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This is to certify that the

thesis entitled

THE CHRONIC EFFECTS OF THE PHOTO-ENHANCED TOXICITY
OF ANTHRACENE 0N DAPHNIA MAGNA REPRODUCTION

presented by

Linda Lee Holst

has been accepted towards fulfillment
of the requirements for .

_M._S_._ degree in FISH . &WILDL .

Major pro

Date APRIL 10, 1987

 

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THE CHRONIC EFFECTS OF THE PHOTO-ENHANCED TOXICITY
OF ANTHRACENE ON DAPHNIA MAGNA REPRODUCTION

BY

Linda Lee Holst

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Fisheries and Wildlife

1987

ABS TRACT

THE CHRONIC EFFECTS OF THE PHOTO-ENHANCED TOXICITY OF
ANTHRACENE ON DAPHNIA MAGNA REPRODUCTION

BY
Linda Lee Holst

The chronic effects of polycyclic aromatic
hydrocarbons (PAH) and‘UV-radiation exposure on zooplankton
reproduction were investigated. Daphnia magma were exposed
to anthracene, a linear 3-ring PAH, in the presence or
absence of ecologically relevant intensities of UV-
radiation for 21 days. Exposure to anthracene in the
absence of UV-radiation significantly reduced the number of
neonates produced by Q. m; however, exposure to UV-
radiation in the absence of anthracene had no significant
effect on the fecundity of Q. m. Concurrent exposure
of 2. means to UV%radiation enhanced the toxicity of
anthracene and caused a reduction in the number of neonates
produced, which was proportional to both anthracene
concentration and UV-radiation intensity. An equation was
developed which predicts the relative percent reduction in
neonate production due to the photo-enhanced toxicity of
anthracene given an anthracene concentration and UV-

radiation intensity.

ACKNOWLEDGMENTS

I would like to express my gratitude to my major
professor, Dr. John P. Giesy, for his guidance and support
during my degree program which has made me a better
scientist. I would also like to thank the other members of
my graduate committee, Dr. Darrell L. King and Dr. Donald
J. Hall, fer their advice and instruction. Special thanks
to Dr. James F. Kitchell whose enthusiasm and encouragement
strongly influenced my pursuance of an advanced degree. I
would also like to thank my family for their love and
encouragement. Appreciation is extended to my friends at
the Pesticide Research Center, past and present, for their
stimulating discussions and close friendships. This
research was supported in part by the Michigan.Sea Grant
College Program Project No. R/TS-Z/ and by the Michigan

Agricultural Experiment Station.

11

LIST OF TABLES . . .
LIST OF FIGURES . .
~INTRODUCTION . . . .
MATERIALS AND METHODS
Zooplankton . .
Algae . . . . .

Exposure System
Experimental Des

TABLE

ign .

Statistical Analysis

RESULTS . . . . . .

Anthracene Effects .
UV-Radiation Effects

Combined Effects of Anthracen

OF CONTENTS

and UV-Radiation .

DISCUSSION . . . . .
CONCLUSIONS . . . .

APPENDIX A . . . . .
APPENDIX B . . . . .

LIST OF REFERENCES .

iii

12
12
13
13
19
24
25

25
25

30

48

54

55
56

62

LIST OF TABLES

Table

1 Modified Woods Hole-MEL Media for Algae
Cultures . . . . . . . . . . . . . . . . . .

2 Experimental Conditions of Light
Intensity, Anthracene Concentration and
Temperature . . . . . . . . . . . . . . . .

3 Average Number of Neonates per Brood
and Total Number of Neonates Within the
First 6 Broods Produced by Q. magna
Exposed to Anthracene in the Absence of
UV-Radiation e e e s e e e e e e s e e e e

4 Average Number of Neonates per Brood
and Total Number of Neonates Within the
First 6 Broods Produced by Q. magga
Exposed to UV-Radiation in the Absence
of Anthracene . . . . . . . . . . . . . . .

5 ANOVA of Model That Predicts the
Relative Percent Reduction in
Production of Neonates by Surviving Q.
magna Given an Anthracene Concentration
andflUV-Radiation.Intensity'.. . .. . . ..

6 ANOVA of Model That Predicts the
Relavive Percent 1Reduction in
Production of Neonates by Q. magna
(Including Adults Which Did Not Surv vs
21 D) Given an Anthracene Concentration
and UV-Radiation Intensity .. . .. .. .

7 Concentrations of Total PAH in Various
River Waters (Neff, 1979) . . . . . . . .

8 Concentrations of PAH (ug/L) in Great
Lakes Water (Eadie, et al., 1982) . . . .

A Neonates per Brood Produced in 21 Days

per 9. magma Fed Chlamydomonas
reinhardti O O O I O O O O I O D O O O O 0

iv

Table

B1

B2

B3

Neonates per Brood Produced per 2.
magna in 21 Days During Experiment 1

Neonates per Brood Produced per 9.
magga in 21 Days During Experiment 2

Neonates per Brood Produced per 2.
magga in 21 Days During Experiment 3

‘7

. . 58

. . 60

Figure

LIST OF FIGURES

Structure of Some Commonly Occurring
Polycyclic Aromatic Hydrocarbons . .

Percent Reduction in TOTLGBRD by
Surviving Q. magna Exposed to
Anthracene LActual Concentrations)
and UV-Radiation Relative to
TOTLGBRD by 2. ma na Not Exposed to
Anthracene or UV-Rad ation . . . . .

Mean Cumulative Number of Neonates
Produced by Surviving Q. magna
Expose to a UV-A Intensity of 117
uW/cm and Anthracene (Actual
Concentrations) ... .. .. .. ..

