oEPA
United States
Environmental Protection
Agency
Environmental Research
Laboratory
Corvallis OR 973^0
EPA-600 3-79-075
July 1979
Research and Development
Effects and
Fate of Sewage
Chlorination
Products in
Phytoplankton
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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 nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
1. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
S^irTqIS aVr ' M ' vt0 the PUbliC thr°ugh the National Technical 'Ca-
bervice, Springfield, Virginia 22161.
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EPA-600/3-79-075
July 1975
EFFECTS AND FATE OF SEWAGE CHLORINATION PRODUCTS IN PHYTOPLANKTON
by
Harish C. Sikka
Knowlton C. Foote
James I. Mangi
Edward J. Pack
Life Sciences Division
Syracuse Research Corporation
Syracuse, New York 13210
Grant No. R 804-938-010
Project Officer: David T. Specht
Marine Division
Corvallis Environmental Research Laboratory
Corvallis, Oregon 97330
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
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DISCLAIMER
Laborl yTs vironLnL Trot ^t^ ^^S Environmental Research
Approval does not ™g™f7?hal the conient*9^' and aPP™ved for publication.
policies of the U.S. Environmental P?S?nnTSSanly reflect the views and
trade names or commercia prS?s coSstitu?^ pSn^' ^ does niention of
for use. nruaucts constnute endorsement or recommendation
ii
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FOREWORD
Effective regulatory and enforcement actions by the Environmental Protection
Agency would be virtually impossible without sound scientific data on pollu-
tants and their impact on environmental stability and human health. Respon-
sibility for building this data base has been assigned to EPA's Office of
Research and Development and its 15 major field installations, one of which
is the Corvallis Environmental Research Laboratory (CERL).
The primary mission of the Corvallis Laboratory is research on the effects
of environmental pollutants on terrestrial, freshwater, and marine ecosystems;
the behavior, effects and control of pollutants in lake systems; and the de-
velopment of predictive models on the movement of pollutants in the biosphere.
This report examines the effects of seven major chloro-organic compounds,
typically formed during chlorination of sewage effluent, on the growth of
seven selected species of marine and freshwater phytoplankton. Despite the
lack of a discernible effect on their growth at a maximum concentration of
0.1 ppm, there was a substantial variance between some of the algae in the
uptake and metabolism of some of the compounds.
This information can be important in assessing the potential for artificially
induced floral changes through species selection or altered grazing pressure
through the transfer of these accumulated compounds through the food chain.
These results more clearly define areas of program interest for further
research that can be useful for determining tolerable levels of these com-
pounds in wastewater discharges.
J. C. McCarty
Acting Director, CERL
iii
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ABSTRACT
The effects of seven stable chloro-organic compounds formed during
chlorination of domestic waste-water on the growth of selected fresh-water
and marine phytoplankton were determined. The uptake and metabolism of
selected chloro-organic chemicals by the phytoplankton were also investigated.
3-Chlorophenol, 3-chlorobenzoic acid, 4-chlororesorcinol, 5-chlorouracil,
5-chlorouridine, 6-chloroguanine or 8-chlorocaffeine at a concentration of
0.1 PPm, alone or in combinations of up to 4 chemicals, had no significant
effect on the yield of Scenedesmus obliquus. Selenastrum capricomutum.
Microcystis aeruginosa. Dunaliella tertiolectirskeleTo^e^o"statum.
Thalassiosira pseudonana. and Porphyridium sp. 4-Chlororesorcinol and
5-chlorouracil were taken up by certain species but neither chemical was
accumulated to a high level. The uptake of chlororesorcinol was considerably
greater than that of chlorouracll. The uptake of 3-chlorobenzoic acid by
the phytoplankton was negligible.
4-Chlororesorcinol was readily degraded in aqueous solution by the
action of simulated sunlight and both Skeletonema and Selenastrum took up
chlororesorcinol as well as its photodegradation products from the medium.
Neither Skeletonema nor Selenastrum were able to metabolize 4-chloro-
resorcinol in the dark but appeared to transform it to some extent into
more polar material(s) in the light.
This report was submitted in fulfillment of EPA Grant R 804^938-010 by
Syracuse Research Corporation under the sponsorship of the U.S. Environmental
Protection Agency. This report covers the period November 1, 1976 to
February 28, 1978, and work was completed as of February 28, 1978.
iv
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CONTENTS
Foreword
Abstract iv
Figures vi
Tables vii
Abbreviations ix
Acknowledgment x
1. Introduction ..... 1
2. Conclusions 3
3. Recommendations 4
4. Materials & Methods 5
5. Results 14
6. Discussion • • • 26
References • • • 28
Appendix 31
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FIGURES
Number
1 Structures of chloro-organic compounds involved
in this study
A6
A7 Calibration curve (cell number vs. optical density) for
Skeletonema costatum . . .
2 Typical protocol for a four-chemical factorial experiment . 9
3 Effect of 3-chlorophenol , 4-chlororesorcinol and
5-chlorouracil at 0.1 ppm on the growth of Scenedesmus
obliquus at two levels of nitrate concentration . . . . . . 16
4 Effect of 3-chlorophenol, 4-chlororesorcinol, and
5-chlorouracil at 0.1 ppm on the growth of Selenastrum
capricormitum as a function of two levels of nitrate —
concentration .........
17
5 Photodegradation of 4-chlororesorcinol in simulated
sunlight 25
Al Calibration curve (cell number vs. optical density) for
low concentrations of Microcystis aeruginosa 32
A2 Calibration curve (cell number vs. optical density) for
high concentrations of Microcystis aeruginosa 33
A3 Calibration curve (cell number vs. optical density) for
Dunaliella tertiolecta 34
A4 Calibration curve (cell number vs. optical density) for
Porphyridium sp 05
A5 Calibration curve (cell number vs. optical density) for
Scenedesmus obliquus 0/:
• ' Jo
Calibration curve (cell number vs. optical density) for
Selenastrum capricornutum
38
A8 Calibration curve (cell number vs. optical density) for
Thalassiosira pseudonana
vi
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TABLES
Number Page
14
1 Uptake of C-chlororesorcinol by phytoplankton 18
14
2 Uptake of C-chlorouracil by phytoplankton 19
14
3 Uptake of C-chlorobenzoic acid by phytoplankton 20
14
4 Distribution of C-chlororesorcinol and its degradation
products in Skeletonema incubated with the chemical
for 10 days 22
14
5 Distribution of C-chlororesorcinol and its degradation
products in Skeletonema and Selenastrum incubated
with the chemical in the light and dark 23
Al The effects of various chloro-organic chemicals at 0.1 ppm
on the yield of Dunaliella fertiolecta 40
A2 The effects of various chloro-organic chemicals at 0.1 ppm
on the yield of Dunaliella tertiolecta 41
A3 The effects of various chloro-organic chemicals at 0.1 ppm
on the yield of Microcystis aeruginosa 42
A4 The effects of various chloro-organic chemicals at 0.1 ppm
on the yield of Microcystis aeruginosa 43
AS The effects of various chloro-organic chemicals at 0.1 ppm
on the yield of Porphyridium sp. 44
i
A6 The effects of various chloro-organic chemicals at 0.1 ppm
on the yield of Porphyridium sp. 45
A7 The effects of various chloro-organic chemicals at 0.1 ppm
on the yield of Scenedesmus* obliquus 46
A8 The effects of various chloro-organic chemicals at 0.1 ppm
on the yield of Scenedesmus obliquus 47
vii
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Tables (continued)
Number
Page
A9 The effects of various chloro-organic chemicals at 0.1 ppm
on the yield of Selenastrum capricornutum 48
A10 The effects of various chloro-organic chemicals at 0.1 ppm
on the yield of Selenastrum capricornutum 49
All The effects of various chloro-organic chemicals at 0.1 ppm
on the yield of Skeletonema costatum 50
A12 The effects of various chloro-organic chemicals at 0.1 ppm
on the yield of Skeletonema costatum 51
A13 The effects of various chloro-organic chemicals at 0.1 ppm
on the yield of Thalassiosira pseudonana 52
A14 The effects of various chloro-organic chemicals at 0.1 ppm
on the yield of Thalassiosira pseudonana 53
A15 Representative data comparing cell numbers obtained by
direct counting and from a calibration curve for
Porphyridium sp 54
viii
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LIST OF ABBREVIATIONS
CB — 3-chlorobenzoic acid
CC — 8-chlorocaffeine
CG — 6-chloroguanine
CP — 3-chlorophenol
CR — 4-chlororesorcinol
CU — 5-chlorouracil
CUD — 5-chlorouridine
±x
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ACKNOWLEDGMENTS
We are grateful to Dr. Sujit Banerjee for synthesizing 14C-labeled
5-chlorouracil and to Mr. Gary Dec for technical assistance.
We thank Dr. K.G. Mehrotra, Professor of Statistics, Department of
Computer and Information Sciences, Syracuse University, for his help in
statistical analysis of the data.
Our sincere appreciation is extended to Mr. David T. Specht of the
Marine Division, Corvallis Environmental Research Laboratory, U.S. Environ-
mental Protection Agency, Corvallis, Oregon, for his constructive suggestions
during the course of these studies.
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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 initiate or upgrade their treatment to
include chlorination as a disinfectant to meet state or local standards in
lieu of not yet widely accepted alternatives such as ozonation or bromination.
Additionally, an increasing number of industries have been required to provide
waste-water treatment which often includes effluent chlorination.
It is conceivable chat some organic compounds may escape secondary treat-
ment or be only partially degraded and as a result they may react with
chlorine, yielding persistent, potentially toxic organic compounds. Recently,
Glaze .et al. (13) and Jolley (17,18) have shown that chlorine-containing
organic compounds are produced when sewage effluents are chlorinated.
Jolley (18) has identified 17 stable, chlorine-containing organic compounds
in domestic waste-water effluent which had been chlorinated to a 1 to 2 mg/Ł
combined chlorine residual. Some of these compounds included chlorinated
phenols, aromatic acids, purines and pyrimidines. Barnhart and Campbell (2),
in studies of industrial waste-water effluents observed that chlorine reacted
readily with phenol, m-cresol, and aniline under conventional effluent treat-
ment conditions. Based on the assumption that a total of 100,000 tons of
chlorine is used annually in the U.S. for disinfecting waste-water, Jolley (18)
estimated that approximately 5,000 tons of stable chlorine-containing organic
compounds would be released to the receiving water ecosystem annually.
The presence of chloro groups is known to render benzenoid compounds
more resistant to microbiological degradation (1), which suggests a potential
for accumulation of these compounds in receiving waters. The introduction
of chlorinated organics into the aquatic environment is of great environ-
mental concern because of their potential toxicity to various organisms. In
order to evaluate fully the impact of waste-water chlorination on the aquatic
environment, it is necessary that we know the effects and fate of the
chlorination products in the biota. The toxicity of residual chlorine on
aquatic life has been extensively investigated and was recently reviewed by
Brungs (5). However, very little information is available on the effects of
stable chlorine-containing organic compounds that may have been produced
during the chlorination process. Gehrs et al. (12) have recently reported
that both 5-chlorouracil and 4-chlororesorcinol, which are among the con-
stituents in chlorinated effluents, decreased the hatchability of carp eggs
at concentrations as low as 1 ppb.
