ABSTRACT
DDT inhibits photosynthesis in natural populations of
marine phytoplankton at levels as low as 10 ppb, and
reduces it to 5% of the normal rate at 1 ppm. The rate
of photosynthesis/unit chlorophyll A decreases with in-
creasing concentration of DDT, with a corresponding in¬
crease in the rate of respiration. In exponentially
growing cultures, the rate of chlorophyll A synthesis
decreases with increasing concentration of DDT. DDE,
the most common analog of DDT in the environment, in-
hibits photosynthesis at much lower concentrations, and
to a greater extent,,than DDT.
-1-
INTRODUCTION
As the concentration of DDT and other polychlori¬
nated hydrocarbons increases in the world ecosystem, the
fact that they are biologically harmful to many non-
target organisms is becoming more and more evident. Harm-
ful effects by these pesticides have been observed on
every trophic level, from man, down through the very
smallest phytoplankters.
DDT is a residual organic compound, readily dis-
solvable in lipid substances, and thus, becomes easily
concentrated in the fatty tissues of organisms. As a
result, it has reached lethal proportions at the highest
trophic levels. The pathway of DDT through the food
chain, and its accumulation at the top, is a major con-
tributing factor to its lethality. How DDT enters the
food chain is an important question, and examination of
the lower trophic levels may provide a clue, since the
marine phytoplankton form the base of many oceanic food
chains, and are known to concentrate DDT from the water
at levels equal to and greater than those of the envir-
onment. Not only is DDT concentrated by these organisms,
but it also exerts a harmful effect on their metabolic
systems. And this fact is of great importance in view
of the impact it might have on the entire oceanic food
supply.
Wurster (1968) found that DDT inhibits photosyn-
-2-
thesis in phytoplankton at concentrations as low as 8 ppb.
However, it is still not known by what mechanism DDT in-
fluences photosynthesis. There was some question as to
whether or not the actual photosynthetic process was being
harmed, or if general inhibition of the entire metabolic
system of the organism was occurring. Therefore, the
objectives of this investigation were: a) to evaluate to
what degree various concentrations of DDT would alter
the photosynthetic rate of a natural marine phytoplankton
community, and, b) to attempt to determine the mechanism
by which the photosynthetic process is altered by DDT.
METHODS AND MATERIALS
All phytoplankton samples were collected in Monterey
Bay, from a depth of fifteen meters, approximately z mile
off shore to the north of Hopkins Marine Station at buoy
no. 4. Two sampling procedures were used during the
study. The first involved taking whole water samples with
a five liter Van Dorn sampler (Van Dorn, 1956), and im-
mediately transferring the sea water to 300 ml. B.O.D.
bottles with a siphon apparatus. The B.O.D. bottles were
kept cool on ice and protected from direct sunlight by
wrapping them in two layers of tin foil, Bottles were
held under these conditions for an average of 1.5 hours be¬
fore subsequent utilization in various experiments conduc¬
ted ashore at the Hopkins Marine Station. The second
sampling procedure involved 20 minute horizontal tows with
a no. 25 phytoplankton net at depths of approximately 15
meters. These phytoplankton samples were also protected
from direct sunlight and kept in glass jars on ice ap-
proximately 1.5 hours before utilization in the laboratory.
The effects of DDT on photosynthetic rates were
measured using two different procedures. The first was the
light-dark bottle method as described by Gran (1927) and
modified by Strickland and Parsons (1968). This pro-
cedure utilized 300 ml. B.O.D. bottles of whole water sam-
ples. In the laboratory, they were inoculated with various
concentrations of DDT. A light bottle and a dark bottle,
wrapped four times with black electrical tape to exclude all
light, were used for each concentration. An initial B.O.D.
bottle was analyzed for O2 concentration to establish the
oxygen content of the water. The bottles were incubated
under flugrescent lighting for six hours. At the end of the
incubation time, they were analyzed for 0 production or
consumption by the Winkler method, as modified by Strickland
and Parsons (1968). The second procedure for photosynthetic
rate determination was the C-uptake method as described by
Strickland and Parsons (1968). Again whole water samples were
used, treated, and incubated in the same manner as those
in the O production method, with the exception that each
bottle received 50 of 5uc/ml radioactive NaC0. At the
end of the incubation period, the samples were killed
with 0.5 ml. 40% Formaldehyde per B.O.D. bottle, and fil-
tered on.45u millipore filters. The filters were then
folded, placed in scintillation viles, covered with Tol-
uene scintillation fluid, and counted on the scintillation
counter. All'controls were run with 95% ethanol, as the
DDT was suspended in that.