Mean Cumulative Number of Neonates
Produced by Surviving Q. magna
Exposed to 8 ug Anthracene/L
(Approximate Concentrations) and UV-
Radiation O O O O O O O O O O O O O 0

Percent Reduction in TOTLGBRD by
2. mafia (Including Adults Which Did
Not Survive 21 D) Exposed to
Anthracene (Actual Concentrations)
and UV-Radiation Relative to
TOTLSBRD by 2. ma na Not Exposed to
Anthracene or UV-Rad ation . . . . .

Mean Cumulative Number of Neonates
Produced by D. magna (Including
Adults Which Did Not Survive 21 D)
Expose to a UV-A Intensity of 117
uW/cm and Anthracene (Actual
Concentrations) .. . .. . .. . ..

Mean Cumulative Number of Neonates
Produced by Q. magna (Including
Adults Which Did Not Survive 21 D)
Exposed to 8 ug Anthracene/L
(Approximate Concentrations) and UV-
Radiation . . . . . . . . . . . . . .

vi

32

34

36

38

44

46

INTMNHKHHON

Polycyclic aromatic hydrocarbons (PAH) are a class of
organic compounds consisting of two or more fused benzene
rings with occasional inclusions of heteroatoms or
cyclopentene rings. The most commonly studied PAH which
are found in the environment include: fluorene, acridine,
anthracene, phenanthrene, fluoranthene, pyrene,
benzo[a]pyrene (BaP), and perylene (Figure 1); however,
many other PAH have been recognized as environmental
contaminants. Although natural processes such as forest
fires and volcanic eruptions release PAH into the
environment, inputs from domestic and industrial pyrolytic
activities are the major sources of PAH (Laflamme and
Hites, 1978). Large quantities of PAH are released into the
environment each year due to human activities, such as oil
spills, fossil fuel combustion, and incineration, as well
as many industrial processes (Mix, 1984; Suess, 1976).
Annual emissions of B[a]P in the United States are
estimated to be between 900 and 1300 tons (Dipple, 1983).
Heat and power generation contribute approximately 38% of
the total, open refuse burning 42 to 46%, coke production
15 to 19% and motor vehicles 1 to 1.5% (Dipple, 1983).

PAH can enter aquatic systems by different routes.

The total annual PAH input into the aquatic environment has

O D Fluorene
O O
N Acridine

30? ......c...

Phenanthrene
Fluoranthene

FYI-'93.

Benzo[a]pyrene

Perylene

 

Figure 1. Structures of Some Commonly Occurring
Polycyclic Aromatic Hydrocarbons.

been estimated to be 230,000 metric tons (Neff, 1979).
This input is expected to increase substantially due to
projected increases in coal benefaction processes (Gehrs,
1976).

Aerial transport is the major source of trace
contaminants to the Great Lakes (Eisenreich et al., 1981).
The atmospheric deposition of PAH to the Great Lakes is
estimated to be 484 metric tons per year (Eisenreich et
al., 1981). The flux of total PAH into southern Lake
Michigan has been measured to be approximately 1x105-106
kg/yr in dry flux and 13:106 kg/yr in wet flux (Strand and
Andren, 1980).

In addition to atmospheric deposition, substantial
amounts of PAH can enter the aquatic environment from oil
spills and runoff. Approximately 6x106 tons of oil enter
the oceans each year (NAS, 1972). Runoff from coal piles
may contain numerous PAH. Simulated rainfall runoffs from
model coal piles contained up to 107 ug/L of certain PAH
(Stahl et al., 1984).

PAH are nonpolar compounds, therefore, they have low
aqueous solubilities, are lipophilic and are readily
bioconcentrated by aquatic organisms. A freshwater
crustacean, Daphnia pulex, accumulated benz(a)anthracene to
concentrations 10,109 times and anthracene 917 times over
that of the water concentrations (Southworth et al., 1978).
Chironomids were able to bioconcentrate anthracene between

47 and 132 times over the amount dissolved in water

(Gerould et al., 1983). In addition, BCF's for anthracene
and B[a]P in bluegill sunfish were determined to be 675 and
490, respectively (Specie et al., 1983).

PAH are of concern. in the aquatic environment
not only because they are ubiquitous contaminants that
can be bioconcentrated, but also because many PAH are
carcinogenic and/or toxic. Much of the research conducted
on PAH has been concerned with their carcinogenic
properties. Many PAH including benzo[a]pyrene (BaP),
dimethylbenz[aJanthracene (DMBA), and methylcholanthrene
are potent carcinogens and have caused malignant skin and
lung tumors in laboratory animals (EPA, 1980).

Some PAH have been shown to be toxic: however; many of
the laboratory tests, which found PAH to be acutely toxic
to organisms, used carrier solvents that resulted in PAH
concentrations well above their respective aqueous
solubility limits (Neff, 1979). Other laboratory tests have
indicated that PAH are not acutely toxic within their
aqueous solubility limits (Herbes et al., 1976; Applegate
et al., 1957), however, many tests of PAH toxicity were
conducted under environmentally unrealistic laboratory
lighting regimes in order to reduce PAH photodegradation.
Recent studies that have incorporated natural or simulated
solar ultraviolet radiation have demonstrated PAH to be
extremely toxic to aquatic organisms at concentrations well

below aqueous solubility limits.

PAH photosensitization in organisms has been well
recognized for many decades. The photo-induced toxicity of

PAH to Paramecium (Mottram et al., 1938) and Drosophila

 

(Maltotsy and Fabian, 1946) by ultraviolet radiation (<400
nm) was observed.more than 40 years ago. IMore recently,
photo-induced lethality by PAH has been demonstrated in
hamster, mouse and human cells (Utsumi and Elkind, 1979).
Photo-toxic reactions of the skin and eyes of outdoor
workers exposed to asphalt, roofing materials and creosote-
treated wood have been well documented (Kochevar et al.,
1982). In addition, products made from crude coal tar
which contain PAH are also used in photo-therapy treatment
of psoriasis (Tanenbaum et al., 1975; Kochevar et al.,
1982).