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™yt°P^nkton 'onf ^ute a vital Part °f the aquatic biota. An adverse
f H6 ST ^^Plankton, which represent the first link in the
? ?* ^ ^ adV6rSe 6ffeCtS °n the entire ecosystem. Algae
.
the ±«r? ^ ^ ^"^J r°le in ^termining the fate of chloro-organics in
adLSttn H/Sy r* Gy ^ rem°Ve the <*emicals from the environment by
accumulatin f°r, °r?tl0n ^ "** subse /\ -P ^"U1 j
aquatic ecosystems. chlorine-treated waste-waters into the
Specific Objectives:
products ori-hffeCt °u selected sewage-effluent chlorination
products on the growth of fresh water and marine phytoplankton.
theSeffLfhnfintera^ti°n am°n8 the test chemicals by examining
effect of one chemical in the presence of the other chemicals,
on the
4.
— — «*• j i.i.ic mi i nice* f\f- 4-i-iA. j- _t_ i -
and
To study the metabolism of the test chemicals by the phytoplankton.
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SECTION 2
CONCLUSIONS
A number of chloro-organic compounds produced during chlorination of
waste-water, such as 3-chlorophenol, 3-chlorobenzoic acid, 4-chlororesorcinol,
5-chlorouracil, 5-chlorouridine, 6-chloroguanine and 8-chloroguanine at a
concentration of 0.1 ppm alone or in combinations of up to four chemicals,
had no significant effect on the yield of several fresh-water and marine
phytoplankton species.
4-Chlororesorcinol and 5-chlorouracil were taken up by certain species
but neither chemical was accumulated to a high level. The uptake of
3-chlorobenzoic acid by the phytoplankton was negligible.
Neither Skeletonema costatum nor Selenastrum capricornuturn were able to
metabolize 4-chlororesorcinol in the dark but appeared to transform it to
some extent into more polar material(s) in the light.
These findings suggest that simple mono-chlorinated organic chemicals,
at the concentrations tested, are not expected to have any significant effect
on growth of phytoplankton in the aquatic environment.
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SECTION 3
RECOMMENDATIONS
^ ^ a UUmber °f 8imPle *°n°-chlorinated organic
ec thv ^lorination of sewage effluents have no significant
in order to fuMv i fre^-^ter and marine phy toplankton . However,
reco±nd thf t : ^ environmental impact of these chemicals we
recond th t :
*"
chemi^lf r" ^ r^"^11 t0 deter***e the toxicity of the
chemicals to aquatic invertebrates and fish.
by fish and ***** Should
Pr?cesses (biodegradation, photodegradation,
bhavior f S ment) WMCh may dete^ne the environmental
of environhe^Cf S, ShOUld be Studled' The rates and
-
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SECTION 4
MATERIALS AND METHODS
CHEMICALS
The chemicals tested for toxiclty to phytoplankton included 3-chloro-
benzoic acid, 8-chlorocaffeine, 6-chloroguanine, 3-chlorophenol, 4-chloro-
resorcinol, 5-chlorouracil, and 5-chlorouridine (Figure 1). These chemicals
were among the 17 stable chlorine-containing organic compounds which
Jolley (18) identified in chlorinated sewage effluents. These chemicals
were specifically selected because their concentrations in chlorinated
sewage-effluents were relatively high compared to those of other chlorine-
containing organic compounds.
3-Chlorobenzoic (99%), 3-chlorophenol (97% or greater), and 4-chloro-
resorcinol (99%) were purchased from Aldrich Chemical Company; 5-chlorouracil
and 5-chlorouridine (both Grade A) from Calbiochem; 6-chloroguanine from
Sigma Co., and 8-chlorocaffeine (98-99%) from ICN. Uniformly ring-labeled
14C-3-chlorobenzoic acid (sp. activity 4.6mCi/mM) and ll*C-4-chlororesorcinol
(sp. activity 9.5mCi/mM) were purchased from the California Bionuclear Corpor-
ation, Sun Valley, California. 14C-5-Chlorouracil was synthesized in our
laboratory by chlorinating ltfC-uracil using the procedure described by West
and Barrett (30). The lifC-chlorouracil produced was then purified by high-
pressure liquid chromatography (HPLC) using a Waters Associates (Milford,
Mass.) liquid chromatograph (model M6000A) equipped with a U.V. detector
(Schoeffel Instrument Corp., Westwood, N.J., model GM770). A 4 mm (i.d.)
X 30 cm column packed with y Bondapak C18 reversed phase medium (Waters
Associates) was utilized. Chlorouracil was detected by its absorption at
282 nm. The solvent system used was methanol:water (1:7 v/v) which provided
excellent separation of the unreacted uracil from 5-chlorouracil. The
reaction solution was injected directly into the HPLC; the fraction containing
5-chlorouracil, as indicated by the UV detector, was collected and stored in
freezer for subsequent use. Analysis of this fraction by HPLC showed that
14C-chlorouracil had a purity of greater than 99%.
ALGAL SPECIES
The fresh-water phytoplankton species tested were Microcystis aeruginosa
(Cyanophyta), Scenedesmus obliquus (Chlorophyta), and Selenastrum capricornutum
(Chlorophyta). The marine phytoplankton were Dunaliella tertiolecta (DUN
clone) (Chlorophyta), Skeletonema costatum (SKEL clone)(Bacillarlophyta),
Thalassiosira pseudonana (CN clone)(Bacillariophyta) and Porphyridium sp.
(Rhodophvta). The stock cultures of the,fresh-water 5algae were obtained
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COOH
3-Chlorobenzoic acid
Cl
O CH,
8—Chlorocaffeine
3-Chlorophenol
Cl
6—Chloroguanine
4—Chlororesorcinol
5—Chlorouracil
5—Chlorouridine
Figure 1. Structures of chloro-organic compounds involved in this study.
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from the Culture Collection of Algae, University of Texas at Austin. The
cultures of marine species were obtained from the Woods Hole Oceanographic
Institute, Woods Hole, Mass. All cultures were axenic except for Microcystis
aeruginosa.
CULTURING OF ALGAE
Scenedesmus obliquus and Selenastrum capricornutum were grown axenically
in the medium specified in the Algal Assay Procedure, EPA, 1971 (28). Non-
axenic Microcystis aeruginosa was grown in a modified Gorham's medium con-
sisfing of the following ingredients (ymoles/Ł) in glass distilled water:
NaN03 (2000), MgCl2 (200), MgSOit*7H20 (200), CoCl2 (200), K2HPOH (100),
FeCl3 (4), NaEDTA (20), H3B03 (40), MnCl2-H2Q (2), ZnCl2 (0.0008), CoCl2
(0.08). The initial pH of the medium was 7.5. All four fresh-water species
were grown on a reciprocating shaker (100 oscillations/min) under controlled
environmental conditions; 21°C ± 1°C, and 400 ft-c continuous illumination.
Skeletonema costatum, Thalassiosira pseudonana., and Dunaliella tertiolecta
were grown axenically in Modified Burkholder's artificial seawater medium
recommended by the Environmental Research Laboratory, USEPA, Corvallis,
Oregon (1974). Porphyridium sp. was grown axenically in Modified Burkholder's
medium supplemented with the following: 16.48 mg N/l as NaN03, 4.45 mg P/l as
K^HPOij, 4.94 mg Si/1 as Na2Si03-9H20, 1 yg/1 biotin, 0.2 mg/1 thiamine-HCl and
1 ml/1 NAAM (29) trace metal mix (0.1856 g H3B03; 0.416 g MnCl2'4H20; 0.032 g
ZnCl2; 1.428 mg CoC^^HgO; 0.0214 mg CuCl2'2H20; 7.26 mg Na2Mo04 • 2H20 per
liter). The salinity was adjusted to 32 ppt and the pH to 7.5 After filtra-
tion through a 0.45 urn membrane filter (pre-rinsed w/H20), 1 ml/1 sterilized
Fe-EDTA solution (33.05 yg Fe/ml as FeCl3+ 300 yg/ml Na2EDTA) was added
aseptically. All three marine species were grown at 17-18°C and 450-550 ft-c
continuous illumination. Dunaliella and Porphyridium cultures were placed on a
reciprocating shaker (100 oscillations/min), while the diatoms Skeletonema
and ^halassiosira were kept static and hand-swirled once/day.
Since we were interested in examining the response of several species of*
algae to a number of chloro-organic compounds, batch culture was the method
of choice. This method, in comparison with continuous-culture assay, provides
a quick and relatively simple method for establishing the relative toxicity of
pollutants as well as the range of toxicity of each pollutant.
The algae were grown in 100 ml aliquots in sterilized 250 ml Erlenmeyer
flaskscapped by• Bellco "Kap-Uts" covers. To initiate an experiment,
sufficient inoculum was added to sterile growth medium to give the following
initial cell concentrations: 1 x 103 cells/ml for Dunaliella and Skeletonema;
1 x 10^ cells/ml for Porphyridium, Thalassiosira, Scenedesmus, and Selenastrum;
and 5 x 104 cells/ml for Microcystis. The test chemicals, dissolved in
absolute ethanol, were then added to the culture aseptically so that the
final concentration of each chemical along with its solvent ethanol in the
medium was 0.1 ppm and 0.01%, respectively. The concentration of the
chemicals to be tested, 0.1 ppm, is considerably higher than expected to be
present in the environment (17). If a given chemical at a concentration
substantially higher than that occurring in the environment proves to have no
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effect on the algae, the safety of the chemical(s) to the algae will have been
established. The ethanol at the concentrations used was shown to have no
effect on the growth of any of the algae except for Microcystis. In non-axenic
cultures of Microcystis, 0.01% ethanol severely repressed algal growth. If
the ethanol was eliminated, the algae exhibited normal growth. Therefore, in
Microcystis experiments, 100 yl of the selected chemical(s) in ethanol was
added to each sterile flask which was then capped by a Bellco "Kap-Uts" cover.
The flasks were placed on a warm surface (30°C) overnight so that the ethanol
evaporated and diffused out of the flasks. The following day, 100 ml of
modified Gorham's medium was added to each flask. The flasks were placed on
a reciprocating shaker (100 oscillations/min) for 8 hr at 22°C to dissolve .the
chemical(s). Tests with one of the chloro-organic chemicals, 5-chloro-
uracil-^C (carbon uniformly labeled), showed that 97.7% of the dried chemical
redissolved in Gorham1s medium in 1 hr and 99.3% by 4 hr.
The growth of the algae was measured every two to three days following
treatment (except in the case of Skeletonema) by measuring the absorbance at
650 nm in a Gary spectrophotometer. The experiments were continued at least
until the stationary plateau had been reached (i.e., the yield), which took
10-13 days in most experiments and up to 40 days in the Porphyridium experi-
ments. Calibration curves relating absorbance at 650 nm and the number of
cells, using a Model B Coulter Countermand the Gary spectrophotometer were
prepared (Figures A1-A8). Approximately 20% of all readings were verified
by counting directly the number of cells with a Coulter Counter Model B or an
American Optical Bright-line hemacytometer and comparing with the calibration
curves (Table A15).
EXPERIMENTAL DESIGN
By means of a factorial experiment (6 ), the effects of a number of
different combinations can be investigated simultaneously. The decision to
use the factorial design was based on the desire to gain as much information
as possible on the interrelationships between the chemicals and their effects
on algal growth, and still work within the time and resource constraints
imposed by the project. To study the effects of all seven chemicals in all
combinations on a single alga would require 27 or 128 flasks with no repeti-
tions. Instead, it was decided to use for each alga four protocols of
16 flasks each, and also repeat some of the chemical combinations two and
three times in the four protocols. Each of the four protocols consisted of
15 treatments plus a control. In another protocol, a different combination
of four chemicals was tested. Not all possible, combinations of four chemicals
were tested, nor was any combination involving more than four chemicals tested.