Cell density measurements were made with the Sedgwick-
rafter cell and a hemocytometer concurrently. Cells making
up long chains were counted individually. Six fields were
counted and then averaged.
Chlorophyll A concentrations were determined by a
fluorescence technique (Holm-Hansen et al, 1968) on 25 ml.
samples of phytoplankton filtered on.45u millipore filters.
The chlorophyll was extracted with 90% acetone, and diluted
36,000x before readings were made on the Florometer. If
readings could not be taken immediately, the filters were
wrapped in tin foil and frozen until extraction with acetone
could be done.
RESULTS
Experiment 1.
Experiment 1 was designed to broadly establish a photo-
synthetic inhibition curve of marine phytoplankton by DDT.
Whole water samples were collected in the manner described above
and incubated in DDT concentrations of 1, 3, 30, 300, and
1000 ppb. The results are shown in Table I, and the inhibi-
tion curve, as well as the respiration rates are shown on
Figure 1.
Experiment 2.
Based on the results of Experiment 1, three concentra¬
tions of DDT, 10, 50, and 100 ppb, were chosen to continue
the study. These were selected because they were relatively
low concentrations of DDT, yet produced clearly discernable
effects on the rate of photosynthesis. Sunsequent testing
confirmed the initial observation that these concentrations
were not lethal to phytoplankton cells. Figures 2 and 3 show
the % dead of cells exposed to these concentrations continu¬
ously over a 96 hour time period. Figure 2 shows the effects
on a pure, uni-alga culture of the dinoflagellate species,
Dunaliella; and Figure 3 shows the effects on a natural pop¬
ulation made up of primarily Nitzchia, Chaetocerous (2 species)
and four or five other diatomsspecies in lesser numbers.
The composition of the culture remained consistant through-
out the study, with only small variations from day to day.
Experiment 3.
Experiment 3 was conducted to establish the long term
effects of DDT on phytoplankton. Four five-liter erlenmeyer
flasks, filled with three liters each of an enriched sea water
broth, were inoculated with a concentrated natural popula-
tion sample to bring the cell density in each flask to ap-
proximately 2.2 x102 cells/ml. Each flask was then exposed
to constant light intensity of 60 footcandles, intermittant
mixing, and a constant temperature of 12°0. After a 24 hour
adjustment period, the flasks were injected with stock
solution DDT to bring their overall concentrations to 10, 50,
-6-
and 100 ppb DDT. The cultures were re-injected with DDT
every six hours. At time 0, 24, 48, and 72 hours, sub¬
samples were taken from each flask, and tested for cell den-
sity (Table II), rate of photosynthesis using the Cjl meth-
od (Table III), and the amount of chlorophyll A/25 ml.
(Table IV). The Cju method was the same as described above
with the exception that 125 ml. samples were used in reagent
bottles instead of 300 ml. samples in B.O.D. bottles.
Experiment 4.
Experiment 4 was designed to determine the effect of
DDT on the rate of chlorophyll A synthesis in exponentially
growing cultures of natural phytoplankton samples. Cultures
were kept in 500 ml. round bottomed flasks, in 300 mls. of
enriched filtered sea water broth. Florometer readings were
made every 12 hours for a 40 hour period on 25ml. sub-sam¬
ples. The results are summarized in Figure 5.
Experiment 5.
A final experiment was conducted to test the inhibitory
nature of DDE, the most common analog of DDT in the natural
environment. Three hundred ml. B.O.D. bottles were filled
with sea water, filtered through a.45u millipore filter, and
inoculated with 50 ml. samples of a concentrated natural
phytoplankton sample. The cultures were brought to 1, 10.
and 100 ppb concentrations of DDE, and innoculated with Ca)
(50 ), (5uc/ml. NaC03). Two controls were run, one with
DDT and one with ethanol. The bottles were incubated for
six hours, filtered and counted. The results are presented
in Figure 6.
DISCUSSION
Inhibition of photosynthesis in marine phytoplankton
communities becomes detectable at a concentration of 10
ppb, is reduced to 50% of the normal at 500 ppb., and at
1000 ppb. is reduced by 95%, as shown in Figure 1 and Table
I. Correspondingly, a very marked increase in the rate of
respiration occurs, the increase being greater at lower
concentrations than higher ones, being observable even at
a DDT concentration of 1 ppb. Figure 4 shows clearly that
the rate of photosynthesis/unit chlorophyll A also de-
creases over a period of lengthy exposure to DDT at various
concentrations. In exponentially growing cultures, the
synthesis of chlorophyll A is retarded, however, the degree
of retardation as a function of DDT concentration is not
clear as evident in Figure 5. DDE, the most common analog
of DDT in the environment, inhibits photosynthesis at a
much faster rate than DDT. Figure 6 shows that it has a
measurable effect at a concentration of 1 ppb, whereas DDT
does not, and its inhibitory capability seems almost three
times as great as that of DDT.