Although the photo-toxic effects of PAH have been
recognized for many years, their effects on aquatic
organisms have been ignored until recently. Recent studies
have demonstrated variable PAH toxicities which were

dependent on the PAH and organism being examined. B(a)P

 

was inhibitory to the growth of the green algae Selenastrum
capricornutum in the'presence of fluorescent black light
(Cody'et.aln, 1984). However, the green alga Chlorella
pyrenoidosa was not adversely affected by anthracene
exposure in the presence of UV-radiation (Oris et al.,
1984). The photo-toxic effects of PAH are much more
dramatic for zooplankton. Photo-induced effects of

anthracene on the water flea Daphnia pulex were

demonstrated (A1 lred and Giesy, 1985). Under natural solar
radiation, 100% of the 2. M were immobilized in less
than 2 minutes at a concentration of 32.7 ug/L, 100% were
immobilized in less than 10 minutes at 7.5 ug/L, and 50%
were immobilized in less than 15 minutes at 1.2 ug/L. In
addition to these studies, 1 h-Lcso values of <20 ug/L were
determined for the photo-toxicity of pyrene, fluoranthene
and anthracene to Q. m (Kagan et al., 1985). Mosquito
larvae (Aedes a_e_gypti) are also sensitive to the photo-
induced toxicity by PAH (Oris et al., 1984: Kagan et al.,
1985, 1986) as well as embryonic forms of the frog ME.
pipiens (Kagan et al., 1984, 1985). Furthermore, the
photodynamic immobilization of brine shrimp nauplii
(Artemia saline) by more than 40 PAH has also been examined
(Morgan and Warshawsky, 1977).

PAH photo-toxicity has been well demonstrated in
fish. Alpha-terthienyl, a natural component of marigolds
and many other plants in the family Compositae, has been
observed to be more toxic than rotenone to fathead minnows
(Pimephales promelas) (Kagan et al., 1986). Acute
mortality of bluegill sunfish (Lepomis macrochirus) exposed
to 12.7 ug/L anthracene and exposed to natural sunlight was
observed in outdoor channels (Bowling et al., 1983).
Extensive laboratory studies conducted under well defined
lighting conditions also demonstrated that anthracene is
acutely photo-toxic to juvenile sunfish and that this

toxicity could be predicted from knowledge of UV intensity
and anthracene concentration (Oris and Giesy, 1985, 1986a).
PAH other than anthracene are also photo-toxic to fish. Of
the 12 PAH tested, six PAH including anthracene caused
photo-induced toxicity to fathead minnow larvae (Oris and
Giesy, 1986b). Anthracene exhibited a median potency among
the PAH that were toxic: therefore, anthracene appears to
be an adequate model compound for the examination of PAH
photo-induced toxicity (Oris and Giesy, 1986b).

The mechanism for photo-induced toxicity of PAH is
unknown, but evidence suggests that disruption of cell
membranes is involved (Landrum et al., 1986). Cellular
damage may result from the direct binding of PAH to
macromolecules in the presence of UViradiation (Sinha and
Chignell, 1983). Exposure to PAH and UV-radiation may
alter membrane function by inactivation of enzymes (Wat et
al., 1980) or by modification of lysosomal membrane
permeability (Allison et al., 1966). Furthermore, upon
absorption of Uviradiation, PAH can be excited to triplet
states which may react with oxygen to form singlet oxygen
(Foote, 1968). Singlet oxygen can react with cell
membranes, modify DNA (Gutter et al., 1977) or cause lipid
peroxidation (Sinha and Chignell, 1983).

Ultraviolet radiation plays an important role in the
photo-induced toxicity of PAH, but UV-radiation alone can
be toxic to aquatic organisms. Exposure to UV-radiation

can cause lesions and sunburning of the epidermis of fish

(Allison, 1960: Bullock, 1982; Bullock and Roberts, 1981).
UV-radiation can also be toxic to zooplankton and can
influence their behavior and occurrence in the environment
(Damkaer et al., 1980; Barcelo and Calkins, 1979; Karanas
et al., 1979). Zooplankton adapt to UV-radiation through
increased pigmentation, increased repair capacity and
avoidance. The avoidance of damaging UV-radiation present
at midday may be an important factor in explaining the
phenomenon of diel migration of these organisms (Damkaer,
1982; Barcelo, 1982). Calkins and Thordardottir (1980)
concluded that tolerance and exposure of a variety of
aquatic organisms (algae, bacteria, protozoa and
anthropods) to solar UVhradiation are approximately equal
which indicates that many aquatic organisms are currently
exposed to UVHradiation intensities at or near thresholds
for adverse effects. Any increases in intensities of UV-
irradiance or PAH concentrations, which effectively'enhance
the photo-induced damage, could have drastic effects on the
abundance of aquatic organisms.

The purpose of the present study was to examine the
chronic effects of sublethal concentrations of anthracene
in the presence of environmentally relevant intensities of
simulated, solar radiation on reproduction of the
microcladoceran, Daphnia magg_. Several studies have
demonstrated that PAH cause acute photo-induced toxicity to
zooplankton, but the chronic effects have not been

elucidated.

Research Rationale

Toxicant

Anthracene is a commonly occurring PAH which is found
in both oil and coal and is a product of various pyrolytic
combustions (Neff, 1979). Anthracene is often used as a
model PAH in laboratory toxicity and fate studies because
it is noncarcinogenic, inexpensive, ubiquitous in natural
environments, and has an intermediate water solubility
relative to other PAH (NAS, 1972). Anthracene exhibits a
median level of photo-induced acute toxicity to aquatic
organisms relative to other PAH which have been tested. In
addition, the anthracene molecule is symmetrical so that
the number of products formed by photolysis, hydrolysis and
biotransformation is smaller than that for other PAH.