The concentration of each chemical was 0.1 ppm. When a mixture was used,
the total concentration of chloro-organic chemicals was 0.2, 0.3, or 0.4 ppm
depending if 2, 3, or 4 chemicals were used in combination.
A typical experimental protocol is shown in Figure 2, where © means
the chemical is present and 0 means it is absent, e.g. combination (D
has chemicals: 3CP, 4CR, and 5CU.
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©
©
©
Vi>
(T
®
©
®
©
@
©
©
3CP© 8CC© 4CR© 5CU©
3CP© 8CC© 4CR© 5CU©
3CP© 8CC© 4CR© 5CU©
3CP© 8CC© 4CR0 5CU©
3CP© 8CC0 4CR© 5CU'©
3CP© 8CC0 4CR(J) 5CU0
3CP© 8CC0 4CR0 5CU©
3CP© 8CC© 4CR0 5CU©
3CP0 8CC0 4CR© 5CU©
3CP0 8CC© 4CR© 5CU©
3CP0 8CC© 4CR0 5CU©
3CP© 8CC© 4CR0 5CU0
3CP0 8CC0 4CR© 5CU©
3CP0 8CC0 4CR© 5CU©
3CP0 8CC0 4CR© 5CU©
3CP0 8CC0 4CR0 5CU0
Figure 2. Typical protocol for a tour-chemical factorial experiment.
(l) — (i?) represent the culture flask number in a given
^-^ experiment. Number 16 is the control.
3CP = 3-chlorophenol
4CR -'4-chloforesorcinol
5CU'• 5-chlorouridine
8CC = 8-chlorocaffeine
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ANALYSIS OF GROWTH DATA
When a combination of chemicals is added to a culture of algae, the
overall growth effect model of a combination is the sum of each chemical
taken alone plus any interactions among the chemicals that may occur. For
the combination of chemicals A and B, the growth effect would be the algebraic
sum of the effect of A alone plus the effect of B alone, plus any interaction
effect of A plus B together beyond the effects of either alone. Thus, the
overall yield, YA fi, will contain the following factorial components:
YA,B = V + k'A + k'B + K'^ + error factor,
where y = average yield of all flasks in a protocol.
k'A = main effect, due to chemical A
k'B = main effect, due to chemical B
k*AB = lnteraction effect, due to chemicals A plus B.
Since the error factor is not known, an estimate of Y. _ is obtained denoted
A A,B
as Y. „. Thus:
The main effects and interactions (k') for each component of Y were obtained
from the yield (k) of each treatment using computer analysis and the methods
of Yates (6). Once the k1 values were obtained, they were tested for signi-
ficance using the F test.
In the analysis of variance using the F test, the error variance was
calculated assuming that three and four factor interactions were negligible
( 6) and could be used as an estimate of the population variance. Thus, for
a.particular protocol, the effect means (k') for all three and four factor
interactions of a protocol were pooled and the error variance calculated.
The F test was then used to determine the significance of each one, two, and
any^large three and four factor interactions. If the F test showed a chemical
or interaction k value to be significant, the overall growth constant, ?,
for that treatment was calculated and tested for significant difference from
the control using the t-test.
UPTAKE OF CHLORO-ORGANIC CHEMICALS BY PHYTOPLANKTON
The chemicals used for these studies were ^C-labeled 3-chlorobenzoic
acid, 5-chlorouracll. and 4-chlororesorcinol. The two chemicals 5-chloro-
uracil and 4-chlororesorcinol were chosen because of their toxic potential (27),
whereas, 3-chlorobenzoic acid was selected since it was readily available in
10
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radioactive-labeled form. Four species were used in this study, two of which
were marine (Dunaliella tertiolecta and Skeletonema costatum) and two fresh-
water (Selenastrum capricornutum and Scenedesmus obliquus). Scenedesmus and
Selenastrum were grown in the medium described earlier. Since the modified
Burkholder's medium used in the growth studies did not give sufficient
biomass needed for the uptake studies, a fortified f/2 medium of Guillard and
Ryther (14) was substituted. The composition of the medium is as follows:
NaN03 (75 mg), NaH2P(VH20 (5 mg), NaSi03-9H20 (15-30 mg), Na2-EDTA (4.35 mg) ,
FeCl3-6H20 (3.15 mg), CuS(V5H20 (0.01 mg) , ZnS04-7H20 (0.022 mg) , CoCl2-6H20
(0.01 mg), MhCl2-4H20 (0.18 mg), NajpMoO^ • 2H20 (0.006 mg) , thiamine-HCl (0.1 mg) ,
biotin (0.5 ug), vitamin B12 (0.5 ug) in one liter of aged sea-water.
The above medium was filtered to remove any suspended material, buffered
with 5 mM glycyglycine, adjusted to pH 7.5, autoclaved, and allowed to stand
over night at room temperature. The following day, the previously described
autoclaved major nutrients and trace elements were added aseptically. A stock
solution of lJiC-chlorobenzoic acid, ^C-chlorouracil, or ll+C-chlororesorcinol
in ethanol or methanol was added to a suspension of exponentially growing
cultures with a cell density of 105 cells/ml for Scenedesmus and Selenastrum
and 106 cells/ml for Skeletonema and Dunaliella. The final concentration of
the ^C-chemical in the cell suspension was 1 ppm. The concentration of
methanol or ethanol did not exceed 0.08%. The rationale for using a higher
concentration of the chemical in the uptake studies was to facilitate detection
of the chemical because its uptake by the cells was low at 0.1 ppm and our
previous studies showed 3-chlorobenzoic acid, 5-chlorouracil, and 4-chloro-
resorcinolto be non-toxic to marine algae at a concentration of 1 ppm (27).
The cultures were agitated on a rotary shaker to keep the cells in suspension.
For determining uptake of chlorobenzoic acid, aliquots of cell suspension
were withdrawn at 0, 1, 2, 4, 7, 24, an,d 48 hr and filtered through a 0.45 ym
cellulose acetate Millipore© membrane filter. The filter and the cells were
rinsed consecutively with 5 ml, 10 ml, and 5 ml of fresh growth medium. The
filter containing the algae was then transferred to scintillation fluid, and
counted for 14C in a Packard Tri-Carb liquid scintillation counter.
In order to determine if the membrane filter retained 3-chlorobenzoic
acid, an aliquot of ll*O-chlorobenzoic acid solution without the algae was
passed through the membrane filter which was then rinsed consecutively with
5 ml, 10 ml, and 5 ml of the fresh medium. The filter was then transferred
to a scintillation vial and counted for radioactivity. Prior to starting the
uptake studies, a quench curve was established for the different species. The
amount of llfc associated with the cells was .corrected for iHC retention
(i.e. cpm) by the filter and for cellular quenching.
The procedure used to determine the uptake of 14C-chlorouracil was
similar to 3-chlorobenzoic acid-UL-14C. Due to thejiigh retention of
5-chlorouracil by the cellulose acetate Millipore ^membrane filter, this
filter was replaced by a Gelman ©DM-450 filter (0.45 \m pore diameter),-a
copolymer of acrylonitrile and polyvinyl chloride, which showed a very low
retention for the chemical. The amount of lt+C uptake was corrected for this,,
filter retention as well as for quenching.
11
-------
In uptake studies studies with ^C-chlororesorcinol, it was necessary,
however, to use centrifugation as a means of collecting and washing cells
since all the membrane filters tested retained a significant amount of the
chemical, which interfered with the measurement of the chemical's uptake by
the cells. At each time point, duplicate 5-ml cell suspensions were centri-
fuged for 5 minutes at 2500 rpm. The supernatant was removed and the pellet
washed twice by suspending it in 10 ml of the fresh medium without the chemical
and then centrifuging the suspension. After the washings, the cells were
extracted with 5 ml of ethanol and then transferred to a scintillation vial
for counting for radioactivity. Separate controls indicated that the adsorp-
tion of the chemical to the glassware was too low to be detected.
METABOLISM OF 4-CHLORORESORCINOL BY PHYTOPLANKTON
In order to obtain sufficient cellular material for studying metabolism
of chlororesorcinol by Skeletonema and Selenastrum. the algae were cultured
in Fernbach flasks in 2 liters of the appropriate growth medium. ^C-Chloro-
resorcinol was added to the cell suspension when it reached a density of
approximately 10b cells/ml. After it was determined that the cells contained
sufficient radioactivity for chromatographic purposes, the cells were separated
from the medium by centrifugation. The pellet was resuspended in the growth
medium, centrifuged, the supernatant discarded. This pellet was then extrac-
ted twice with methanol. The methanol extract was concentrated under vacuum
and spotted on thin- layer silica-gel and cellulose plates. The plates were
developed in the following systems:
Cellulose plates
1) water-saturated toluene race tic acid (4:1)
2) ethanol: water: ammonium hydroxide (16:3:1)
Silica-gel plates
1) chloroform: ethyl acetate: acetic acid (100:5:3)
2) toluene :methanol: acetic acid (45:8:4)
Authentic ^C-chlororesorcinol was co chroma to graphed for comparison with the
III? Were SCanned for radioactivity on a Nuclear Chicago
i-onH contai?lng the labeled chemical but no algae was acidified
to PH 2 and extracted with ethyl ether. The ether extract was counted for
C, concentrated under vacuum, and chromatographed as described above.
PHOTODEGRADATION OF 4-CHLORORESORCINOL
H*nn f aq^OUS S°lutl0n °f Chlororesorcinol was irradiated with a 450-Watt
ex^nX/tf ^ PfeSSUr? ***?? lamp fitted with a py«* 7740 filter which
amotnl oV^i^f WT ng^ fess than 280 nm. As there is no significant
amount of radiation in sunlight with a wavelength less than 280 run, it is
12
-------
assumed that the photodegradation of the chemical in aqueous solution in
these studies would be similar to that occurring in the sunlight. The
irradiation was carried out in a photochemical reactor (Ace Glass Company),
which consists of a jacketed borosilicate glass vessel and is equipped with a
side arm for withdrawing samples. A double-walled water-cooled quartz well,
housing the light source and filter., is fitted into the vessel and is immersed
in the solution to be irradiated. Aliquots of photolyzed solution were with-
drawn at appropriate intervals and analyzed for chlororesorcinol by high-
pressure liquid chromatography. The instrument and column used were the same
as those described for the purification of 5-chlorouracil. A solvent system
consisting of acetonitrile:5% glacial acetic acid in water (50:50 v/v) was
used to analyze the chemical. The retention volume of chlororesorcinol on
the HPLC column was 3.8 ml. Chlororesorcinol was detected by its absorbance
at 283 nm.
13
-------
SECTION 5
RESULTS
EFFECTS OF CHLORO-ORGANIC CHEMICALS ON THE GROWTH OF PHYTOPLANKTON
It is quite likely that algae in the bodies of water receiving chlor-
inated waste-waters will be exposed to several chloro-organic chemicals
simultaneously. As a result, there may be either synergistic or antagonistic
effects produced by the interaction of these chemicals. Individually, a
given chemical may not be toxic to certain species. However, the response
of a species to a particular chemical may be altered by the presence of
another chemical(s). Therefore, in evaluating the effects of chloro-organic
chemicals, the interaction among various chemicals should also be considered.
