Ecological Research Series
EFFECTS OF SELECTED WASTEWATER
CHLORINATION PRODUCTS AND
CAPTAN ON MARINE ALGAE
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32581
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
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This report has been assigned to the ECOLOGICAL RESEARCH series. This series
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tion Service, Springfield, Virginia 22161.
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EFFECTS OF SELECTED WASTEWATER CHLORINATION
PRODUCTS AND CAPTAN ON MARINE ALGAE
by
Harish C. Sikka and Gary L. Butler
Syracuse Research Corporation
Syracuse, New York 13210
Grant No. R803943010
Project Officer
Gerald E. Walsh
Environmental Research Laboratory
Gulf Breeze, Florida 32561
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory,
Gulf Breeze, 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 endorse-
ment or recommendation for use.
ii
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FORWARD
Organochlorine compounds such as pesticides have been known to be pollu-
tants in water for many years. Biological effects of similar compounds that
are formed during chlorination of waste water are poorly known. The .Environ-
mental Research Laboratory, Gulf Breeze, has long supported research on ef-
fects of organochlorine compounds on living things. Data from that research
has been used for setting water quality standards in estuaries.
The research reported here demonstrates effects of organochlorine com-
pounds formed during the waste water treatment process, and the fungicide
captan, on growth of marine unicellular algae. Synergistic effects of some
of the compounds demonstrates, in a simple way, the complex nature of pollu-
tant interactions and their effects on algal productivity.
Gerald E. Walsh
Project Officer
Environmental Research Laboratory
Gulf Breeze, Florida
iii
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ABSTRACT
The investigation was undertaken with the overall objective of determin-
ing for marine phytoplankton the effects of both certain stable chloro-organic
compounds produced during chlorination of sewage effluents and the pesticide
captan.
3-Chlorobenzoic acid (1 or 10 ppm) had either slight or no effect on
growth of Dunaliella or Porphyridium. It inhibited growth of Skeletonema at
10 ppm, but had no marked effect at 1 ppm.
5-Chlorouracil at 1 or 10 ppm did not affect Skeletonema, but stimulated
growth of Dunaliella initially.
4-Chlororesorcinol had no effect on Dunaliella at 1 ppm, but 10 ppm of
the chemical caused a small decrease in growth. The chemical produced an
initial stimulation in growth of Forphyridium, followed by an inhibition.
Growth of Skeletonema was inhibited by 4-chlororesorcinol at concentrations
ranging from 1 to 10 ppm.
Treatment with 3-chlorophenol stimulated growth of Dunaliella.
Skeletonema growth was inhibited at concentrations higher than 2.5 ppm, but
showed some stimulation at 1 ppm. At 1 ppm it stimulated growth of
Porphyridium, but was slightly inhibitory at 5 ppm.
A combination of 3-chlorophenol and 4-chlororesorcinol interacted
synergistically to reduce Skeletonema growth.
Captan suppressed growth of Dunaliella and Porphyridium at a concen-
tration of 5 ppm. Slight stimulation in growth of the two organisms was
noticed in the presence of 0.1 and 1 ppm of the fungicide. Captan was
inhibitory to Skeletonema at concentrations ranging from 0.25 to 5 ppm.
Treatment of Skeletonema with 0.5 ppm of captan for 30 minutes caused a
substantial reduction in photosynthetic C02 fixation.
This report was submitted in fulfillment of Grant No. R803943010 by the
Syracuse Research Corporation under the sponsorship of the U.S. Environmental
Protection Agency. This report covers the period July 15, 1975 to July 14,
1976, and work was completed as of July 31, 1976.
iv
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CONTENTS
Foreword ill
Abstract iv
Figures vi
EFFECT OF SELECTED WASTEWATER CHLORINATION
PRODUCTS ON MARINE ALGAE
1. Introduction 1
2. Conclusions 3
3. Recommendations 4
4. Materials and Methods ..... 5
5. Results 7
6. Discussion 12
References 14
EFFECT OF CAPTAN ON MARINE ALGAE
1. Introduction 27
2. Conclusions and Recommendations 28
3. Materials and Methods _. 29
4. Results . . . 30
5. Discussion 32
References 33
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FIGURES
No. Page
1. The effect of 3-chlorobenzoic acid on the growth of
Dunaliella 15
2. The effect of 3-chlorobenzoic acid on the growth of
Porphyridium 16
3. The effect of 3-chlorobenzoic acid on the growth of
Skeletonema 17
4. The effect of 5-chlorouracil on the growth of Dunaliella
and Skeletonema 18
5. The effect of 4-chlororesorcinol on the growth of
Dunaliella and Skeletonema 19
6. The effect of 4-chlororesorcinol on the growth of
Skeletonema 20
7. The effect of 4-chlororesorcinol on the growth of
Porphyridium 21
8. The effect of 3-chlorophenol on the growth of Dunaliella ... 22
9. The effect of 3-chlorophenol on the growth of Skeletonema . . 23
10. The effect of 3-chlorophenol on the growth of Skeletonema . . 24
11. The effect of 3-chlorophenol on the growth of Porphyridium . . 25
12. The effect of 3-chlorophenol and 4-chlororesorcinol singly and
in combination on the growth of Skeletonema 26
13. The effect of captan on the growth of Dunaliella 34
14. The effect of captan on the growth of Porphyridium 35
15. The effect of captan on the growth of Skeletonema 36
16. The effect of captan on the growth of Skeletonema 37
vi
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EFFECT OF SELECTED WASTEWATER CHLORINATION
PRODUCTS ON MARINE ALGAE
SECTION 1
INTRODUCTION
Chlorination is a widely used practice for disinfecting municipal waste-
waters or combined municipal-industrial wastes before they are discharged
into receiving waters. The quantity of chlorine used for sewage treatment
is expected to increase as municipalities not presently treating their sewage
are required to comply with the treatment standards mandated by the Federal
Water Pollution Control Act by July 1, 1977 (U.S. Government Public Law,
92-500; 1972). Additionally, an increasing number of industries have been
required to provide waste-water treatment facilities which often include
effluent chlorination.