These products are also well described.

Organism

Zooplankton are important components of aquatic food
chains because they are a food link between primary
producers and predators of higher trophic levels. Any
reduction in zooplankton abundance can significantly alter
the structure of aquatic communities. Decreases in
zooplankton reproduction due to toxicant exposure are
commonly studied using Daphnia in chronic toxicity tests.

Daphnia have been used as indicator organisms for
aquatic pollution in both acute and chronic toxicity tests
for many years. The advantages of Daphnia as a test

10

organism are: small size; ease of culture, maintenance and
testing; relatively short life-cycle; uniformity of
cultures and sensitivity to toxicants (Anderson, 1944;
Leonhard, 1979). Q. lam, the largest and easiest species
of Daphnia to handle, is a standard test organism in
toxicity tests and has been shown to be one of the most
sensitive aquatic species (Adema, 1978). Daphnia are
particularly useful in studying anthracene because they
rapidly bioconcentrate anthracene (Eastmond et al., 1984:
Herbes and Risi, 1978) but do not biotransform this PAH
(Herbes and Risi, 1978). Q. m accumulate anthracene to
steady state concentrations in approximately 4 h (Herbes

and Risi, 1978).

Chronic Photo-toxicity 32535

Acute toxicity tests provide information about the
relative toxicity of a compound to the test organism but do
not provide information about the chemical's long-term
effects on populations of the organism. Chronic toxicity
tests are designed to assess the effects of a compound on
the survival, reproduction and growth of an organism in
order to assess and predict the possible impact of a
compound on populations of organisms in natural
environments. Short-term tests have shown anthracene to be
acutely toxic to Daphnia in the presence of UV light,
however, no studies on the chronic toxicity of anthracene

and UV light to Daphnia have been conducted prior to the

11

work presented here. The study reported on here provides

information on both lethality and reduced fecundity of Q.

magna due to chronic exposure to anthracene and UV-

radiation.

Objectives 9; Research

The objectives of the present study were to:

1.

2.

Determine the effects of anthracene exposure on 2.
mega; survival and reproduction in the absence of
'ultraviolet radiation.

Determine the effects of chronic exposure to low
intensity, simulated, solar radiation on the
survival and reproduction of Q. m.

Examine the dose-response relationships between
anthracene concentration and UV-radiation
intensity necessary to reduce fecundity of 9.
22922-

Use the chronic dose-response information to
determine the hazard of present concentrations of
PAH and intensities of UV-radiation and set
criteria for the protection of aquatic organisms
exposed to PAH under ecologically relevant

conditions of solar radiation.

MATERIALS AND METHODS

Zooplankton

Daphnia m were cultured in 20 liter glass aquaria
filled with 15-17 liters of aerated well water (temperature
- 23 +/- 1°C, pH - 8.1 +/- 0.2, 0.0. -7.8 +/- 0.4 mg Oz/L,
hardness - 230 mg CaCOa/L, alkalinity - 236 mg Cacog/L).
The Q. m were maintained on a 18 h:6 h (light:dark)
photoperiod under a bank of two fluorescent white lamps
(Sylvania Gro-luxR F48T12-GRO-VHO). Q. m were fed
Chlamydomonas reinhardti once a day. Approximately 400 m1
of algae were centrifuged at 6000 .rpm for 15 minutes,
resuspended in 100 ml of well water, and added to each
aquarium. Q. m cultures were gently aerated to keep
the algae suspended and the water well oxygenated. In
order to maintain healthy cultures, 9. m populations
were reduced when necessary (every 3-5 days) and the tanks
were drained, scrubbed and refilled with aerated well water
once a week.

The Q. m cultures were characterized as follows:
time to first brood - 7.8 +/- 0.4 days, number of broods in
21 days - 6 per female, number of neonates per brood - 15.9
+/- 7.4, interbrood period - 2.4 +/- 0.5 days and total

number of neonates per female in 21 days - 95.2 +/- 8.5.

12

13

Algae

Cultures of Chlamydomonas reinhardti were grown in
sterile 4 L Erlenmeyer flasks filled with 3 liters of
autoclaved modified Woods Hole-MEL medium (Table 1:
Nichols, 1973). The neck of each flask was fitted with a
rubber stopper that contained 2 glass pipettes: one each
for air inlet and outlet. The algae cultures were
continuously aerated to keep the cells suspended and the
media well oxygenated. Air was filtered by a 25 mm extra
thick glass fiber filter placed in-line between the air
pump and rubber stopper. The top of each flask was loosely
covered with aluminum foil to keep foreign matter from
collecting on the stoppers and contaminating the cultures.
The cultures were grown in a greenhouse.

_C_. reinhardti cultures were diluted with fresh medium
every 2 to 5 days, depending on the density of algal cells.
.All but approximately 500-1000 ml of algae were harvested
and refrigerated. The unharvested cells were rediluted
with 2-2.5 liters of medium, and the refrigerated cells
were fed to Q. m. All algae transfers and dilutions
were performed under a laminar flow hood to minimize

contamination.

Exposure System

Three 21-day static renewal tests were conducted.to

determine the effects of various anthracene concentrations

14

Table 1. Modified Woods
Cultures.