The results show that DDT does affect the actual photo¬
synthetic process rather than merely slowing the entire or¬
ganism down, as shown in Figures 1 & 4, and suggests that
the synthesis of chlorophyll A is also inhibited in some man¬
ner, as shown in Figure 5, in the presence of DDT. One ex¬
planation compatable with the above results, and with re-
sults obtained from other organisms, is that DDT could be
damaging the cell membrane systems. The fact that the rate
of respiration increases as the photosynthetic rate de-
creases, as clearly shown in Figure I and Table I, and
that the ability to photosynthesize of the chlorophyll A
also decreases, (Figure 4), in the cell suggest that the
cell membranes are being affected. If the external membrane
system of the cell is being damaged in any way, respiration
rates may increase to maintain osmotic balance with the
environment. At high concentrations of DDT, cells were found
to plasmolize, further indicating that the ability to
regulate osmotic balance might be damaged. If energy was
being utilized to maintain osmotic balance and/or repair
membranes (as indicated by the increased respiration rate), the
synthesis of chlorophyll A, along with any other energy requir¬
ing process, may be slowed down, as indicated in Figure 5.
Furthermore, the chloroplasts of the cell are also sur-
rounded by a relatively sensitive membrane, and if DDT were
damaging it, the ability to photosynthesize would be les-
soned, as my data indicates in Figure 4. It has been found
also that DDT inhibits Hill reaction activity in barley
chloroplasts (Lawler & Rogers, 1967), and this could happen
in phytoplankton chloroplasts as well.
Another interesting phenomena is that when cells were
exposed to a given concentration of DDT once, and then their
photosynthetic rate measured over a long period of time, they
regained the ability to photosynthesize normally after ap-
proximately 20 hours. Possibly DDT could be blocking or
clogging the cell membranes, but not permanently damaging
them, or possibly the cell is able to effectively repair the
DDT damaged membranes at the expense of stored food reserves.
The fact that DDE has a greater inhibitory ability
than DDT is very significant. It has been demonstrated that
DDT is reduced to DDE by ultra-violet rays. Therefore, due
to sunlight alone, most of the DDT residues in the environ-
ment may be in the form of DDE. The fact that DDE is a more
potent substance could have dangerous biological reper¬
cussions in the environment.
CONCLUSION
DDT does affect the photosynthetic process of marine
phytoplankton, but the actual mechanism by which damage is
incurred remains unknown. It is known that respiration
increases, the synthesis of chlorophyll A decreases, and at
high concentrations of DDT, cells plasmolize. Further studies
on this subject are needed. Possible approaches to the
problem might involve using metabolic poisons with known
inhibition abilities, and comparing results of these to
those gained from DDT. Investigation of the phytoplankton
ability to regain photosynthetic prowess could reveal pos-
sible inhibition mechanisms. Long term respiration studies
might also shed some light on the subject.
The fact remains though, that much work is needed. The
C
-10.
phytoplankton in the oceans are a major food source in
our environment. If their ability to produce food is being
seriously inhibited by chemical pesticided, such as DDT and
DDE, which are being constantly dumped into the ocean from
river run-off, rains and winds, the result could prove
disasterous for many, if not all, biological communities.
BIBLIOGRAPHY
1. Gran,H.H., Rept. Norwegian Fishery & Marine Invest.,
3(8):1-74, (1927).
2. Lawler,P.D. and L.J. Rogers, Nature 215, 1515 (1967).
3. Riseborough, R.W., "Chlorinated Hydrocarbons in Marine
Ecosystems", Rochester Conf. on Toxicity; June,
1968, Rochester University.
4. Strickland, J.D.H., and Parsons, A Practical Handbook of
Sea Water Analysis; Fis. Res. Bd. of Canada; Ottawa
1968.
5. Fogg,G.E.; "Algal Cultures and Phytoplankton Ecology"
Univ. of Wisconsin Press, 1965.
6. Pringsheim,E.G.; "Pure Cultures of Algae, Their Prepar-
ation and Maintainance". Univ. of Cambridge Press,
1949
7. Strickland; "Measuring the Productivity of Marine
Phytoplankton". Fis. Res. Bd. of Canada; Ottawa, 1960.
8. Wurster, Charles f, "DDT Reduces Photosynthesis by
Marine Phytoplankton"; Science, 159(3822), 1474-1475,
Illus. 1968.
AMOUNT
DDT:
CONTROL
1 ppb
3 ppb
30 ppb
300 ppb
1000 ppb
TRIAL:
TABLE I.
Accomparison of the amount of 0 produced or
consumed by whole water samples incubated in
300 ml. B.O.D. bottles for six hours, at var-
ious concentrations of DDT. The second num¬
ber in each box is the% of that amount of 02
to the control in each respective trial.
LIGHT BOTTLE: PHOTOSYN.
DARK BOTTLE: RESPIRAT'N
mg-/Liter % of CONTROL
mg-Og/Liter
% of CONT.
+2
7073
700
705
127,
065
02,
178,
/1
1100.
1100.
1100
00
1100.
/
00.
702
21
1112
181
129
013,
063
139/
112
110
20
12.
o
703
7
102
135
022
00
Ab
10
125
17
11
1113
17
103,
/.