Some organic compounds may escape secondary treatment or be only par-
tially degraded and as a result may react with chlorine, yielding persistent,
potentially toxic organic compounds. Gla2e et al. (1973) and Jolley (1974,
1975) have shown that chlorine-containing organic compounds are produced
when sewage effluents are chlorinated. Jolley (1975) identified seventeen
stable, chlorine-containing organic compounds in domestic waste-water effluent
which had been chlorinated to a 1 to 2 mg/£ chlorine residual. Some of these
compounds included chlorinated phenols, aromatic acids, purines, and pyrimi-
dines. Barnhart and Campbell (1972) studied the reaction of chlorine with
organic chemicals which are known to be present in industrial waste-water
effluents. They observed that chlorine reacted readily with phenol, m-cresol,
and aniline under conventional effluent treatment conditions. Based on the
assumption that 90,700 metric tons of chlorine are used annually in the U.S.
for disinfecting waste-water, Jolley (1975) estimated that approximately
4,535 metric tons of stable chlorine-containing organic compounds would be
released to the receiving water.
The presence of chloro groups is known to render benzenoid compounds
more resistant to microbiological degradation (Alexander and Lustigman, 1966),
which suggests a potential for accumulation of these compounds in receiving
waters. The introduction of chlorinated organics into the aquatic environ-
ment is of great environmental concern because of their potential toxicity
to various organisms. In order to fully evaluate the impact of waste-water
chlorination on the aquatic environment, it is necessary that we know effects
and fate of chlorination products in the biota. The toxicity of residual
chlorine on aquatic life has been extensively investigated and was recently
reviewed by Brungs (1973). However, very little information is available on
effects of stable chlorine-containing organic compounds that may have been
produced during the chlorination process. Gehrs et al. (1974) reported
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that 5-chlorouracil and 4-chlororesorcinol, which are among the constituents
of chlorinated effluents, decreased the hatchability of carp eggs at con-
centrations as low as 1 ppb.
Phytoplankton constitute a vital part of the aquatic biota. They con-
tribute to the oxygen supply in the aquatic environment through their photo-
synthetic activity. An adverse effect on growth of phytoplankton, which
represent the first link in the aquatic food chain, may have adverse effects
on the entire ecosystem. Algae may also play an important role in determin-
ing the fate of chloro-organics in the aquatic ecosystem. They may remove
the chemicals from the environment by adsorption and/or absorption and may
subsequently metabolize them. The metabolites may be more toxic and/or more
persistent than the parent chemical and so may present additional pollution
problems. Chemicals which are accumulated by algae may be transferred to
higher trophic levels with potentially deleterious effects on man.
The present study was undertaken to investigate in marine phytoplankton
the effects of selected stable organic compounds produced during chlorination
of sewage effluents. The results of this study will provide information
needed for establishing guidelines for the discharge of chlorine-treated
waste-waters into the aquatic ecosystems.
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SECTION 2
CONCLUSIONS
Our studies have shown that certain chloro-organic compounds produced
during chlorination of sewage effluents such as 3>-chlorophenol, 3-chloro-
benzoic acid, 4-ehlororesorcinbl and 5-chlorouracil affect the growth of
Dunaliella tertiblecta, Skeletonema costatum. and Porphyridium SJL. at
concentrations considerably higher than those expected to be found in the
environment. A combination of 3-chlorophenol and 4-chlororesorcinol
interacted synergistically to inhibit the growth of Skeletonema. As our
results are based on the effects on a limited number of species, it is not
possible to make a general statement concerning the toxicity of these
chemicals to phytoplankton.
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SECTION 3
RECOMMENDATIONS
1. To fully assess the impact of chloro-organic compounds on phytoplankton,
we suggest that the effect of these chemicals on additional algal
species be examined.
2. It is likely that algae in the bodies of water receiving chlorinated,
waste-waters will be exposed to several chloro-organic chemicals
simultaneously. As a result there may be synergistic or antagonistic
effects produced by the interaction of these chemicals. Therefore,
it is recommended that the effects of chloro-organic compounds be
studied in the presence of other similar compounds.
3. It is quite likely that the chloro-organic compounds may be accumulated
by phytoplankton. An accumulation of these chemicals by phytoplankton
is of ecological significance because the chemicals can be trans-
ferred to higher trophic levels. We suggest that the uptake and
metabolism of these chemicals by phytoplankton be examined.