Hole-MBL Media for Algae

 

Macronutrients

Cac12 - 2320
ugso - 7320
NaHC 3
KZHPO4
NaNO

Na28103 ° 9320
Micronutrients

Na ' EDTA

F3 13 . 682°
2nso4 - 7nzo
c6c12 - 6820
Mnc12 ° 4320

Vitamins
Thiamine

Cyanocobalamin
Biotin

Concentration (ngL)

36.76
36.97
12.60

8.71
85.01
28.42

Concentration (mg1L)

4.36
3.15
0.02
0.01
0.18

Concentration (ugAL)
100

100
5

 

15

and ultraviolet radiation intensities on the survival and
fecundity of Q. megn_a_. These chronic tests were conducted
under different UV-radiation intensities on a 16 ms h
(light:dark) photoperiod. Lights were mounted on a 1122 x
1.37 m frame on 15.24 cm centers, and the light bank was
divided in half by a black plastic curtain. One half of
the light bank contained GEE. Chroma F40C50 white
fluorescent bulbs, above a 3 mm thick sheet of plexiglasR
that eliminated ultraviolet wavelengths of light (<400 nm).
The other half of the light bank contained a combination of
G.E. Chroma F40C50 white and FS40T12 ultraviolet
fluorescent bulbs. Sheets of 0.005 mm thick MylarR plastic
were used to eliminate‘wavelengths less than 315 nm and
obtain a UV-A to UV-B ratio similar to what occurs outdoors
(approximately 8:1). The desired light intensities were
obtained by varying both the height of the light bank over
the water bath and the number of MylarR sheets.

During each chronic test, 9. megme were exposed to
various concentrations of anthracene. Stock solutions of
anthracene were obtained from a once-through, aqueous,
elution column which obviated the need for a carrier
solvent. These "generator" columns were prepared by
dissolving anthracene crystals in acetone and pouring the
solution onto a thin layer of silica sand at 0.2% wt/wt.
The solvent was then allowed to evaporate overnight. When
dry, the sand was packed into a glass column (400 x 75 mm)

and flushed with water to remove any loose anthracene

16

crystals. Water eluted from the column containing
anthracene at a concentration of 33 ug/L and was diluted to
the desired concentration before being added to the test
vessels.

The test vessels in the chronic exposures were 1.8 L
glass baking dishes (250 x 200 x 45 mm) with fiberglass-
coated wood covers that contained two rows of five evenly
spaced holes (Gersich, 1984). A glass tube (51 x 54 mm)
with a 368 um pore size stainless steel mesh bottom was
placed in each hole and suspended in the test solution
contained in the glass dish. Each vessel contained ten 9.
megme (l/tube) which were exposed to the same anthracene
concentration. The vessels were randomly placed under the
lighting system in a water bath in order to maintain the
temperature of the test solutions between 22 and 23°C.

Temperature fluctuations occurred during experiment 1
which resulted in fecundity of Q. megrg which was less
than expected. Therefore, the raw data obtained during
(experiment 1 were multiplied by a weighting factor of
1.3529 in order to allow comparisons of production of
neonates among experiments 1, 2 and 3. The weighting
factor was calculated by dividing the mean total number of
neonates produced in the first 6 broods (TOTL6BRD) by m.
megme not exposed to UV-radiation or anthracene in
experiments 2 and 3 by the mean of TOTL6BRD by Q. megme not
exposed to UV-radiation or anthracene in experiment 1. The

weighted data were used in all statistical tests.

17

Q. meg_n_e were fed 9. reinhardti during each test at a
concentration of 2.5 x 105 cells/ml/day which was
determined to be an adequate concentration to yield healthy
2. megme (Appendix A). Before addition of algae to the
test vessels, an excess amount of algae was placed in
anthracene solution eluted from the “generator" column and
was allowed to sorb anthracene overnight in order to
prevent rapid depletion of anthracene from the water by
algae when added to the test vessels. Although the algal
cells were probably at steady state with the saturated
anthracene solution (Herbes and Risi, 1978) before being
added to the test vessels, the cells were not an
appreciable source of anthracene for Q. m. Even though
algal cells rapidly depurate anthracene (Giesy et al.,
1978) and quickly come into equilibrium with anthracene
concentrations in the surrounding water, measured
concentrations of anthracene indicated that the amount of
anthracene added to the water by algae was minimal. In
addition, ingested algal cells were not a significant
source of anthracene for 2. M because 2. meQe are only
able to assimulate approximately one-third or less of PAH
which are ingested in food (McCarthy, 1983). Therefore,
the major source of anthracene for Q. megme was the
anthracene dissolved in water.

Cell density of the anthracene-algae solution was

determined daily by using a standard regression equation

18

previously calculated from absorbance readings vs. algal
cell densities. Absorbance of algal cells was determined
with a Gilford Model 2600 spectrophotometer with the
wavelength set at 665 nm. The number of cells necessary to
yield a concentration of 2.5 x 105 cells/ml in each test
vessel was calculated, and the appropriate volume of algal
solution was centrifuged at 6000 rpm for 15 minutes. The
cells were resuspended with well water and evenly dispensed
to vessels containing anthracene. The control vessels
received algal cells that were placed in anthracene-free
well water overnight and were centrifuged, resuspended and
dispensed in the same manner as the anthracene containing
cells.

UV-A (365 +/- 36 nm) and UV-B (310 +/- 34 nm) were
quantified with a Macam Photometrics (Livingstone,
Scotland) Model UV-103 radiometer equipped with Model
80104, cosine-corrected, photodiodes fitted with water-
tight, wavelength selective filters. Photosynthetically
active radiation (PAR) was measured by a Techtum
Instruments QSM-2500 scanning quantum spectrometer, which
was coupled to a LI-cor model LI 188-B integrating quantum
meter. All light measurements were taken at the surface of
the zooplankton test chambers.