Interaction among certain organic pollutants in marine algae has been
recently reported by Mosser et al. (22) . They observed that DDT counteracted
the toxicity of PCB's in a marine diatom, whereas DDE and PCB's acted syner-
gistically in inhibiting the growth of the alga. In these studies, we
examined the effects of the chemicals alone, as well as in combination with
other chloro-organic chemicals to determine if the chemicals interact with
each other.
It is well known that phytoplanktonic species vary greatly in their
response to pollutants. Dunstan (8) recently reported that different
phylogenetic groups of marine phytoplankton and species within these groups
varied considerably in their response to effluent from the same sewage
treatment plant. Since the results based on the effects on one or two
species can be misleading, we examined the effects of the chemicals in
several algal species.
Yield data of algae with selected chemicals are shown in Tables A1-A14.
Of the 278 different treatments representing single chemicals and their
combinations with 7 algal species, only 21 were found to either significantly
stimulate or inhibit algal growth. Of these 21 treatments, the effects due
to 9 treatments, when tested in other protocols, were found to be non-signi-
ficant. Chloroguanine may possibly have an effect on Thalassiosira. It
reduced the growth by 6% in one protocol and by 8.5% in another. The effects
of the remaining 11 significant treatments were tested only once, with six
treatments being stimulatory and five being inhibitory.
Because of these inconsistent results, most if not all of the significant
results must be treated as being inconclusive. As a result, it is concluded
that these chemicals at Q.lppm alone or in combinations of up to four
chemicals had little effect, if any, on the yield of algae tested. It is
14
-------
suggested, however, that further selected testing should be carried out to
substantiate one way or another the effects of some of these chemicals on
algal growth.
EFFECT OF NITRATE CONCENTRATION ON THE TOXICITY OF THE TEST CHEMICALS
Environmental factors such as temperature, salinity, light, pH and
nutrients influence the metabolic activity of algae. It is expected that
the response of the organisms to toxicants may vary with changes in these
factors. An organism may become more susceptible to outside stress under
environmental conditions which do not favor its optimal growth (3, 11,
19,20,21). Hannan and Patouillet (16) observed that the toxicity of mercury
to algae increased with decreasing nutrient concentrations. Fisher e± al. (11)
reported that the growth of the diatom Thalassiosira was unaffected by PCBs
in high nitrate media, but was substantially reduced in the media containing
lower nitrate levels.
In order to assess the influence of nutrient level on the toxicity of
the chloro-organic chemicals, the algae were grown in the media containing
the test chemical(s) and varying amounts of nitrate. Two nitrate levels
were examined: (i) that specified in the Algal Assay Procedure, EPA (1971)
and (ii) 10% of this level. These studies included the effects of three
chloro-organic chemicals on Selenastrum and Scenedesmus.
As shown in Figures Sand 4, nitrate deprivation does not appear to
have any influence on the effects of the chloro-organic chemicals.
UPTAKE OF CHLORO-ORGANIC COMPOUNDS BY PHYTOPLANKTON
These studies were done using ll4C-labelled chemicals. The chemicals
used for these studies were 3-chlorobenzoic acid, 4-chlororesorcinol, and
5-chlorouracil. The species included in these studies were Dunaliella
tertiolecta. Skeletonema costatum (both marine), Selenastrum capricornutum
and Scenedesmus obliquus (both fresh-water).
The uptake studies were carried out at an initial concentration of 1 ppm
of the test chemical in the medium and a cell density of 10 - 10 cells/ml.
The rationale for using a higher concentration of the chemicals and a higher
cell density compared to the growth experiments was to facilitate detection
of the chemicals if their uptake by the organisms was low.
4-Chlororesorcinol; The uptake of l4C-4-chlororesorcinol by all four species
was rapid (Table 1). In the cases of Scenedesmus, Selenastrum, and Skeletonema,
uptake of the chemical reached its maximum within one hour of treatment,
whereas the maximum uptake in Dunaliella was observed 24 hours after treat-
ment. The four species varied in their ability to accumulate chlororesorcinol
from the medium. They accumulated the chemical in the following order:
Skeletonema > Dunaliella > Selenastrum > Scenedesmus.
15
-------
I
CO
X10~1
o
IS
XI0
p-2
Q
1
4>rf
a
O
X10~a
D Control With Full N03 (4.2 mg N/1)
• Growth With Full IM03 + All 3 Chemicals
O Control With 1/10 NO3 (0.42 mg N /1)
A Growth With 1/10 NO3 + All 3 Chemicals
_L
1
1
6 8
Days After Treatment
10
12
14
Figure 3.
l ™ 3-chi°r°Phenol» 4-chlororesorcinol and 5-chlorouracil
0.1 ppm on the growth of Scenedesmus obliguu» at two levels
of nitrate concentration."
16
-------
I
CN
O)
(O
X10
D Control With Full NO3 (4.2 mg N /1)
• Growth With Full N03 + All 3 Chemicals
O Control With 1 /10 N03 (0.42 mg N /1)
A Growth With 1/10 N03 + All 3 Chemicals
X10
-3
, I , I.I. I .
I 1 1 1
6 8
Days After Treatment
10
12
14
Figure 4. Effect of 3-chlorophenol, 4-chlororesorcinol, and 5-chlorouracil
at 0.1 ppm on the growth of Selenastrum capricornuttim as a
function of two levels of nitrate concentration.
17
-------
TABLE 1. UPTAKE OF 14C-CHLORORESORCINOL BY PHYTOPLANKTON
Concentration of 14C-Residue in the cells-ppm ,
(expressed as chlororesorcinol equivalent) —
Hours after
treatment
0
1
2
4
.7±
24
0.
0.
0.
0.
0.
0.
Scenedesmus
obliguus
074^
039
048
048
040
036
(73.7)^
(38.7)
(47.1)
(47.9)
(39.7)
(36.1)
Selenastrum
capricornutum
0.102
0.107
0.151
0.093
(101.6)
(107.1)
(151.3)
(93.0)
Skeletonema
costatum
0.119
0.294
0.277
0.296
0.309
0.206
(118.
(293.
(277.
(295.
(308.
(206.
6)
7)
4)
7)
9)
3)
Dunaliella
tertiolecta
0.
0.
0.
0.
0.
083
074
097
130
182
(82.9)
(73.7)
(97.3)
(130.2)
(182.3)
00 a./ Calculated from the specific activity of C-chlororesorcinol.
b_/ Each value represents the mean of 4 replications.
Ł/ Values in parentheses indicate bioconcentration factors.
-------
TABLE 2. UPTAKE OF 14C-CHLOROURACIL BY PHOTOPLANKTON
VD
14
Concentration of C-Resldue in the cells-ppm .
(expressed as chlorouracil equivalent) —
Hours after
treatment
0
1
2
4
8
24
48
Scenedesmus
obliquus
0.011^(11)^
0.012 (12)
0.049 (49)
0.067 (67)
0.018 (48)
0.050 (50)
Selenastrum
capricornutum
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
Skeletonema
costatum
0.034 (33.7)
0.025 (24.9)
0.026 (25.7)
0.025 (25.1)
0.046 (45.6)
0.042 (42.3)
— ™ T-JL 1 "
Dunaliella
tertiolecta
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
___ _
aj Calculated from the specific activity of C-chlorouracil.
bj Each value represents the mean of 4 replications.
cj Values in parentheses indicate bioconcentration factors.
-------
14
TABLE 3. UPTAKE OF C-CHLOROBENZOIC ACID BY PHYTOPLANKTON
NJ
O
14
Concentration of C-Residue in the cells-ppm
(expressed as chlorobenzoic equivalents) —
Hours after
treatment
0
1
2
4
7
24
48
Scenedesmus
obliquus
0.000^(0)-'
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
0.000 CO)
0.000 (0)
Selenastrum
capricornutum
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
Skeletonema
costatum
0.007 (7.0)
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
Dunaliella
tertiolecta
0.004 (3.8)
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
0.000 (0)
a/ Calculated from the specific activity of C-chlorobenzoic acid.
b_/ Each value represents the mean of 4 replications.
c/ Values in parentheses indicate bioconcentration factors.
-------
5-Chlorouracil: Skeletonema and Scenedesmus appeared to remove small amounts
of chlorouracil from the medium, whereas Dunaliella and Selenastrum showed
no capacity for taking up the chemical (Table 2), In the case of Skeletonema,
maximum uptake of chlorouracil was noticed within one hour of treatment. How-
ever, in Scenedesmus maximum uptake was noticed 8 hours after treatment.
3-Chlorobenzoic Acid: None of the species appeared to take up the chemical
from the medium (Table 3).
Although the algae accumulated both chlororesorcinol and chlorouracil
from the medium, bioaccumulation of chlororesorcinol was greater than that
of chlorouracil. The concentration of ! ^-chlororesorcinol and its degrada-
tion products in the algae at equilibration ranged from 38 to 308 times the
concentration in the medium. In the case of algae treated with ^C-chloro-
uracil, the bioaccumulation ranged from about 26 to 67. The bioaccumulation
of chlororesorcinol and chlorouracil by phytoplankton is considerably lower
than that reported for chlorinated hydrocarbons (25,26). The lower uptake of the
two chemicals may be explained by their relatively high water-solubility
(lower lipophilicity)and their ,low pKa's (4,23,24). Since at the pH of the
medium used the chemicals are expected to be present mostly in an ionized
form, they are less likely to partition from the water into the algal cells.
METABOLISM OF 4-CHLORORESORCINOL BY PHYTOPLANKTON
For metabolism studies, only those chemical-algal systems were investi-
gated which showed sufficient uptake of the ltfC-chemical so that the radio-
activity in the cells could be characterized by chromatographic procedures.
For this reason, we examined only the metabolism of ^C-chlororesorcinol in
two species. Skeletonema and Selenastrum. Although 14C-chlorouracil was taken
up by the algae, the uptake was not sufficient to permit the characterization
of ^-material in the cells. To obtain sufficient amounts of * ^-materials
for chromatographic analysis, the metabolism studies were done with algae
grown in large batch-cultures.
In our initial studies on the metabolism of 14C-chlororesorcinol by
_Skeletonema. the cells were incubated with the chemical for 10 days. The
cells were then extracted and the extract chromatographed on thin-layer
silica-gel and cellulose plates.
Thin-layer chromatography of the cell extract on the cellulose plates
showed 3-4 major peaks. However, the peaks were poorly resolved and showed
considerable tailing. Because silica-gel plates showed better resolution
they were used for chromatography in the subsequent studies.