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SECTION 4
MATERIALS AND METHODS
CHEMICALS
The chemicals tested for toxicity to phytoplankton included 3-chloro-
phenol, 3-chlorobenzoic acid, 4-chlororesorcinol and 5-chlorouracil. They
are among the several stable chlorine-containing organic compounds which
Jolley (1975) identified in chlorinated sewage effluents. These chemicals
were selected because their concentrations in chlorinated sewage effluents
were relatively high (1-5 yg/£) compared to those of other chlorine-
containing compounds and because nothing was known about their toxicity
to phytoplankton. 3-Chlorobenzoic acid (99+%), 3-chlorophenol and 4-chloro-
resorcinol (99%) were purchased from Aldrich Chemical Company, and 5-chloro-
uracil (A-grade) from Calbiochem.
CULTURING OF ORGANISMS
The three species representing different algal divisions used in this
study were Skeletonema costatum (Bacillariophyta), Dunaliella tertiolecta
(Chlorophyta), and Porphyridium sp. (Rhodophyta). They were obtained from
the University of Rhode Island, Narragansett Marine Laboratory. The
cultures were bacteria-free and grown axenically on Modified Burkholder's
artificial seawater medium recommended by the Environmental Research
Laboratory, EPA, Corvallis, Oregon (1974). The medium was supplemented
with the following: 16.48 mg N/l as NaN03, 4.45 rag P/l as K2HPO(t, 4.94 mg
Si/1 as Na2Si03.9H20, 1 yg/1 vitamin Bi2, 1 Pg/1 biotin, 0.2 mg/1 thiamin-HCl
and 1 ml/1 NAAM trace metal mix (0.1856 g H3B03; 0.416 g MnCl2.4H20; 0.032 g
ZnCl2; 1.428 mg COC12.6H20; 0.0214 mg CuCl2.2H20; 7.26 mg Na^oO^.2^0 per
liter). The salinity was adjusted to 30 ppt and the pH to 7.2 After
filtration through a 0.45 urn membrane filter, 1 mlA sterilized Fe-EDTA
solution (33.05 ug Fe/ml as Feds + 300 ug/ml Na2 ,EDTA) was added aseptically
The cultures were grown on a reciprocating shaker under controlled environ-
mental conditions, 16 hours of light per day at 4304 lux and a temperature
of 22° + 1°C.
GROWTH STUDIES
Exponentially growing cells were inoculated into 100 ml of sterile
medium to give an initial density of 101* cells per ml. Stock solutions of
the chemicals were prepared in acetone. The chemicals in acetone solution
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were added to the medium so that the final concentration of carrier solvent
was 0.01%. The control consisted of growth medium containing 0.01% acetone.
Each treatment was replicated 5-6 times. Aliquots of cell suspension were
removed at various intervals following treatment and growth of algae was
determined by measuring absorbance at 650 nm in a Gary spectrophotometer.
The results were analyzed statistically by using the paired difference test
(Mendenhall, 1975).
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SECTION 5
RESULTS
In our initial studies on chloro-organic chemicals and phytoplankton,
the organisms were cultured in aged, synthetic seawater medium (Rila Products)
supplemented with nutrients recommended by Guillard and Ryther (1962).
Growth of untreated algae varied considerably in these experiments. In an
attempt to achieve reproducibility in growth rates, we cultured the algae
in a medium of better-defined composition than that prepared from the Rila
Marine Mix. For this purpose, the artificial seawater-medium recommended
by the Environmental Research Laboratory, EP, Corvallis, Oregon, was used in
subsequent studies.
EFFECT OF THE TEST CHEMICALS ON PHYTOPLANKTON GROWTH
The results concerning effects of 3-chlorobenzoic acid, 5-chlorouracil,
4-chlororesorcinol and 3-chlorophenol on growth of Dunaliella, Skeletonema,
and Porphyridium are reported below. Inhibition or stimulation of growth,
when reported, is statistically significant at the 95% confidence level.
3-Chlorobenzoic Acid
The chemical at a concentration of 1 ppm produced no effect on growth
of Dunaliella or Porphyridium (Figures 1 and 2). It did not affect
Skeletonema within the first week of treatment, but inhibited its growth
by 10-12% during the second week (Figure 3). At a concentration of 10 ppm,
Porphyridium was not affected; however, the growth of Dunaliella was inhib-
ited 4-12% during the first five days following treatment. Subsequently,
the Dunaliella cells treated with 10 ppm of 3-chlorobenzoic acid showed the
same growth as the control.
5-Chlorouracil
Figure 4 shows the effect of 1 and 10 ppm of 5-chlorouracil on the
growth of Skeletonema and Dunaliella. The chemical, at concentrations of
1 and 10 ppm, did not significantly affect the growth of Skeletonema until
7 days following treatment. However, at both concentrations a significant
stimulation of growth (3-9%) was noticed on days 10 through 14. 5-Chloro-
uracil, at concentrations of 1 and 10 ppm, stimulated the growth of Dunaliella
through day 10; subsequently, the stimulatory effect was eliminated and after
14 days the treated cultures showed the same growth as the control.