/220
26
52
111
07
1
103
158)
tb
1
910
1930
810

1152


1
111/
010
3/
08/
128,
S33)
6

2
113
/720
1122
015
158
35
/
0/
08,
025/
0287

Ob
/ 123
1165

130
1250 20
112
125
1
1
1 3 4
2
19
SAMPLE:
CONTROL
10 ppb
50 ppb
100 ppb
TABLE II. A comparison of the of cells alive in the
natural population phytoplankton cultures,
incubated at various DDT concentrations, for
each given amount of time. Cells in long
chains were counted individually. The en-
tire population was counted with a Sedgwick-
rafter cell and a hemocytometer.
TIME IN HOURS
24
48
72
2.2 x10
1.9 x10
1.4 x10
1.3 x10
cells/ml
cells/ml
cells/ml
cells/ml
0.8 x10
1.0 x105
2.1 x10
1.9 x10
cells/ml
cells/ml
cells/ml
cells/ml
—
1.5 x10
2.2 x10
2.3 x10
1.0 x10
cells/ml
cells/ml
cells/ml
cells/ml
1.4 x10
2.2 x105
1.0 x10
0.6 x10°
cells/ml
cells/ml
cells/ml
cells/ml
TABLE IV. A comparison of the amount of chlorophyll A found
in 25 ml. aliquots of the natural population phy-
toplankton cultures incubated in various concen-
trations of DDT, at given points in time. Chloro-
phyll measurements were made with the Florometer.
TIME IN HOURS
48
72
24
CONCENTRATION:
SAMPLE
u-gram CHLOROPHYLL. A/25 m1.
2.02 x10
2.02 x10
1.43 x10
CONTROL
1.50 x10
1.50 x10
1.50 x10
10 ppb DDT
1.42 x10
2.34 x10
1.17 x10
50 ppb DDT
1.59 x10
1.70 x10
1.04 x105
100 ppb Dor
C
TABLE III. A comparison of the rate of photosynthesis of
the natural population phytoplankton cultures
incubated in various concentrations of DDT for
giveh lengths of time, as measured by the up-
take of radioactive Naco, (specific activ-
ity: 5uc/ml.), The uptake was over a period
of six hours.
ATTMEIN HOURS
48
24
72
0.
CONCENTRATION:
RADIOACTIVITY ABSORBED
AMIOUNT
3491
3013
1560
3840
CONTROL
DPM
DPM
DPM
DPM
5129
3698
1523
705
DPM
DPM
10 ppb DDT
DPM
DPM
2859
1551
2520
1325
DPM
50 ppb DDT
DPM
DPM
DPM
3583
3546
1043
1530
DPM
100 ppb DDT
DPM
DPM
DPM
TABLE V.
CONCENTRATION
CONTROL
10 ppb DDT
50 ppb DDT
100 ppb DDT
A comparison of the rate of photosynthesis / unit
chlorophyll A of the natural population phytoplan-
kton cultures, incubated at various concentrations
of DDT, for given lengths of time, as measured by
the uptake of radioactive NaCO3 in six hours time.
(Specific activity: 5uc/ml.).
TIME IN HOURS
72
48
24
DPM'S per UNIT CHLOROPHYLL A% OF NORMAL
1.680 x
1.490 3
2.375 x
-2
-2
-2
10
10
762.9
100
170.8
1
2.460x
1.015 X)
3.419 x
-2
-2
10 7100
10 772.0
129.7
927 x
2.160 x
1.220 x
2
10
-2
10
56.5
100