Table 4 shows that Skeletonema either extensively metabolized the
4-chlororesorcinol in vivo or took up its photodegradation products from the
medium. In order to distinguish between these possibilities, metabolism of
14C-chlororesorcinol was examined in Skeletonema Incubated with the chemical
in light as well as in the dark. In these studies the algae were incubated
with the ^C-chemical for 24 hours. Similar experiments were conducted to
21
-------
study the metabolism of lt+C-chlororesorcinol by Selenastrum. We also investi-
gated if chlororesorcinol was degraded by light in the culture medium without
the algae, x
TABLE 4. DISTRIBUTION OF UC-CHLORORESORCINOL AND ITS DEGRADATION PRODUCTS
IN SKELETONEMA INCUBATED WITH THE CHEMICAL FOR 10 DAYS
Compound
Unknown
4-chlororesorcinol
Unknown
Unknown
Unknown
14
% of 1Łlc in the
methanol extract
66
8
4
3
19
I/
Rf~
0.00
0.31
0.48
0.66
0.86
I/ Silica-gel; chloroform: ethyl acetate: acetic acid (100:5:3)
Thin-layer chromatography of the methanol extract of Skeletonema or
Sflen"t5um incubate<* with ^C-chlororesorcinol for 24 hours in the light
showed the presence of two ^C-materials (Table 5). One ^C-compound
co-chromatographed with authentic chlororesorcinol while the other radio-
er rao-
thafchlZr (S Tal^f nea*?* Ori8in indicating that it was more polar
than chlororesorcinol. All the "c in the extract from algae incubated with
^C-chlororesorclnol in the dark was present as the parent chemical Thln-
reaLerc5noTfo0r824aPhiC ^^ °f ^ Ster±le «"» *c«b.f d ^S' ^C- hlor
-
.
These results indicate that chlororesorcinol is degraded by Sght to compound (s)
resorclnol photoproducts from the medium. Further studies are needed to
distinguish between these possibilities. neeaea to
PHOTODEGRADATION OF 4-CHLORORESORCINOL
?"! r8^^ °f the ab°Ve exPeriments showed that chlororesorcinol is
degraded by light. These studies were done in a growth chlmber under light
° mSor:sofotflurerent larps- "
does not simulate sunlicht
22
-------
ro
OJ
TABLE 5. DISTRIBUTION OF C-CHLORORESORCINOL AND ITS DEGRADATION PRODUCTS IN SKELETONEMA AND SELENASTRUM INCUBATED
WITH THE CHEMICAL TN THE LIGHT AND DARK
Skeletonema Selanastrum
Compound . , 14
f % of C in 1
Light
4-chlororesorcinol 65
Unknown 35
the Cell Extract % of C in the Cell Extract
Dark Light Dark
100 89 100
11
Sterile Medium R^ Value — '
f
14
% of C in the Medium System I System II
Light Dark
96 100 0.41 0.43
4 — 0.00 0.00
~U Solvent System I: Chloroform:ethyl acetate:acetic acid (100:5:3)
Solvent System II: Toluene:methanol:acetic acid (45:8:4)
-------
It was noticed that 24% of the chemical had degraded within 5 hours of
irradiation with a 450-watt medium pressure mercury lamp fitted with a
Pyrex 7740 filter (Figure 5). After 24 hours, only 33% of the original
chemical was present in the solution. These findings confirm our earlier
results that chlororesorcinol is readily degraded by the action of light.
Further studies are recommended for characterizing the products resulting
from photodegradation of chlororesorcinol.
Since chlororesorcinol readily undergoes photodegradation, one would
expect that in bodies of water contaminated with chlororesorcinol, phyto--
plankton would be exposed to its photodegradation products. In order to
have comprehensive information on the interaction of chlororesorcinol with
phytoplankton, it is important to assess the toxicity and bioaccumulation
of the photodegradation products of the chemical by the organisms.
24
-------
NS
I I I I I I I I
24
Figure 5. Photodegradation of 4-chlororesorcinol in simulated sunlight
-------
SECTION 6
DISCUSSION
nrnHn^HH y M S*OWn.that several <*able chloro-organic chemicals
produced during chlorination of domestic waste-water do not affect the
growth of a number of fresh-water and marine phytoplankton species. The
chemicals were ineffective when added alone at a concentration of 0.1 ppm
JL™ °?mblnat!0Yf 2 5° 4 chemi<^s, indicating a lack of interaction
Se Llnrn , ^ f^"88 SU8g6St that at the concentrations tested,
on nrii^ Sr^- m±?alS *" nOt exPected fc° have any significant effect
on primary productivity in the aquatic environment.
aauatcor *"%*** ^ PhytoPlankton are less sensitive than other
tha? both f MmS Chloro-0^ani- compounds. Gehrs et al. (12) observed
earn e± J:chl°r°^acl1 and ^-chlororesorcinol decrea^dThe hatchability of
effect of ^pC°^Centratlon! as low as 1 Ppb. Xn order to full assegs th*
to aouatL ?nvf J0J°-°rganic chemicals on aquatic organisms, their toxicity
are Tnerallv'uch and.fi^es should be determined since these organisms
are generally much more sensitive to low concentration of chemicals.
appear to have no adverse effect on the phytoplankton themselves at the
"
a
chain may become exposed to this potentially hazardous compound?
resistanttomcroM^°r0 f T" iS ^^ tO make aromatic compounds more
resistant to microbial degradation which suggests that chlorine-containine
~s^rtf^r^
to determine the envirc^enta!
SLn^ t8 ?f, Łound "« to read«y ^grade, their effect in atc
organisms should be studied at concentrations higher than those used In these
26
-------
studies. Furthermore, the chloro-organic chemicals may be converted to other
stable products as a result of biological and/or nonbiological transformation
in the aquatic environment. To fully assess the effects of chloro-organic
chemicals on phytoplankton, we suggest that the effects of their transformation
products be determined.
27
-------
REFERENCES
1. Alexander, M. and B.K. Lustigman. 1966. Effect of chemical structure
on microbial degradation of substituted benzenes. J. Agr. Food Chem.,
14:410-413.
2. Barnhart, D.R. and G.R. Campbell. 1972. Effect of chlorination on
selected organic chemicals. Environmental Protection Agency, Water
Pollution Control Research Series 12020, EXG 03/72.
3. Batterton, J.C., G.M. Boush and F. Matsumura. 1972. DDT: Inhibition
of sodium chloride tolerance by the blue-green alga Anacystis nidulans.
Science, 176:1141-1143. ~~
4. Brown, D.J. 1970. The Pyrimidines. Chemistry of Heterocyclic Compounds
V. 16 (A. Weissberger, Ed.), Interscience, N.Y., 774 pp.
5. Brungs, W.A. 1973. Effect of residual chlorine on aquatic life. J.
Water Pollut. Cont. Fed., 45:2180.
6. Cochran, W.C. and C.M. Cox. 1957. Experimental Designs. 2nd ed.,
Wxley, N.Y., pgs. 122-139.
7. Cummings, R.B. 1976. The potential for increased mutagenic risk to
the human Copulation due to the products of water chlorination. Proc.
Conf. Environmental Impact of Water Chlorination Conf. 751096, Oak
Ridge National Laboratory, Oak Ridge, Tenn.
8. Dunstan, W.M. 1975. Problems of measuring and predicting influence of
effluents on marine phytoplankton. Environ. Sci. Technol., 9:635-638.
9. Federal Water Pollution Control Act, Amendments, Public Law 92-500,
U.S. Government. October 18, 1972.
10. Fisher, N.S., R.L. Guillard and C.F. Wurster. 1976. Effects of a
chlorinated hydrocarbon pollutant on the growth kinetics of a marine
diatom. In: Modeling Biochemical Processes in Aquatic Ecosystems
(R.P. Canale, ed.). Ann Arbor Science Publishers, Inc., p. 305.
11. Fisher, N.S. and C.F. Wurster. 1973. Individual and combined effects
of temperature and polychlorinated biphenyls on the growth of three
species of phytoplankton. Environ. Pollut., 5:205-212.
28
-------
1'2. Gehrs, C.W. , L.D. Eyman, R.L. Jolley and J.E. Thompson. 1974. Effect
of stable chlorine-containing organics on aquatic environments. Nature,
249:675.
13. Glaze, W.H., J.E. Henderson, J.E. Bell and V.A. Wheeler. 1973. Analysis
of organic materials in waste water effluents after chlorination.
J. Chromatogr. Sci., 11:580.
14. Guillard, R.R.L. and J.H. Ryther. 1962. Studies on marine planktonic
diatoms. Can. J. Microbiol., 8:229-239.
15. Guillard, R.R.L. 1975. Division rates. In: Handbook of Phycological
Methods: Culture Methods and Growth Measurements (JsR.Stein, ed.).
Cambridge Univ. Press.
16. Hannan, P.J. and C. Patouillet. 1972. Effects of mercury on algal
growth rates. Biotech. Bioeng., 14:93-101.
17. Jolley, R.L. 1974. Determination of chlorine-containing organics in
chlorinated sewage effluents by coupled 36C1 tracer-high-resolution
chromatography. Environ. Lett., 7:321-340.
18. Jolley, R.L. 1975. Chlorine-containing organic constituents in chlor-
inated effluents. J. Water Pollut. Control Fed., 47:601-618.
19. Kanazawa, T. and K. Kanazawa. 1969. Specific inhibitory effect of copper
on division in Chlorella. Plant Cell Physiology, 10:495-502.
20. Mandelli, E.F. 1969. The inhibitory effect of copper on marine
phytoplankton. Contrib. Mar. Sci., Univ. of Texas, Mar. Sci. Inst.,
14:47-57.
21. McFarlane, R.B., W.A. Glooschenko and R.C. Harris. 1972. The inter-
action of light intensity and DDT concentration upon the marine diatom
Nitzschla delacatissiroa. Hydrobiologia, 39:373-382.
22. Mosser, J.L., T.C. Teng, W.C. Walther, and C.G. Wurster. 1974. Inter-
action of PCBs, DDT and DDE in a marine diatom. Bull Envxron. Contam.
Toxicol., 12:665-668.
23. Prager, B., P. Jacobson and F. Richter. 1936. Beilstein Handbuch der
Orgfnischen Chemie. XXIV:318. Verlag Von Julius Springer. Berlin.
24. Richter, F. 1944. Beilstein Handbuch der Organischen Chemie. VI
(2nd ed.):818. Springer-Verlag, Berlin.
25. Rice, C.P. and B.C. Sikka. 1973. Uptake and metabolism of DDT by six
species of marine algae. J. Agr. Food Chem.,21:148-152.
29
-------
26. Rice, C.P. and H.C. Sikka. 1973. Fate of dieldrin in selected species
of marine algae. Bull. Environ. Contain. Toxicol. , 9:111-123.
27. Sikka, H.C. and G.L. Butler. 1977. Effect of selected wastewater
chlorination products on marine algae. EPA-600/3-77-029. U.S.
Environmental Protection Agency, Gulf Breeze, Florida. 38 pp.
28. U.S. EPA. 1971. Algal Assay Procedure Bottle Test. National Eutro-
phication Research Program, Corvallis, Oregon. U.S.GPO:1972-295
146/1.
29. U.S. EPA. 1974. Marine Algal Assay Procedure Bottle Test. Eutro-
phication and Lake Restoration Branch, Corvallis, Orgeon. EPA-660/3-
75-008.
30. West, R.A. and H.W. Barrett. 1954. Synthesis of chloropyrimidines by
reaction with N-chlorosuccinimide and by condensation methods.
J. Amer. Chem. Soc., 76:3146-3149.
30
-------
APPENDIX
31
-------
10'
K3
m
OD65Q 10-2
10
-3
1 1—r-rr
T 1—i i i i i n
106
Cells/ml
Al. Caption curve (eel! number „. optlcal denslty) for
Microcvstis aeruainosa
32
-------
D
I
§
10C
OD650 10
10
,-2
107
108
Cells/ml
109
Figure A2. Calibration curve (cell number vs., optical density) for
Microcystis aeruginosa.