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4-Chlororesorcinol
This chemical, at the concentration of 1 ppm, did not inhibit growth of
Dunaliella (Figure 5). In the cultures treated with 10 ppm of 4-chloro-
resorcinol, a 13% reduction in growth was observed on day 3; however, after
day 6, the treated cultures showed the same growth as the controls. Chloro-
resorcinol was more toxic to Skeletonema. Figures 5 and 6 show the effect
of 4-chlororesorcinol on Skeletonema at concentrations ranging from 1 to
10 ppm. Growth decreased with an increase in concentration of the chemical.
4-Chlororesorcinol at concentrations of 1.0, 2.5, 5.0, 7.5, and 10 ppm
reduced growth of Skeletonema by 22, 39, 62, 72, and 100%, respectively after
3 days of treatment. Subsequently, the cells in all treatments except 10 ppm
recovered from the effect of the chemical so that growth proceeded at almost
the same rate as those in the controls when the study was terminated. The
growth of Skeletonema was completely suppressed in the presence of 10 ppm
of 4-chlororesorcinol.
Figure 7 shows the effect of chlororesorcinol on growth of Porphyridium.
A stimulation (38-33%) in the growth of the organism was noticed on days 3
and 4 after treatment with 1 ppm of the chemical, followed by a small
inhibition (5-14%) on days 6 through 14. In the cultures treated with
10 ppm of chlororesorcinol, an increase (18-34%) in growth was noticed on
days 3 and 4. This initial stimulation was followed by an inhibition in
growth (6-21%) after day 6.
3-Chlorophenol
A significant stimulation (8-18%) in the growth of Dunaliella was
noticed on day 4 through day 14 after treatment with 1 ppm of 3-chlorophenol
(Figure 8). Treatment with 10 ppm of the chemical also stimulated growth,
but the maximum increase in growth was only 12%.
Growth of Skeletonema was significantly increased (9-13%) on day 4
through day 6 following treatment with 1 ppm of 3-chlorophenol (Figure 9);
however, after day 6, growth in the treated cultures was the same as in the
controls. The concentration of 10 ppm completely suppressed growth of
Skeletonema. At concentrations ranging from 2.5 to 7.5 ppm, growth of
Skeletonema decreased with increase in concentration (Figure 10). At con-
centrations of 2.5, 5.0, and 7.5 ppm the chemical caused decrease in growth
of the organism by 20, 82, and 97%, respectively, following 7 days of
treatment.
Figure 11 shows the effect of 3-chorophenol on growth 'of Porphyridium.
It did not significantly affect growth of the alga on the concentration of
0.1 ppm. The cultures treated with 1 ppm of the chemical showed a 12%
increase in growth after one week as compared to the control. However, a
slight inhibition in growth (5%) was observed in the cells exposed to
5 ppm of chlorophenol.
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INTERACTION OF 3-CHLOROPHENOL AND 4-CHLORORESORCINOL
It is quite likely that the bodies of water receiving chlorinated waste-
waters, phytoplankton may be exposed to several chloro-organic chemicals
simultaneously. As a result, there may be synergistic or antagonistic
effects produced by their interaction. We observed that 3-chlorophenol and
4-chlororesorcinol are toxic to Skeletonema individually. In order to
ascertain if the two chemicals interacted, we examined their effect in
combination with one another on growth of alga.
At a concentration of 2.5 ppm, 3-chlorophenol, and 4-chlororesorcinol
reduced growth of Skeletonema by 26 and 17%, respectively, on day 3 after
treatment; by day 10, inhibition had decreased to 5 and 8%, respectively.
When algae were exposed to a combination of 2.5 ppm each of 3-chlorophenol
and 4-chlororesorcinol, the cells showed a time lag in growth until the
7th day after treatment, but growth subsequently proceeded at almost the
same rate as those in the control (Figure 12). In cultures treated with 5 ppm
of either of the chemicals alone, a lag in cell growth was noticed until
day 7 following treatment, but afterwards the growth proceeded at a rate
close to that of the control. However, treatment with a combination of
5 ppm of each of the two chemicals completely inhibited growth, and the
cells failed to recover for the duration of the experiment. These findings
indicate an interaction between 3-chlorophenol and 4-chlororesorcinol in
inhibiting the growth of Skeletonema.
The following formula described by Colby (1967) was used to ascertain
whether the effect produced by a combination of the two chemicals was
synergistic or additive: E = XY/100; where
E = expected growth as a percentage of control with the test
chemicals
X = growth as a percentage of control with chemical A
Y = growth as a percentage of control with chemical B
When the observed value is less than the expected value, the combination is
synergistic. When the observed and expected values are equal, the effect
is additive. If the observed value is greater than the expected value,
antagonism is indicated. When Skeletonema was exposed to a combination of
3-chlorophenol and 4-chlororesorcinol, the growth-was reduced below the
expected value indicating that the two chemicals interacted synergistically
(Table 1).
COMPARATIVE EFFECTS OF CHLORINATED AND NON-CHLORINATED ORGANIC COMPOUNDS
ON THE GROWTH OF PHYTOPLANKTON
A wide variety of organic chemicals including phenolic compounds are
present in municipal water supplies and waste-water treatment plant effluents.