43.0
655 X
2.095 X
3.455 %
-2
-2
10
10
100
60.5
1.9
12
C
Fig. 1. A comparison between the photosynthetic
rate and the respiration rate of whole
water samples in the presence of DDT.
O
+++
8



++
++
++

+

++++++

+++++
+++
++++
+++++
+


++
L
+


+
+

+


35 100 150 20 958 5 380 10 70 g00 30 1o0 410 7o 750 0 886 700.

RAI
ONCE
D

S6
+

+
H

+++
++++
++++
++
+
+
E
++


++
000
++
I
+++
C
C
Fig. 2. A comparison of the % cells of Dunaliella
alive in various concentrations of DDT,
after given amounts of time.
O
H
RE
+
+
++++++
100
+++++++++
+

++
++


75
+++
+++
++


++
+++
++
10
+

++
11

A
+++++++
+++
+
2
+

H
+

—

tt
++++


++++++


CONTROE
JOPPE DDT
--
m 5Opp» DDT
DOE
— 100
+
++++++++
+
—
ug

+—
U
E
+
O
Fig. 3.
A comparison of the  of cells of natural
phytoplankton samples, alive after exposure
to various concentrations of DDT, after
a given amount of time.
+
++
+++
1
+

++



O
+
L

+
e

+
L
+
1
5)
t
++

crp
+
DOT
—


enn
51
—
2-
18

+
+
HOURS
+++
++

+
90
+++
+
++
+
+++

1
+
++
++

++++L

+++
H
+++
+++
+++++

+
+
C
0
Fig. 4. A comparison of photosynthetic rates /
unit chlorophyll A, of natural population
phytoplankton samples, exposed to various
concentrations of DDT, after given a¬
mounts of time
O
2
++
00
307
70
10

1504



40



30


++++
0
20
++
O
+
öt
+
1-
++++++
+++
++
++
+0
+

+
+++
++
+
++
+++++

++++

++++
24
X
++++
+++
—Co
++++++
++1
DDI
—-—- pob
+
un
+
+++++
—— DD
+

+


—
18

EHE
Houre

+
+++
+++


++
+
+++
+26
++++
+

+
8
O
O
Fig. 5. The relative amounts of chlorophyll A /
unit cell of natural population phyto-
plankton samples, incubated in various
concentrations of DDT, for a 96 hour
time period.
O

IL
Q
+

I6UR
19.
1
+
++
++
112

S

10


+


++
++++
+

++
C
++
+++
+

+
++
1
++++
++++++
H

+

+1
+
+++
+
+

++

++
+
+++

+

+
++
+
—
+++
+

+++

++++++
++++++
+



COTRO
PE DDT
——
50ppb DDT
pp D
+
72
2S



t
t
+
—
++++

++
++
48



+++

++
+++
++
+
+

+++
+

+


+++

—
+
O
ig. 6. A comparison of the effect of DDT and its
analog, DDE, on the rate of photosynthesis
on natural population phytoplankton sam-
ples.
++
+
+++
++

+
+++
++++
+++
++
+

+++++
At
+
++++++++
++++++
++++
++

++
++
++
20
a

S
++++

90-
++++++

++
a
++++++++
1+

+
++

+
D
O
0

10+
—CONTEOL
++++
—--


20
++E
—
+
DDE
+


p
H ESTICIDELOC
+++++++++
++
+++

pot
+++++++
LL


++++


++++
+++++
+++
+
++4

+
8
+++++

+

++
+++
0

+
++
+++
100
NTON


—
++
+
+
++
++
++
H
++
++
+
+++