33
-------
X101
X10°
E
c
o
in
-------
10°
1-1
10
-2
105
« ' ' ' '
1
106
Cells/ml
i" ' ' "i '
10'
Figure A4V 'Calibration curve (cfell number vs.' optical density) for
Porphyridiuitt sp.
35
-------
X10°
X10"1
E
c
o
in
0*
O
X10
-2
XI O-
X104
XI O5
XI O6
X107
Cells/ml
- Figure A5.
Calibration curve (cell number vs. optical density) for
Scenedesmua pbliquus.
36
-------
a
i
X10°
X10
1-1
E
c
o
in
Q*
O
X10-2
1—i i i i i 1 r 1—i i i i i
X10~3
X104
X105
_L_U L.
X106
Cells/ml
X107
Figure A6..
Calibration curve (cell number vs. optical density) for
Selenastrum capricorhutum.
37
-------
D
I
X10°
X10
,-1
o
ID
CO
Q
O
X1
-------
X10°
X10
,-1
E
e
o
ID
(O
Q
O
X10
-2
X10~3
X104
i i i r i
' ' 1 I I
X10S
X106
xto7
Cells/ml
Figure A8. Calibration curve (cell number vs. optical density) for
Thalassiosira pseudonana.
39
-------
TABLE Al. THE EFFECTS OF VARIOUS CHLORO-ORGANIC CHEMICALS AT 0.1 ppm ON THE YIELD OF
DUNALIELLA TERTIOLECTA
Experiment 1
Treatment
Biomass Main effects
Experiment 2
& Treatment
(cells/ml) interactions
CR
CP
CG
CC
CP,
CG,
CC,
CG,
CC,
CC,
CG,
CC,
CC,
CC,
CC,
7.4 x IO4 -.04 x IO4
8.2 x IO4 -.09 x IO4
CR
CR
CR
CP
CP
CG
CP, CR
CP, CR
CG, CP
CG, CR
CG, CP, CR
Control
7.0 x 10* -.14 x
7.4 x IO4 -.21 x
7.5 x IO4 -.24 x
7.8 x 10 .21 x
7.4 x 10 .14 x
7.8 x IO4 .24 x
7.5 x 10* -.12 x
7.6 x HT .09 x
7.4 x IO4 -.19 x
7.4 x 10 .14 x
7.0 x 10 -.21 x
8.0 x 10* -.16 x
/
6.9 x 10* .04 x
7.8 x IO4
io4
io4
io4
io4
io4
io4
IO4
IO4
io4
io4
io4
io4
io4
CC
CU
CUD
CB
CC,
CC,
CB,
CU,
CB,
CB,
CC,
CB,
CB,
CB,
CB,
CU
CUD
CC
CUD
CU
CUD
CU, CUD
CC, CU
CU, CUD
CC, CUD
CC, CU, CUD
Control
Biomass
(cells /ml)
7.2 x IO4
7.6 x IO4
7.2 x IO4
7.4 x IO4
7.6 x IO4
7.6 x IO4
7.6 x IO4
7.2 x IO4
7.4 x IO4
7.2 x IO4
7.2 x IO4
7.6 x IO4
7.8 x IO4
7.0 x IO4
7.4 x IO4
7.4 x IO4
Main effects &
interactions
.00
.15
-.15
.05
-.05
-.05
-.05
.00
.10
.00
-.10
.00
.25
-.20
.05
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
* Denotes significance at 5% level of significance.
-------
TABLE A2. THE EFFECTS OF VARIOUS CHLORO-ORGANIC CHEMICALS AT 0.1 ppm ON THE YIELD OF
DUNALIELLA TERTIOLECTA
Experiment 3
Treatment
CR
CB
CUD
CP
CB,
CR,
CP,
CB,
CB,
CP,
CB,
CB,
CB,
CP,
CB,
CR
CUD
CR
CUD
CP
CUD l
CR, CUD
CP, CR
CP, CUD
CR, CUD
CP, CR, CUD
Control
. .• -
Biomass
(cells/ml)
7.2
7.2
7.6
7.2
6.5
7.2
7.0
7.0
6.9
6.9
7.5
6.5
6.9
7.2
6.1
7.0
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
x IO4
Experiment A
Main effects & Treatment
interactions
-.19 x
-.33 x
.11 x
-.31 x
-.16 x
.09 x
-.09 x
-.01 x
-.13 x
-.24 x
.11 x
-.16 x
-.06 x
-.06 x
-.33 x
IO4
IO4
IO4
IO4
IO4
IO4
IO4
IO4
IO4
IO4
IO4
io4
io4
IO4
IO4
CR
CP
CG
CU
CP,
CG,
CR,
CG,
CP,
CG,
CG,
CP,
CG,
CG,
CG,
CR
CR
CU
CP
CU
CU
CP, CR
CR, CU
CU, CP
CR, CU
CP, CR, CU
Control
Biomass
(cells/ml)
6.7 x IO4
6.9 x IO4
7.3 x IO4
6.9 x IO4
7.0 x IO4
6.5 x IO4
6.9 x IO4
6.5 x IO4
6.9 x IO4
7.0 x IO4
6.9 x IO4
6.9 x IO4
7.0 x IO4
7.0 x IO4
7.6 x IO4
6.6 x IO4
Main effects &
interactions
.05
.10
.13
.23
.23
.00
.10
-.05
.05
.13
.23
-.08
.20
.15
-.08
x
x
X
X
X
X
X
X
X
X
X
X
X
X
X
IO4
IO4
IO4
IO4
IO4
IO4
io4
IO4
IO4
IO4
IO4
IO4
IO4
io4
IO4
* Denotes significance at 5% level of significance.
-------
TABLE A3. THE EFFECTS OF VARIOUS CHLORO-ORGANIC CHEMICALS AT 0.1 ppm ON THE YIELD OF
MICROCYSTIS AERUGINOSA
4s
N3
Experiment 1
Treatment
CR
CP
CG
CC
CP, CR
CG, CR
CC, CR
CG, CP
CC, CP
CC, CG
CG, CP, CR
CC, CP, CR
CC, CG, CP
CC, CG, CR
CC, CG, CP, CR
Control
Biomass
(cells/ml)
2.4 x 107
2.4 x 107
2.7 x 107
2.7 x 107
2.3 x 107
2.7 x 107
2.5 x 107
2.7 x 107
1.9 x 107
2.3 x 107
2.7 x 107
2.5 x 107
2.3 x 107
2.3 x 107
3.0 x 107
3.3 x 107
Main effects
interactions
.01 x 10?
-.13 x 107
.09 x 107
-.21 x 107
.29 x 107*
.16 x 107
.26 x 107
.31 x 107*
.11 x 107
.01 x 107
-.11 x 107
.09 x 107
.06 x 107
.09 x 107
.09 x 107
Experiment 2
& Treatment
CC
CU
CUD
CB
CC, CU
CC, CUD
CB, CC
CU, CUD
CB, CU
CD, CUD
CC, CU, CUD
CB, CC, CU
CB, CU, CUD
CB, CC, CUD
CB, CC, CU, CUD
Control
Biomass
(cells /ml)
2.7 x 107
2.2 x 107
2.8 x 107
2.9 x 107
2.6 x 107
2.6 x 107
2.7 x 107
2.7 x 107
2.7 x 107
2.5 x 10?
2.7 x 107
2.7 x 107
2.7 x 107
3.0 x 107
2.7 x 107
2.0 x 107
Main effects &
interactions
.08 x 108
-.11 x 108
.08 x 108
.12 x 108
.02 x 108
-.02 x 108
-.01 x 108
.07 x 108
.01 x 108
-.08 x 108
-.11 x 108
-.09 x 108
-.07 x 108
.19 x 108
-.07 x 108
* Denotes significance at 5% level of significance
-------
TABLE A4. THE EFFECTS OF VARIOUS CHLORO-ORGANIC CHEMICALS AT 0,1 ppm ON THE YIELD OF
MICROCYSTIS AERUGINOSA
to
Experiment 3
Treatment
CR
CP
CG
CU
CP,
CG,
CR,
CG,
CP,
CG,
CG,
CP,
CG,
CG,
CG,
CR
CR
CU
CP
CU
CU
CP, CR
CR, CU
CP, CU
CR, CU
CP, CR, CU
Control
Biomass
(cells/ml)
2.1
1.8
2.2
2.1
2.2
2.0
1.8
1.9
1.8
1.9
2.3
1.9
2.0
2.3
2.1
2.0
x IO7
x IO7
x 107
x 10'
x 10
x IO7
x 10 7
x IO7
x IO7
x 10
x 1Q7
x 10
x 107
x 10
x 10 '
x 107
Exoeriment 4
Main effects & Treatment
interactions
.13
-.05
.13
-.08
.13
.05
-.05
.03
-.03
.05
-.05
-.10
.00
.13
-.13
x 107
x 10?
x 107
x 107
x 107
x 107
x 107
x 107
x IO7
x 107
x 10?
x 107
x 107
x 107
x 107
CR
CB
CUD
CP
CB,
CR,
CP,
CB,
CB,
CP,
CB,
CB,
CB,
CP,
CB,
CR
CUD
CR
CUD
CP
CUD
CR, CUD
CP, CR
CP, CUD
CR, CUD
CP, CR, CUD
Control
Biomass
(cells /ml)
3.4 x
2.9 x
3.0 x
2.8 x
3.9 x
3.0 x
3.4 x
3.4 x
3.5 x
3.1 x
3.4 x
3.6 x
3.3 x
2.4 x
3.6 x
3.4 x
107
107
io7
io7
io7
io7
io7
io7
io7
io7
io7
io7
io7
io7
io7
io7
Main effects &
interactions
.16
.39
-.21
-.09
.19
-.26
-.09
.16
.19
-.01
.06
-.06
-.04
-.01
.31
x IO7
x IO7
x IO7
x IO7
x IO7
x IO7
x IO7
x IO7
x IO7
x IO7
x IO7
x IO7
x IO7
x IO7
x IO7
* Denotes significance at 5% level of significance.
-------
TABLE A5. THE EFFECTS OF VARIOUS CHLORO-ORGANIC CHEMICALS AT 0.1 ppm ON THE YIELD OF
PORPHYRIDIUM SP.
Experiment 1
Treatment
CR
CP
CG
CC
CP,
CG,
CC,
CG,
CC,
CC,
CG,
CC,
CC,
CC,
CC,
CR
CR
CR
CP
CP
CG
CP, CR
CP, CR
CG, CP
CB, CR
CG, CP, CR
Control
Biomass
(cells/ml)
3.7
3.5
4.8
3.3
2.5
3.0
3.7
4.7
4.1
4.9
4.6
3.5
2.7
3.9
2.5
2.9
5
x 10
x 10
5
x 10
x 105
x 105
x 10
5
x 10
5
x 10
x 10
x 105
c
x 10
5
x 10
5
x 10
x 105
x 105
x IO5
Main effects &
interactions
-.44
-.26
.49
-.14
-.04
-.34
.09
-.26
-.49
-.64
.66
-.01
-.79
.09
-.21
5
x 10
x 105
5
x 10
x 105
x 10
x 105
5
x 10
s
x 10
x 105
x 10
r
x 10
5
x 10
5
x 10
x 10
x 105
Experiment 2
Treatment
CC
CU
CUD
CB
CC,
CC,
CB,
CU,
CB,
CB,
CC,
CB,
CB,
CB,
CB,
CU
CUD
CC
CUD
CU
CUD
CU, CUD
CC, CU
CU, CUD
CC, CUD
CC, CU, CUD
Control
Biomass
(cells/ml)
6
1.6 x 10
1.5 x 106
6
1.3 x 10
1.9 x 106
1.8 x 106
1.6 x 106
6
1.5 x 10
6
1.4 x 10
1.4 x 106
1.4 x 106
r
1.5 x 10
g
1.3 x 10
A
1.5 x 10
1.1 x 106
1.6 x 106
1.7 x 106
Main effects &
interactions
-.01
-.01
-.16
-.09
.11
.06
-.16
.16
-.01
.04
-.06
.06
.16
.01
.09
x
x
X
X
X
X
X
X
X
X
X
X
X
X
X
6
10
io6
5
10
IO6
IO6
IO6
6
10
g
10
io6
io6
6
10
6
10
6
10
io6
io6
* Denotes significance at 5% level of significance.