In order to determine if the chlorination of organic compounds present in
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waste waters alters their toxicity to phytoplankton, we compared the effects
of 3-chlorophenol and 4-chlororesorcinol, which were found to be toxic in
our studies, with their non-chlorinated analogs, i.e., phenol and resorcinol.
Treatment of Skeletonema with 5 ppm of phenol or resorcinol caused a small
inhibition (less than 5%) over a two-week period. In contrast, the growth
of the organism was reduced by 83 and 77% after 3 days of treatment with —
3-chlorophenol and 4-chlororesorcinol, respectively, which suggests that
chlorine-containing compounds present in sewage effluents may be more
toxic to phytoplankton than parent compounds that would be present in the
effluent prior to chlorination.
10
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TABLE 1. EFFECT OF 3-CHLOROPHENOL AND 4-CHLORORESORCINOL AND
THE COMBINATION OF 3-CHLOROPHENOL AND 4-CHLORORESORCINOL
ON GROWTH OF SKELETONEMA
Chemical
3-chlorophenol
"
"
3-chlorophenol
"
11
4-chlororesorc inol
"
"
4-chlororesorcinol
"
"
3-chlorophenol
4-chlororesorc inol
3-chlorophenol
4-chlororesorcinol
2 . 5 ppm
2.5 ppm
2 . 5 ppm
5 . 0 ppm
5.0 ppm
5 . 0 ppm
2.5 ppm
2 . 5 ppm
2.5 ppm
5.0 ppm
5.0 ppm
5.0 ppm
2.5 ppm
2.5 ppm
5 . 0 ppm
5.0 ppm
Days after
Treatment
3
7
14
3
7
14
3
7
14
3
7
14
3
7
14
14
Growth
% of
74
85
96
14
9
58
83
81
94
22
10
51
0
0
75
0
Response
Control3
(61)
(69)
(90)
(29)
Expected responses for combinations are shown in parentheses following
each observed response.
11
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SECTION 6
DISCUSSION
Our findings have shown that certain chloro-organic compounds present
in effluents of sewage treatment plants affect the growth of phytoplankton
at concentrations considerably higher than those expected to be found in
the environment. The toxicities of chlorophenol, chlororesorcinol, chloro-
benzoic acid, and chlorouracil to phytoplankton varied among the species.
The chemicals, up to 10 ppm, either had no effect or caused stimulation in
growth of Dunaliella and Porphyridium. However, Skeletonema was found to be
more sensitive to the effect of the chloro-organic chemicals than the other
two organisms. At 1 ppm, none of the chemicals except 4-chlororesorcinol
appeared to inhibit growth of Skeletonema. However, at a higher concentra-
tion (10 ppm), 3-chlorobenzoic acid, and 3-chlorophenol were found to be toxic,
These findings suggest that phytoplankton are less sensitive to the
chloro-organic compounds than other aquatic organisms. Gehrs et al. (1974)
observed that both 5-chlorouracil and 4-chlororesorcinol decreased the
hatchability of carp eggs at concentrations as low as 1 ppb. A lack of
effect of the test chemicals on phytoplankton growth does not rule out the
possibility that the chemicals may have ecologically significant effects.
It is quite likely that these chemicals may be accumulated by phytoplankton.
An accumulation of the chemicals by phytoplankton is of ecological signifi-
cance because these chemicals may be transferred to higher trophic levels.
Therefore, the uptake and accumulation of the chloro-organic chemicals by
phytoplankton need to be investigated in order to fully access their impact.
It was observed that 3-chlorophenol and 5-chlorouracil caused an initial
stimulation in growth of the organisms, followed by a decline in the growth
rate. The reason for this stimulation is not known; however, the subsequent
decrease may be explained by assuming that biological or non-biological
transformations of the chemicals resulted in formation of products which
caused inhibition of growth. In the case of the other chemicals, an initial
lag in the growth of the organisms was observed, but subsequently the cells
recovered and growth resumed at the same rate as in the untreated cells. In
nature, these brief periods of growth stimulation or inhibition may have a
profound effect on species diversity in phytoplankton communities. During
these periods, the faster-growing, less susceptible organisms may gain a
competitive advantage over the more sensitive species and may eventually
become the dominant species in the community. A change in species composi-
tion of phytoplankton communities may produce an impact on organisms at
higher trophic levels since many marine animals, particularly zooplankton
and oyster and clam larvae, graze selectively.
12
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3-Chlorophenol and 4-chlororesorcinol acted synergistically in inhibiting
growth of Skeletonema, which suggests that interaction among chloro-organic
chemicals should be taken into account in evaluating their effects. In
nature, where several chloro-organic chemicals may be present simultaneously
in bodies of water receiving chlorinated waste-waters, the combined effect
of these chemicals will be expected to be different from that of the
individual chemicals.
13
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REFERENCES
Alexander, M. and B.K. Lustigman. Effect of chemical structure on microbial
degradation of substituted benzenes. J. Agric. Food Chem., 14:410-413,
1966.
Barnhart, D.R. and G.R. Campbell. Effect of Chlorination on Selected
Organic Chemicals. NTIS Publ. No. PB 211 160, 1972, 105 pp.
Brungs, W.A. Effect of residual chlorine on aquatic life. J. Water Pollut.
Control Fed., 45:2180-2193, 1973.