Anthracene concentrations in water samples were
determined directly by reverse-phase HPLC. Fifty
microliters of sample were injected onto a 5 cm, C-18, 2 um

guard column that was directly connected to a 15 cm x 4.6

19

mm Supelcosil LC-PAH, 5 um column, at 22°C. .An isocratic
elution was performed with 90% acetonitrile: 10% water with
a flow rate of 1.0:m1/minute. A Kratos FS-970 fluorescence
detector was used at an excitation of 252 nm with a 370 nm
emission filter. Peak areas were integrated by a Hewlett-
Packard 3390-A integrator. Values for anthracene
concentrations in the water samples were obtained by using
a regression equation of peak areas vs. concentrations of

anthracene in standards.

Experimental Desigm

At the beginning of each test, ten neonates (<48 h
old) were placed in each test vessel (1/tube) and remained
in the same tubes for the test duration. Survival of adults
and number of neonates produced were monitored and recorded
daily (Appendices Bl-B3). After enumeration, the young were
removed from the test chamber, and the adults were
transferred to fresh solutions of anthracene and food.
Adults were transferred to clean baking dishes every other
day and to clean exposure tubes once a week.

Adult 9. m in the three experiments did not all
produce equivalent numbers of broods during 21 day periods.
Seventy-eight percent of the adults produced 6 broods
during 21 days but 15% of the adults had 7 broods. This
difference in number of broods was due to slight

differences in the ages of the Q. magma, which were placed

20

in the test vessels at the beginning of each experiment.
All of the adult 2. mm were <48 h old, but the Q. m_agr_1e
born earlier in the 48 h period had more time to have an
additional brood than 2. megme born later in the 48 h
period. In order to compare production of neonates within
or between experiments, fecundity was defined as the total
number of neonates produced in the first 6 broods
(TOTL6BRD) rather than total number of neonates produced
during the entire 21 d duration of the experiment.

During each chronic test, half of the Q. m were
exposed to four anthracene concentrations, including a
control (0.0 ug/L) in the absence of UV-radiation, while
the other half were exposed to the same anthracene
concentrations under UV-irradiation. The three experiments
differed by the UV intensity used, which was either 31, 60
or 117 uW/cm2 UV-A. However, the nominal concentrations of
anthracene, which were 0 (control), 2.5, 5 or 10 ug/L,
remained the same. Actual concentrations appear in Table
2.

Anthracene concentrations in each vessel were measured
at least twice a day by HPLC. The daily transfer of adults
to fresh test solution occurred at t-0 h. Appropriate
volumes of anthracene solution were added to the vessels at
t-4, 8, 12 and 14 h to replenish the anthracene removed
from the water. Before fresh anthracene solution was added

to the test vessel, an equivalent volume was removed and

21

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22

discarded from the dishes to maintain a volume of 1.6
liters. An appropriate amount of algae was added.with the
replacement anthracene solution to replace the discarded
algal cells.

Anthracene concentrations fluctuated in a cyclic
manner with the maximum concentrations occurring at the
beginning and the minimum concentrations occurring at the
end of every four-hour renewal period. The maximum
anthracene concentration in each vessel at the beginning of
every four-hour cycle was targetted at 20% above the
nominal concentration: the minimum concentration in each
vessel at the end of every four-hour cycle was targetted at
20% below the nominal concentration. The loss of
anthracene during these cycles occurred at a predictable
rate which was first-order with respect to anthracene
concentration and was described by a rate constant that was
specific for each experiment and determined for each test
vessel. Since the rate of anthracene loss was first-order,
anthracene concentrations could.be predicted.at any time

during the cycle (equation 1).
Ct - COG-rt (1)

where: r-rate constant (1/h), t-time (h), CO-initial
concentration (ug/L), and Ct-predicted concentration (ug/L)
at time t. Predicted values were calculated for each
treatment concentration of anthracene before renewal in

order to determine the amount of anthracene to be added to

23

each test vessel so that concentrations of anthracene could
be maintained within the range of +/- 20% of the desired
nominal concentrations. In addition, actual concentrations
of anthracene were measured and compared to predicted
values to verify that the predicted concentrations
calculated from equation 1 were accurately modelling the
cyclic fluctuations of anthracene concentrations. The
predictive equation did accurately model the cyclic
fluctuations of anthracene because the predicted and
measured anthracene concentrations were not significantly
different (Chi-square, p>0.3) for all three experiments.
This allowed the actual concentration of anthracene to be
maintained within +/- 20% of the desired. nominal
concentrations.

Concentrations of anthracene were continuously
changing with time but were only measured 2 to 4 times
daily. Therefore, the actual concentration of anthracene
that each 9. megme was exposed to for 21 days could not be
determined. However, an actual concentration of anthracene
was estimated for each nominal concentration (2.5, 5 and 10
ug/L) by averaging 24 predicted values (1/h) that modelled
the daily cyclic fluctuation of anthracene under the
specified experimental conditions. It is this integrated,
predictive value, which is reported for each anthracene

concentration (Table 2) .

24
Statistical.Analysis

All data were analyzed with the computer program
Statistical Analysis Systems (SAS: SAS Institute Inc”
1986). Means, variance, standard deviation, range,
corrected sums of squares, uncorrected sums of squares,
standard error of the mean and coefficient of variation
were calculated with the MEANS procedure for the dependent
variables: number of neonates produced per brood (BRD): the
total number of neonates produced within the first 6 broods
(TOTL6BRD): and percent decreases of BRD and TOTL6BRD
(relative to Q. megme not exposed to UV-radiation and
anthracene).

Analyses of variance (ANOVA) of all dependent
variables were performed with the General Linear Models
(GLM) procedure of SAS. The statistical models contained
fixed main effects of anthracene and UV-A radiation. The
GLM procedure gave results in the form of sums of squares
for main effects, interactions, and.error,IE-values, and
associated probabilities. Comparisons of means were
performed by Tukey's Studentized Range (HSD) with the

MEANS/GLM procedures of SAS.