-------
TABLE A6. THE EFFECTS OF VARIOUS CHLORO-ORGANIC CHEMICALS AT 0.1 ppm ON THE YIELD OF
PORPHYRIDIUM SP.
Experiment 3
Treatment
CR
CP
CG
CU
CP, CR
CG, CR
CR, CU
CG, CP
CP, CU
CG, CU
CG, CP, CR
CP, CR, CU
CG, CP, CU
CG, CR, CU
CG, CP, CR, CU
Control
Biomass
(cells/ml)
2.5 x 105
6.8 x 105
4.0 x 105
4.8 x 105
• " 5
2.5 x 10
5.2 x 105
4.2 x 105
5.5 x 105
4.7 x 105
3.5 x 105
5.0 x 105
4.1 x 105
5.4 x 105
5
4.9 x 10
4.6 x 105
3.5 x 105
Main effects &
interactions
.65 x 105
.75 x 105
.63 x 105
.15 x 105
c;
-.90 x 10
.98 x 105
.50 x 105
-.03 x 105
-.40 x 105
-.48 x 105
-.08 x 105
.35 x 105
.48 x 105
"5
-.53 x 10 •
-.48 x 10
Experiment 4
Treatment
CR
CB
CUD
CP
CB, CR
CR, CUD
CP, CR
CB, CUD
CB, CP
CP, CUD
CB, CR, CUD
CB, CP, CR
CB, CP, CUD
CP, CR, CUD
CB, CR, CP, CUD
Control
Biomass
(cells/ml)
9.7 x 105
7.9 x 105
9.7 x 105
8.5 x 105
c
9.4 x 10
11.7 x 105
9.9 x 105
9.9 x 105
9.2 x 105
9.0 x 105
10.9 x 105
7.5 x 105
9.4 x 105
«;
9.6 x 10
9.3 x 105
8.4 x 105
Main effects &
interactions
.75 x 105 *
-.38 x 105
5 *
1.13 x 10
-.70 x 105
c
-.58 x HT
.13 x 103
-.70 x 105
.25 x 105
-.03 x 105
-.58 x 105
.15 x 105
-.38 x 105
.20 x 105
c
.08 x 10
.45 x 105
* Denotes significance at 5% level of significance.
-------
TABLE A7. THE EFFECTS OF VARIOUS CHLORO-ORGANIC CHEMICALS AT 0.1 ppm ON THE YIELD OF
SCENEDESMUS OBLIQUUS
Experiment 1
Treatment
CU
CR
CP
CR, CU
CP, CU
CP, CR
CP, CR, CU
Control
Experiment 2
Biomass Main effects & Treatment
(cells/ml) interactions
2.2 x 10 6 .06 x 106 CG
1.8 x 106 .11 x 106 CUD
2.0 x 106 .04 x 106 CC
1.8 x 106 -.04 x 106 CB
1.9 x 10 .01 x 106 CUD, CG
1.6 x 106 .04 x 106 CC, CG
2.0 x 106 .09 x 106 CB, CG
CC, CUD
2.0 x 10 CB, CUD
CB, CC
CC, CG, CUD
CB, CG, CUD
CB, CC, CUD
CB, CC, CG
CB, CC, CUD, CG
Control
Biomass
(cells/ml)
1.9 x 106
2.0 x 106
1.7 x 106
2.0 x 106
1.8 x 106
2.0 x 106
2.2 x 106
2.1 x 106
1.8 x 106
2.0 x 106
1.9 x 106
1.8 x 106
1.8 x 106
1.9 x 106
2.0 x 106
1.8 x 106
Main effects &
interactions
.04 x 106
-.04 x 106
.01 x 106
• .04 x 106
-.09 x 106
.01 x 106
.04 x 106
.09 x 106
-.14 x 106
-.04 x 106
.04 x 106
.11 x 106
.04 x 106
-.04 x 106
.09 x 106
* Denotes significance at 5% level of significance.
-------
TABLE A8. THE EFFECTS OF VARIOUS CHLORO-ORGANIC CHEMICALS AT 0.1 ppm ON THE YIELD OF
SCENEDESMUS OBLIQUUS
JS
-J
Experiment 3_
Treatment
CU
CP
CG
CB
CP,
CG,
CB,
CG,
CB,
CB,
CP,
CB,
CB,
CB,
CG,
CU
CU
CU
CP
CP
CG
CG, CU
CP, CU
CG, CP
CG, CU
CG, CP, CU
Control
Biomass
(cells/ml)
1.9
2.0
1.9
2.1
2.2
2.0
1.9
1.9
2.2
1.8
1.9
1.9
1.9
2.1
1.9
1.9
x IO6
x IO6
x IO6
6
x 10°
x IO6
x IO6
x IO6
xlO6
A
x 10b
x IO6
x IO6
6
x 10b
x IO6
A
x 10°
x IO6
x IO6
Main effects &
interactions
.01 x
.04 x
-.09 x
.01 x
-.04 x
.09 x
-.06 x
-.09 x
-.04 x
-.01 x
.06 x
-.06 x
.04 x
.11 x
.01 x
io6
IO6
IO6
6
10b
io6
io6
io6
io6
A
iob
io6
io6
A
iob
io6
g
iob
io6
Experiment 4
Treatment
CUD
CC
CR
CP
CC,
CR,
CP,
CC,
CC,
CP,
CC,
CC,
CC,
CP,
CC,
CUD
CUD
CUD
CR
CP
CR
CR, CUD
CP, CUD
CP, CR
CR, CUD
CP, CR, CUD
Control
Biomass
(cells /ml)
8.2
9.2
8.2
8.8
8.8
8.8
8.8
8.2
9.2
10.0
8.2
8.8
9.2
9.0
8.0
8.8
x IO5
x IO5
x IO5
*
x 10
x IO5
x IO5
x IO5
x IO5
c
x 10
x IO5
x IO5
c
x 10
x IO5
Q
x 10
x IO5
x IO5
Main effects &
interactions
-.38 x
-.13 x
-.13 x
.43 x
-.13 x
-.03 x
.28 x
.48 x
.23 x
.28 x
.08 x
-.03 x
.08 x
.44 x
.13 x
1C5
IO5
io5
"i
IO5
ia5
io5
io5
io5
c
IO5
io5
IO5
c
IO5
io5
c
IO5
io5
* Denotes significance at 5% level of significance.
-------
TABLE A9. THE EFFECTS OF VARIOUS CHLORO-ORGANIC CHEMICALS AT 0,1 ppm ON THE YIELD OF
SELENASTRUM CAPRICORNUTUM
Experiment 1
Treatment
CU
CR
CP
CR, CU
CP, CU
CP, CR
CP, CR, CU
Control
Experiment 2
Biomass Main effects & Treatment
(cells/ml) interactions
6.3 x 106 -0.14 x 106 CG
6.1 x 106 .11 x 106 CUD
5.9 x 106 .24 x 106 CC
6.1 x 106 .01 x 106 CB
4.9 x 106 .24 x 106 CG, CUD
6.1 x 106 .11 x 106 CC, CG
5.6 x 106 .11 x 106 CB, CG
CC, CUD
5.9 x 10 CB, CUD
CB, CC
CC, CG, CUD
CB, CG, CUD
CB, CC, CUD
CB, CC, CG
CB, CC, CG, CUD
Control
Biomass
(cells /ml)
4.0 x 106
3.6 x 106
4.1 x 106
4.1 x 106
3.7 x 106
5.2 x 106
3.7 x 106
3.9 x 106
4.1 x 10
4.1 x 106
4.0 x 106
4.8 x 106
3.8 x 106
3.7 x 106
3.6 x 106
4.3 x 106
Main effects &
interactions
.09 x 106
-.21 x 106
.01 x 106
-.11 x 106
.09 x 106
.06 x 106
-.16 x 106
-.24 x 106
.39 x 106
-.39 x 106
-.29 x 106
.24 x 106
-.14 x 106
-.29 x 106
.06 x 10
* Denotes significance at 5% level of significance,
-------
TABLE A10. THE EFFECTS OF VARIOUS CHLORO-ORGANIC CHEMICALS AT 0,1 ppm ON THE YIELD OF
SELENASTRUM CAPRICORNUTUM
q
Experiment _
Treatment
CU
CP
CG
CB
CP,
CG,
CB,
CG,
CB,
CB,
CG,
CB,
CB,
CB,
CB,
CU
CU
CU
CP
CP
CG
CP, CU
CP, CU
CG, CP
CG, CU
CG, CP, CU
Control
Biomass
(cells/ml)
6
4.3 x 10
4.3 x IO6
4.5 x IO6
ft
4.3 x 10
4.3 x IO6
ft
4.9 x 10
4.5 x IO6
4.0 x IO6
ft
4.2 x 10
4.4 x IO6
ft
4.5 x 10
6
4.5 x 10
ft
4.3 x 10
4.2 x IO6
4.3 x IO6
4.5 x IO6
Experiment 4
Main effects & Treatment
interactions
.13
-.15
.03
-.08
.08
.05
-.05
-.08
.13
-.10
.00
.00
.10
-.23
.03
ft
x 10
x IO6
x IO6
ft
x 10b
x IO6
ft
x 10b
x IO6
x IO6
6
x 10
x IO6
6
x 10
ft
x 10°
ft
x 10
x IO6
x 10
CUD
CC
CR
CP
CC,
CR,
CP,
CRr
CC,
CP,
CC,
CC,
CC,
CP,
CC,
CUD
CUD
CUD
CC
CP
CR
CR, CUD
CP, CUD
CP, CR
CR, CUD
CP, CR, CUD
Control
Biomass
(cells/ml)
2.2 x
2.4 x
2.5 x
2.6 x
2.5 x
2.5 x
2.4 x
2.4 x
2.5 x
2.4 x
2.4 x
2.5 x
2.5 x
2.4 x
2.4 x
2.4 x
ft
iob
IO6
io6
ft
iob
io6
ft
iob
io6
io6
ft
iob
io6
ft
iob
ft
10
ft
iob
io6
io6
io6
Main effects &
interactions
-.05
.03
.00
.05
.05
.03
-.03
-.05
.00
-.08
-.08
-.03
.08
.00
.00
x
x
X
X
X
X
X
X
X
X
X
X
X
X
X
ft
10b
IO6
IO6
ft
io6
io6
ft
iob
io6
io6
ft
iob
io6
ft
iob
ft
iob
ft
10b
io6
IO6
* Denotes significance at 5% level of significance.