Colby, R.S. Calculating synergistic and antagonistic responses of herbicide
combinations. Weeds, 15:20-22, 1967.
"Federal Water Pollution Control Act, Amendments," Public Law 92-500, U.S.
Government, October 18, 1972.
Gehrs, C.W., L.D. Eyman, R.L. Jolley, and J.E. Thompson. Effect of stable
chlorine-containing organics on aquatic environments. Nature,
249:675-676, 1974.
Glaze, W.H., J.E. Henderson, J.E. Bell, and V.A. Wheeler. Analysis of
organic materials in waste water effluents after chlorination.
J. Chromatogr. Sci., 11:58-584, 1973.
Guillard, R.R.L. and J.H. Ryther. Studies on marine planktonic diatoms.
Can. J. Microbiol., 8:229-239, 1962.
Jolley, R.L. Determination of chlorine-containing organics in chlorinated
sewage effluents by coupled 36C1 tracer-high-resolution chromatography.
Environ. Lett., 7:321-340, 1974.
Jolley, R.L. Chlorine-containing organic constituents in chlorinated
effluents. J. Water Pollut. Control Fed., 47:601-618, 1975.
Mendenhall, W. Introduction to Probability and Statistics. Duxbury Press
North Scituate, Massachusetts, 1975, 228 pp.
U.S. Environmental Protection Agency, National Environmental Research Center,
Corvallis. Marine Algal Assay Procedure Bottle Test, 1974, 43 pp.
14
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Days After Treatment
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Figure 2. The effect of 3-chlorobenzoic acid on the growth of Porphyridium.
16
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0.5
0.4 -
0-3
a>
o
o
J5 0.2
0.1
• Control
o 1 ppm 3—Chlorobenzoic Acid
& 10ppm 3—Chlorobenzoic Acid
8 9 10
Days After Treatment
11
12 13 14 15
Figure 3. The effect of 3-chlorobenzoic acid on the growth of Skeletonema.
-------
1.
0.
0.
0.8
E
c
o
s
I
S 0.5
1
J3
<
0.4
0.3
0.2
0.1
5—Chlorouracil
Dunaliella:
• Control
A 1 ppm
A 10ppm
Skeletonema:
o Control
V 1 ppm
T 10ppm
3 4
8 9 10 11
Days After Treatment
12
13
14
15
Figure 4. The effect of 5-chlorouracil on the growth
of Dunaliella and Skeletonema.
18
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1 I
4 —Chlororesorcinol
Dunaliella :
• Control
A 1 ppm
10ppm
Skeletonema:
» Control
v 1 ppm
10ppm
3456
7 8 9 10 11
Days After Treatment
12 13 14 15
Figure 5. The effect of 4-chlororesorcinol on the growth
of Dunaliella and Skeletonema.
19
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to
0.2 —
E
a
in
10
I
8
1
2
0.1
i r
o Control
a 2.5 ppm
v 5.0 ppm
v 7.5 ppm
J
J L
4—Chlororesorcinol
4—Ch lororesorcinol
4—Chlororesorcinol
7 8 9 10
Days After Treatment
11 12 13 14 15
Figure 6. The effect of 4-chlororesorcinol on the growth of Skeletonema.
-------
° Control
A 1 ppm
• 10ppm
4—Chlororesorcinol
4—Ch lororesorcinol
7 8 9 10 11 12 13
Days After Treatment
15
Figure 7. The effect of 4-chlororesorcinol on the growth of Porphyridium.
21
-------
0.2
E
o
8
i
8
I
o
JS 0.1
• Control
A 1 ppm
A 10ppm
_L
_L
3 — Chlorophenol
3,—Chlorophenol
7 8 9 10
Days After Treatment
11
12 13
14
15
Figure 8. The effect of 3-chlorophenol on the growth of Dunaliella.
-------
ro
0.2 -
E
c
o
IT)
co
I
0}
u
c
«3
J3
<
0.1
o Control
• 1 ppm
* 10ppm
3—Chlorophenol
3—Chlorophenol
8 9 10
Days After Treatment
11 12 13 14
15
Figure 9. The effect of 3-chlorophenol on the growth of Skeletonema,
-------
Control
2.5 ppm
A 5.0 ppm
• 7.5 ppm
3 —Chloropnenol
3—Chlorophenol
3 —Chlorophenol
8 9 10
Days After Treatment
Figure 10. The effect of 3-chlorophenol on the growth of Skeletonema,
-------
ro
Ui
0.2 -
e
c
o
in
co
I
0)
o
£ 0.1
o Control
* 0.1 ppm
A 1.0 ppm
• 5.0 ppm
3 —Chlorophenol
3—Chlorophenol
3 —Chlorophenol
1
1
7 8 9 10
Days After Treatment
11
12
A
o
13 14
15
Figure 11. The effect of 3-chlorophenol on the growth of Porphyridium.
-------
o Control
• 2.5 ppm 3—Chlorophenol
A 5 ppm 3—Chlorophenol
A 2.5 ppm 4 — Chlororesorcinol
5 ppm 4—Chlororesorcinol
T 2.5 ppm 3—Chlorophenol + 2.5 ppm 4—Chlororesorcinol
n 5 ppm 3—Chlorophenol + 5 ppm 4—Chlororesorcinol
7 8 9 10 11
Days After Treatment
13 14 15
Figure 12. The effect of 3-chlorophenol and 4-chlororesorcinol
singly and in combination oh the growth of Skeletonema.