 

RESULTS

Anthracene Effects

Adult 9. m exposed to anthracene in the absence of
UV-radiation produced significantly fewer neonates than
adult 9. megme which were not exposed to anthracene (Table
3). The total number of neonates produced within the first
6 broods (TOTL6BRD) was significantly (p<0.05) less when 2-
megme were exposed to 2.1, 4.0 or 8.2 ug anthracene/L,
relative to adults which were not exposed to anthracene.
The number of neonates produced in 6 broods by Q. m
exposed to an anthracene concentration of 2.1, 4.0 or 8.2
ug/L were 5.3, 8.0 or 13.8% less than the TOTL6BRD by _D_.
megme which were not exposed to anthracene. In addition,
significantly (p<0.05) fewer neonates were produced in
broods 2 through 6 by Q. m__ag_me exposed to 8.2 ug
anthracene/L, relative to adult 2. megfl which were not

exposed to anthracene.

UV-Radiation Effects

Exposure to UV-radiation did not affect the production
of neonates by adult 2. magna (Table 4). TOTL6BRD produced
by 9. mafia exposed to a UV-radiation intensity of 31, 60

25

 

Table 3.

26

Average Number of Neonates per Brood and Total
Number of Neonates Within the First 6 Broods
Produced by Q. magna Exposed to Anthracene in the
Absence of‘UV-Rad ation. IN-120. ‘Value Within
Parentheses is Standard Deviation.

27

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m m e n a a Aa\osv
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noose

 

.n OHQMH.

Table 4.

28

Average Number of Neonates per Brood and Total
Number of Neonates Within the First 6 Broods
Produced by 2. magna Exposed to UV-Radiation in
the Absence of Anthracene. N-120. Value Within
Parentheses is Standard Deviation.

29

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30

or 117 uW/cm2 UV-A were not significantly (p>0.05)
different from m. m which were not exposed to UV-
radiation. Although the mean TOTL6BRD produced by adult 9.
megme exposed to an intensity of 117 uW/cm2 UV-A was 3.5%
less than the TOTL6BRD produced by adults not exposed to
UV-radiation, the smaller production of neonates by Q.
megme exposed to this intensity of UV-A was not
statistically significant (p>0.05).

Combined Effects 95 Anthracene and UV-Radiation

Survival of adult 2. megme was decreased by concurrent
exposure to anthracene and UV-radiation. Exposure to 8.5
ug anthracene/L and 60 uW/cm2 UV-A resulted in 10%
mortality of Q. megme, and exposure to 7.2 ug anthracene/L
and 117 uW/cm2 UV-A resulted in 70% mortality. No
mortality was observed at the other treatment combinations
of anthracene and UV-radiation. In all cases, mortality
occurred after the _D_. we had reached reproductive age
and had produced at least one brood of neonates. Due to
the occurrence of mortality at two treatment combinations,
fecundity of _D. megme at a specific treatment combination
can be examined in two ways: as production per surviving
adult or as production which includes adults that died
within 21 days. Both cases are included below.

Exposure of Q. megme to anthracene in the presence of

ecologically relevant intensities of UV-radiation caused a

31

reduction in the number of neonates produced by Q. megme,
which was proportional to both anthracene concentration and
‘UV-radiation intensity (Figures 2, 3 and 4). Q. megme,
which were exposed to 7.2 ug anthracene/L and 117 uW/cm2
UV-A and survived for 21 d, had 69.4% fewer neonates than
did 2. megme which were not exposed to anthracene or UV-
radiation (Figure 2). However, if production by 12° m_ag_r£
which did not survive the entire 21 d is included, 9. m
exposed to 7.2 ug anthracene/L and 117 uW/cm2 had 84.3%
fewer neonates, relative to Q. teem which were not exposed
to anthracene or UV-radiation (Figure 5).

An equation was derived to predict the percent
reduction in the number of neonates produced within the
first 6 broods (TOTL6BRD), relative to production by Q.
megme which were not exposed to anthracene or UV-radiation,
from anthracene concentrations and UV-A radiation
intensities (equation 2). This predictive equation was
obtained from the Model option of the GLM procedure in SAS
(SAS Institute Inc., 1986).

Y--0.212 + 1.972(X) + 0.0226(2) + 0.0592(X*Z) (2)

where: Y - % reduction of TOTL6BRD, X - anthracene
concentration (ug/L), z - UV-A intensity (uW/cmz) and (x*2)
is a term for the interactive effects of UV-A radiation and
anthracene. The independent variables of the model
explained 78% of the variation of the dependent variable
(Table 5). An equivalent equation was derived to predict

Figure 2.

32

Percent Reduction in TOTL6BRD by Surviving _D.
magna Exposed to Anthracene (Actual
Concentrations) and UV-Radiation Relative to

TOTL6BRD by D. magna Not Exposed to Anthracene
or UV-Radiati—on.

33

 

 

  

/////////
m m m .4.
558.844.

Figure 2.

Figure 3.

34

Mean Cumulative Number of Neonates Produced by
Surviving 9. ma na Exposed to a UV-A Intensity
of 117 uW/cm and Anthracene (Actual
Concentrations). Vertical Bars Correspond to
(Gill. 1978). (a - 0.05): n-120 and r-30 for
UV-A-O: n-40 and r-lo for UV-A-3l, 60 or 117
uW/cm ).

35

 

I I T l I I

e—e 7.2 Anth ,ug/L
.... 3.6 Anth ug/L
Au-A 1.9 Anth ,ug/ L
140 —

em. 0 Anth ,ug/L ,8
omo o Anth [.Lg/L [.3
12° — o UV-A ILW/cmz ,{I-‘

Number of Neonates
O) a: E3
0 C) O
I I I

3
l

20—

 

 

 

 

Figure 3.