-------
TABLE All. THE EFFECTS OF VARIOUS CHLORO-ORGANIC CHEMICALS AT 0.1 ppm ON THE YIELD OF
SKELETONEMA COSTATUM
Experiment _1
Treatment
Blomass
(cells/ml)
CR
CP
CG
CC
CP,
CG,
Oi
0 CC,
CG,
CC,
CC,
CG,
CC,
CC,
CC,
CC,
CR
CR
CR
CP
CP
CG
CP, CR
CP, CR
CG, CP
CG, CR
CG, CP, CR
5.0
4.5
4.6
2.7
3.2
4.0
1.7
4.5
3.0
3.2
2.3
2.0
3.8
2.3
x 105
x 105
x 105
x 105
x IO5
x 105
x 105
x 105
x 105
x 105
x 105
x 105
x 105
x 105
2.3 x 105
Main effects &
Experiment 2
Treatment
interactions
-1.00
-.30
.05
-1.45
-.50
-.30
-.10
.00
.60
.50
-.05
.35
.00
.20
-.10
5*
x 10
x 105
x 105
xlO5*
xlO5*
x 105
x 105
x 105
5*
x 10
5*
x 10
x 105
x 105
x 105
x 105
x 105
CR
CB
CUD
CP
CB,
CR,
CP,
CB,
CB,
CP,
CB,
CB,
CB,
CP,
CB,
CR
CUD
CR
CUD
CP
CUD
CR, CUD
CP, CR
CP, CUD
CR, CUD
CP, CR, CUD
Biomass
(cells /ml)
3.3
3.3
3.1
2.8
2.6
2.8
2.6
2.8
2.6
2.1
2.6
2.6
1.9
2.1
2.8
x 105
x 105
x 105
x 105
x 105
x 105
x 105
x 105
x 105
x 105
x 105
x 105
x 105
x 105
x 105
Main effects &
interactions
-.21 x
-.26 x
-.50 x
-.69 x
.21 x
.31 x
.39 x
.26 x
.34 x
.09 x
.04 x
.06 x
-.09 x
-.04 x
.14 x
5 *
10
5 *
10J
5 *
10°
io5*
io5
5 *
10
5 *
10
5 *
10
IO5*
IO5
IO5
IO5
IO5
IO5
IO5
Control
4.5 x 10-
Control
4.5 x 10"
* Denotes significance at 5% level of significance,
-------
TABLE A12. THE EFFECTS OF VARIOUS CHLORO-ORGANIC CHEMICALS AT 0.1 ppm ON THE YIELD OF
SKELETONEMA COSTATUM
Experiment 3
Treatment
CR
CP
CG'
CU
CP, CR
CG, CR
CR, CU
CG, CP
CP, CU
CG, CU
CG, CP, CR
CP, CR, CU
CG, CP, CU
CG, CR, CU
CG, CP, CR, CU
Control
Biomass
(cells/ml)
4.0 x 105
5
3.8 x 10
5
4.3 x 10
4.5 x 105
3.8 x 105
4.5 x 105
4.5 x 105
5.0 x 105
2.1 x 105
5.0 x 105
4.0 x 105
5
4.7 x 10
4.5 x 105
5.0 x 105
3.8 x 105
3.2 x 105
Experiment
Main effects & Treatment Biomass Main effects &
interactions (cells/ml) interactions
.24 x 105
5
-.41 x 10
5
.69 x 10
.19 x 105
-.01 x 105
-.61 x 105
.24 x 105
.04 x 105
-.56 x 105
-.06 x 105
-.46 x 105
s
.49 x 10
.09 x 105
-.21 x 105
-.36 x 105
* Denotes significance at 5% level of significance.
-------
TABLE A13. THE EFFECTS OF VARIOUS CHLORO-ORGANIC CHEMICALS AT 0.1 ppm ON THE YIELD OF
THALASSIOSIRA PSEUDONANA
Experiment
Treatment
Biomass
(cells/ml)
CR
CP
CG
CC
CP,
CG,
CC,
CG,
CC,
CC,
CG,
CC,
CC,
CC,
CC,
CR
CR
CR
CP
CP
CG
CP, CR
CP, CR
CG, CD
CG, CR
CG, CP, CR
Control
2.6
3.1
3.1
3.0
3.1
2.7
2.6
2.8
3.1
2.8
3.1
3.3
2.6
2.5
2.6
2.9
x IO6
x IO6
ft
x 10b
x IO6
xlO6
x IO6
x IO6
x IO6
x IO6
6
x 10°
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
Main effects &
o
Experiment
Treatment
interactions
-.13
.18
-.18
-.13
.23
.03
.03
-.18
.03
-.18
.03
.03
-.03
-.03
-.08
x IO6
6 *
x 10°
Ł *
x 10
x IO6
6 *
x 10°
x IO6
x IO6
6 *
x 10
x IO6
A *
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
CC
CU
CUD
CB
CC,
CC,
CB,
CU,
CB,
CB,
CC,
CB,
CB,
CB,
CB,
CU
CUD
CC
CUD
CU
CUD
CU, CUD
CC, CU
CU, CUD
CC, CUD
CC, CU, CUD
Control
Biomass
(cells /ml)
2.5
2.3
2.6
2.5
2.2
2.3
2.8
2.6
2.4
2.8
2.7
2.5
2.4
2.5
2.4
2.5
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
Main effects &
interactions
-.03
-.13
.08
.08
.05
-.10
.05
.10
-.10
-.10
.13
-.03
-.13
-.08
.00
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
* Denotes significance at 5% level of significance.
-------
TABLE A14. THE EFFECTS OF VARIOUS CHLORO-ORGANIC CHEMICALS AT 0.1 ppm ON THE YIELD OF
THALASSIOSIRA PSEUDONANA
Experiment 3
Treatment
Biomass
(cells/ml)
CR
CP
CG
CU
CP,
CG,
CU,
CG,
CP,
CG,
CG,
CP,
CG,
CG,
CG,
_
CR
CR
CR
CP
CU
CU
CP, CR
CR, CU
CP, CU
CR, CU
CP, CR, CU
Control
3.1 x
3.4 x
3.1 x
3.0 x
3.0 x
2.8 x
3.1 x
2.8 x
2.8 x
2.8 x
2.9 x
2.9 x
2.9 x
2.6 x
2.4 x
3.1 x
IO6
IO6
IO6
IO6
IO6
IO6
IO6
IO6
IO6
IO6
IO6
IO6
io6
io6
IO6
IO6
Main effects &
Experiment 4
Treatment
interactions
-.14
-.06
-.26
.21
-.04
.09
.01
-.01
-.06
-.01
.06
-.04
.09
-.14
-.14
x IO6
x IO6
6 *
x 10
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
x IO6
CR
CB
CUD
CP
CB,
CR,
CP,
CG,
CB,
CP,
CB,
CB,
CB,
CP,
CB,
CR
CUD
CR
CUD
CP
CUD
CR, CUD
CP, CR
CP, CUD
CR, CUD
CP, CR, CUD
Control
Biomass
(cells/ml)
3.4 x IO6
3.4 x IO6
3.4 x IO6
3.6 x IO6
3.4 x IO6
3.6 x IO6
3.5 x IO6
3.7 x IO6
3.6 x IO6
3.3 x IO6
3.6 x IO6
3.6 x IO6
3.3 x IO6
3.3 x IO6
3.3 x IO6
5.0 x IO6
Main effects &
interactions
-.20
-.18
-.23
.28
.18
.23
.18
.25
.15
.00
-.25
-.15
-.23
-.20
.23
x
x
X
X
X
X
X
X
X
X
X
X
X
X
X
IO6
IO6
IO6
IO6
io6
io6
IO6
io6
io6
io5
io6
io6
io6
io6
io5
* Denotes significance at 5% level of significance.
-------
TABLE A15. REPRESENTATIVE DATA COMPARING CELL NUMBERS OBTAINED BY DIRECT
COUNTING AND FROM A CALIBRATION CURVE FOR PORPHYRIDIUM SP.
Cells/ml
I/ 2/
Direct cell count— Calibration curve—
1.0 x 105 1.2 x 105
3.2 x 105 3.2 x 105
3.6 x 105 3.6 x 105
8.0 x 105 9.0 x 105
1.1 x 106 9.6 x 105
1.1 x 106 1.1 x 106
1.1 x 106 1.2 x 106
1.2 x 106 1.4 x 106
2.0 x 106 2.1 x 106
2.5 x 106 2.6 x 106
I/ Direct cell count made with an A-0 Bright-Line hemacytometer.
"if Calibration curve related cell number to optical density
measurements.
54
-------
r
TECHNICAL REPORT DATA
(Please read In'tructions on the r,.\wsc before completing)
1. REPORT NO.
EPA-600/3-79-075
3. RECIPIENT'S ACCESSION NO.
4. TITLE ANDSUBTITLE
EFFECTS AND FATE OF SEWAGE CHLORINATION
PRODUCTS IN PHYTCPLANKTON
REPORT DATE
.inly 1975 issuing date
,. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Sikka, Harish C., Knowlton C. Foote, James
I. Mangi, and Eduiard J. Pack.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Life Sciences Division
Syracuse Research Corporation
Syracuse, New York 13210
8. PERFORMING ORGANIZATION REPORT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Marine Division
Corvallis Environmental Research Laboratory
U. S. Environmental Protection Agency
Corvallis, Oregon 97330
10. PROGRAM ELEMENT NC
13A819
11. CONTRACT/GRANT NO.
R-804-936-010
13 TYPE OF REPORT AND PEPIOD COVERED
Final r
. SPONSORING AGENCY CODE
EPA/600/02
15. SUPPLEMENTARY NOTES
16. ABSTRACT
3-Chlorophenol. 3-chlorobenzolc acid, 4-chlororesorcinol, 5-chlorouracil,
5-chlorouridine, 6-chloroguanine or 8-chlorocaf feine at ""^"Jj^
0.1 ppm, alone or in combinations of up to 4 chemicals, had no **S«J"cant
effect on the yield of Scenedesmus obliquus, Selenastrum capricornutum.
~ • Skeletonema cotatum,
sp.
5-chlorouracil^e-Eak^n up by certain species but .
accumulated to a high level. The uptake of chlo^o "sorcinol "**
greater than that of chlorouracll. The uptake of 3-chlorobenzoic acid by
the phytoplankton was negligible.
4-Chlororesorcinol was readily degraded in aqueous so lution by the
action of simulated sunlight and both Skeletonema and Selenastrum took up
chlororesorcinol as well as its photodegradation products from the medium.
17.
Neither Skeletonema nor Selenastrum were able to «*tabo"" J-
resorcinol in the dark but appeared to transform it to some extent into
more polar material(s) in the light.
KEY WORDS AND DOCUMENT ANALYSIS
!______ DESCRIPTORS •
Phytoplankton, chlorine organic
compounds, chlorination, sewage
treatment, metabolism.
, |
b.lDENTIHERS/OPEN ENDED^TERMS
'875771 HI BUTION STATEMENT
release unlimited
uptake, 3-chloro-
phenol, 3-chloroben-
zoic acid, 4 chloro-
resorcinol, 5-chlcro-
uracil, 5-chlorouri-
dine, 6-chloroguanine
8-chlorocaffeine
20 SECURITY CLASS (TMspage)
unclassified
°'m 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
55
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