26
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EFFECT OF CAFTAN ON MARINE ALGAE
SECTION 1
INTRODUCTION
Captan (N-trichloromethylthio-4-cyclohexane-l,2-dicarboximide) is one
of the fungicides widely used in coastal areas. Usage of this chemical may
increase in the future as it is a registered substitute for certain fungi-
cides which have been reported to be ecologically hazardous. Captan may be
introduced into the estuarine environment through run-off from treated
coastal lands or drift from fungicide application. The effects of captan
on terrestrial organisms have been investigated, but relatively little
research has been done concerning effects of the fungicide on phytoplankton
which represent the first link in the aquatic food chain. Knowledge of the
effects of pesticides on primary producers is important in predicting their
impact on the marine environment. We have investigated the effects of
captan on growth and photosynthesis in selected species of marine phyto-
plankton.
27
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SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
The results indicate that marine phytoplankton vary in their sensitivity
to the fungicide captan. Skeletonema is considerably more sensitive to the
fungicide than Dunaliella and Porphyridium. Treatment with 0.5 ppm of captan
caused a substantial inhibition of photosynthesis in Skeletonema.
Our results indicate the following areas of interest: (1) the effect
of captan on additional algal species should be examined, (2) the effect
of captan in phytoplankton should be studied in the presence of other
pesticides and environmental contaminants, and (3) the uptake and metabolism
of captan by phytoplankton should be investigated.
28
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SECTION 3
MATERIALS AND METHODS
GROWTH STUDIES
The effect of captan was examined in Skeletonema costatum (Bacillario-
phyta), Dunaliella tertiolecta (Chlorophyta), and Porphyridium sp. (Rhodophyta),
The procedures for culturing the organisms and measuring their growth were
similar to those used in determining the effects of chloro-organic compounds.
Desired volumes of captan solution in acetone were added to the cell sus-
pension so that the final concentration of acetone was 0.01%. The control
cell suspension contained a similar amount of acetone.
PHOTOSYNTHESIS
The photosynthetic activity of Skeletonema was determined by measuring
lkC02 fixation by the cells. The cell suspension (101* cells/ml) was pre-
incubated with captan for 30 minutes on a reciprocating shaker at 22°C under
a light intensity of approximately 10,750 lux. Following preincubation with
the fungicide, NaH11+C03 was added to the cell suspension and the cells were
allowed to fix 1£*C02 for 60 minutes. Aliquots of cell suspension were
removed at selected times after addition of NaH^COs, filtered through a
0.45 my Millipore filter and the cells were washed with the growth medium.
To determine the amount of ltfC02 fixed by the cells, the filter discs with
the cells were transferred to a scintillation fluid and counted for 11+C
in a Nuclear Chicago liquid scintillation counter.
29
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SECTION 4
RESULTS
EFFECT ON GROWTH
The effect of captan on the growth of Dunaliella is shown in Figure 13.
The fungicide, at 0.1 and 1 ppm, significantly stimulated growth by 6 and
20%, respectively, after 7 days of treatment. However, 5 ppm of captan
completely inhibited growth of the alga.
Figure 14 shows the effect of captan on growth of Porphyridium. The
fungicide, at 0.1 ppm, stimulated growth by 23% through day 4, but sub-
sequently growth proceeded at almost the same rate as that in the controls.
Treatment with 1 ppm of the chemical did not significantly affect growth.
Growth of cultures exposed to 5 ppm captan was completely suppressed
during the first 3 days following treatment; however, the cells recovered
subsequently and the inhibition decreased to 18% by day 12.
Skeletonema was more sensitive to captan than either Dunaliella or
Porphyridium (Figures 15 and 16). The fungicide inhibited growth of the
organism at concentrations ranging from 0.25 to 5 ppm, inhibition increasing
with an increase in captan concentration. Cells exposed to 0.25-1 ppm of
captan showed a time lag of growth with the duration of the lag period
increasing with an increase in concentration of the fungicide. Following
the lag period, the growth of the treated cells proceeded at almost the
same rate as that in the controls. However, at 5 ppm, captan damaged the
cells so severely that they never resumed growth.
EFFECT ON PHOTOSYNTHESIS
To ascertain the mechanism by which captan causes toxicity in
Skeletonema, the effect of the fungicide on photosynthesis was examined.
Photosynthetic CQ^ fixation by Skeletonema was used as a measure of photo-
synthetic activity. Since C02 fixation is the result of both light and dark
reactions of photosynthesis, an adverse effect on any of the photosynthetic
reactions will be reflected in reduced C02 fixation. Exposure of Skeletonema
to 0.5 and 1 ppm of captan for 30 minutes reduced llfC02 fixation by 83 and
93%, respectively (Table 1).
30
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14
TABLE 1. LFFECT OF CAPTAN ON PHOTOSYNTHETIC C02 FIXATION
BY SKELETONEMA
14 -3
Treatment Total C fixed (dpm x 10 ) after
30 min. 60 min.
