces of filters, and samples were counted for 10 minutes
each in a Packard Tri Carb Liquid Scintillation Counter.

Radioactivity counting standards were prepared as follows: Cells
from the 10 liter continuous culture at the same turbidity as experi-
mental cultures were collected on a glass fiber filter. The filter was
dried and cut up into a scintillation vial containing 10 ml of scintil-
lation cocktail, 0.4 grams of Cabosil and a standard quantity of
labeled pesticide.

By passing 10 ml of medium containing labeled pesticide but no
algae through glass fiber filters, the radioactivity held by the fil-
ters was determined. This value was subtracted from counting rates
obtained from each cell sample in order to obtain counts associated
only with the cells. By comparing the counts of each sample with
counts obtained with the standard, the total number of nanograms of
pesticide associated with the cells was determined. Finally, utiliz-
ing a cell turbidity reading (indicating cell density) all pesticide
uptake was expressed as nanograms of pesticide per gram (wet weight) of
algal cells.

In order to determine whether or not the labeled pesticide taken
up by the cells could be exchanged with unlabeled pesticide at various
time intervals after adding cells to the medium containing labeled
pesticide, some of these cells were re-collected and re-suspended in
10 volumes of medium containing the same concentration of unlabeled
pesticide. Samples were then taken at prescribed time intervals from
the suspension of labeled cells in medium containing unlabeled pesti-
cide and the amount of labeled pesticide still associated with the
cells was determined as before. This was expressed as nanograms per
gram (wet weight) of algal cells. The amount of labeled pesticide
that exchange with unlabeled pesticide was then calculated, and the
quantity of initial radioactivity in the cells that was lost by ex-
change was determined.

MALATHION DEGRADATION IN LARGE SCALE CULTURES

The organisms used in this study were Chlorella sp. (isolate #1)
Nitzschia sp. (isolate #35) and Anacystis nidulans.

These studies involved 4-liter cultures prepared as were the
10-liter cultures previously described in this report. Cultures were
utilized for an experiment when the cell density for Chlorella and
Anacystis reached 4 g/1 and for Nitzschia reached 0.9 g/l. At this