Control 16.7 37.4
Captan 0.5 ppm 2.8 2.9
Captan 1.0 ppm 1.0 1.2
31
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SECTION 5
DISCUSSION
Our findings indicate that different species of marine phytoplankton
vary in their response to captan. Skeletonema is considerably more sensi-
tive to the fungicide than Dunaliella or Porphyridium. Captan was toxic to
Skeletonema at a concentration as low as 0.25 ppm. The toxic action of
captan has been suggested to be due to its ability to react readily with
cellular sulfhydryl groups (Lukens, 1971; Lukens and Sisler, 1958; Owens
and Blaak, 1960; Richmond and Somers, 1966). The trichloromethylthio
moiety produced as a result of the reaction of thiolls with captan is also
capable of reacting with sulfhydryl and amino groups. Because captan may
inhibit enzymes and coenzymes with functional sulfhydryl and amino groups
known to operate in many areas of cell metabolism, the toxicity of the
fungicide to phytoplankton may result from its interference with any number
of biochemical processes. Our findings indicate that inhibition of photo-
synthesis by captan is an important mechanism by which the fungicide causes
growth inhibition in Skeletonema. The present studies do not identify the
mechanism(s) by which the fungicide inhibits photosynthesis. However, on
the basis of the chemical reactivity of captan, it may be postulated that
the fungicide inhibits both the light-mediated reactions and the reactions
in the carbon-reduction cycle of photosynthesis.
32
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REFERENCES
Lukens, R.J. Chemistry of Fungicidal Action. Springer-Verlag, New York,
1971, 136 pp.
Lukens, R.J. and H.D. Sisler. Chemical reactions involved in- the fungi-
toxicity of captan. Phytopathology, 48:235-244, 1958.
Owens, R.G. and G. Blaak. Chemistry of the reactions of dichlone and
captan with thiols. Contrib. Boyce Thompson Inst., 20:475-497, 1960.
Richmond, D.V. and E. Somers. Studies of the fungitoxicity of captan. IV.
Reactions of captan with cell thiols. Ann. Appl. Biol., 57:231-240,
1966.
33
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0.2
. E
o
1 0.1
• Control
A 0.1 ppm
A 1.0 ppm
o 5.0 ppm
Captan
Captan
Captan
-Ql
=O=
--<*-•
8 9 10
Days After Treatment
11
12
13
14
15
Figure 13. The effect of captan on the growth of Dunaliella.
-------
1 - 1 - 1 - 1
U)
Ul
° Control
A 0.1 ppm
A 1.0 ppm
• 5.Q ppm
1 - r
Captan
Captan
Captan
8 9 10
Days After Treatment
11 12
Figure 14. The effect of captan on the growth of Porphyridium.
-------
0.2
I
o
8
o
JS 0.1
o Control
A 0.1 ppm
• 1.0 ppm
A 5.0 ppm
8 9 10
Days After Treatment
11
12
13
15
Figure 15. The effect of captan on the growth of Skeletonema.
-------
o Control
• O.IOppm
7 0.25 ppm
A 0.50 ppm
A 0.75 ppm
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing/
1. REPORT NO.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Effects of Selected Wastewater Chlorination Products
and Captan on Marine Algae
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Harish C. Sikka and Gary "L. Butler
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Syracuse Research Corporation
Syracuse, New York 13210
10. PROGRAM ELEMENT NO.
1EA714
11. CONTRACT/GRANT NO.
R803943010
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
13. TYPE OF REPORT AND PERIOD COVERED
July 15, 1975 - July 14, 1976
14. SPONSORING AGENCY CODE
EPA-OKD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Effects of stable chloro-organic compounds formed during chlorination of sewage
effluents on growth of marine unicellular algae were determined. Captan suppressed
growth of Dunaliella tertiolecta and Porphyridium cruentum at 5 ppm. Growth of
Skeletonema costatum was inhibited by 0.25 ppm captan.
3-Chlorobenzoic acid inhibited growth of S_. costatum at 10 ppm but had no effect
on I), tertiolecta or P_. cruentum. There was no effect of 1-10 ppm 5-chlorouracil on
§.' costatum, but growth of I), tertiolecta was stimulated initially. Growth of S_.
costatum was inhibited by 1 ppm 4-chlororesorcinol, and 10 ppm inhibited growth of
P_. cruentum. At 1 ppm, 3-chlorophenol stimulated growth of all three species, but
growth of j^. costatum was inhibited by 2.5 ppm.
A combination of 3-chlorophenol and 4-chlororesorcinol interacted synergistically
to reduce growth of j^. costatum.
It is concluded that chloro-organic compounds formed during chlorination of
sewage effluent are not an immediate threat to marine unicellular algae.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Algae, Marine, Phytoplankton, Growth,
Sewage Treatment, Water Pollution
b.lDENTIFIERS/OPEN ENDED TERMS
Dunaliella, Skeletonema,
Porphyridium, Chloroben-
zoic acid, chlorophenol,
chlororesorcinol, captan
c. COSATI I ic!d/Group
13. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report)
unclassified
21. NO. OF PAGES
38
20. SECURITY CLASS fTIiis page)
unclassified
22. PRICE
EPA Form 2220-1 (9-73)
*U.S. GOVERNMENT PRINTING OFFICE: 1977 - 740-114/1596
38
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