Figure 4 .

36

Mean Cumulative Number of Neonates Produced by
Surviving Q. magma Exposed to 8 ug Anthracene/L
(Approximate Concentrations) and UV-Radiation.
Vertical Bars Correspond to Minimum Significant
Differences (Gill, 1978). ( all-0.05: n-60 and
r210) .

37

 

I I I I T I
O—0117 uv-A )uw/cm2
I:I--c1 so LIV-A ,uw/cm2
Ame 31 UV-A )uwnzm2

14° _ o-«o o UV-A ,chm2 ,
.... O UV-A pW/cmz f
120 - o Anth (Lg/L ,1 o

'2‘

Number of Neonates
8 8

8

h)
C)

 

 

 

 

Figure 4.

Figure 5.

38

Percent Reduction in TOTL6BRD by D. magma
(Including Adults Which Did Not Survive:21 D)
Exposed to Anthracene (Actual Concentrations)
and UV-Radiation Relative to TOTL6BRD by Q.
magma Not Exposed to Anthracene or UV-Radiation.

39

 

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41

the percent reduction in the total number of neonates
produced within the first 6 broods which included
production of neonates by Q. m which did not survive
for 21 d (equation 3). This predictive equation contains

the same variables as equation 2.
Y = 0.0435 + 1.790(X) - o.oozso(2) + 0.0773(x*2) (3)

The independent variables of the model explained 84% of the
variation of the dependent variable (Table 6).

Decreases in production of neonates were manifested
in the first brood as well as in successive broods produced
by Q. m which were exposed to anthracene and UV-
radiation. Rather than including the cumulative effects of
all treatment combinations of anthracene and UV-radiation
on production of neonates, only the effects of exposure to
anthracene concentrations of 0, 1.9, 3.6 or 7.2 ug/L at the
greatest [JV-radiation intensity (117 uW/cm2 UV-A) (Figure
3), and exposure to UV-radiation intensities of o, 31, 60
or 117 uW/cm2 UV-A and the greatest anthracene
concentration (8 ug/L) (Figure 4) are presented. At a UV-
radiation intensity of 117 uW/cm2 UV-A, significantly fewer
neonates were produced in the first brood by Q. m which
were concurrently exposed to either 3.6 or 7.2 ug
anthracene/L (Figure 3) relative to Q. m which were not
exposed to anthracene or UV-radiation. Q. magna exposed to
1.9 ug anthracene/L and 117 uW/cm2 UV-A did not have

significantly fewer cumulative number of neonates than 9.,

 

42

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43

m which were not exposed to anthracene or UV-radiation
until brood 3. At an anthracene concentration of 8 ug/L,
significantly (p<0.05) fewer neonates were produced in the
first brood when 2. Lam were exposed to 31, 60 or 117
uW/cm2 UV-A (Figure 4) relative to Q. m which were not
exposed to UV-radiation or anthracene. Q. m exposed to
8 ug anthracene and 117 uW/cm2 UV-A ug had significantly
(p<0.05) fewer cumulative number of neonates in broods 4
through 6 than 9. m exposed to either 0 or 60 uW/cm2
UV-A. Cumulative production of neonates by _D_. was;
exposed to 8 ug anthracene/L and either 31 or 60 uW/cm2 UV-
A were not significantly (p>0.05) different from each
other at any brood.

Greater decreases in the mean cumulative number of
neonates produced are evident when the production by Q.
m which did not survive for 21 d is included (Figures 6
and 7). Q. magna exposed to 8 ug anthracene/L and 117
uW/cm2 UV-A had significantly fewer cumulative number of
neonates in broods 3 through 6 than 2. m exposed to o,
31 or 60 uW/cm2 UV-A and 8 ug anthracene/L. TOTL6BRD for
9. 13.993 exposed to 8 ug anthracene/L and 117 uW/cmz,
including production by 9. £933.! which died during the 21d,
was 21.5 (Figure 7) compared to 42.0 by Q. m which
survived for the entire 21 d (Figure 4).

. VIVID-{Du .
f.

 

Figure 6.

44

Mean Cumulative Number of Neonates Produced by
9. mafia (Including Adults Which Did Not Survivg
21 D) Exposed to a UV-A Intensity of 117 uW/cm

and Anthracene (Actual Concentrationsp
Vertical Bars Correspond to Minimum Significant
Differences (Gill, 1978). (a- 0.05: n-120 and
r-30 for‘Ungso; n-4O and r-lo for‘UV-Ap31, 60
or 117 uW/cm ).

45

 

140

120

100

80

Number of Neonates

60

4O

20

 

Figure 6.

l I r I

H 7.2 Anth pg/L
.--. 3.6 Anth [Lg/L
H 1.9 Anth [Lg/L

Om. O Anth [lg/L
O-«O O Anth [Lg/L
o UV-A )uwIcm2

I I I I

 

 

 

Figure 7.

46

Mean Cumulative Number of Neonates Produced by
D. magna (Including Adults Which Did Not Survive
21 D) Exposed to 8 ug Anthracene/L (Approximate
Concentrations) and UV-Radiation. ‘Vertical Bars
Correspond to Minimum.significant Differences
(6111,1978). (oz-0.05; n-so and r-lO).

47

 

 

F T I l I 1
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D—-Cl so UV-A [.LW/cm2
A.---A 31 UV-A ,uW/cm2
gm. 0 UV-A )LW/cum2 g"
120 — o Anth p.9/L f" O
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8 .d
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Figure 7.

 

DISCUSSION

Although PAH have received considerable attention in
recent decades, most of the studies have focused primarily
on the carcinogenic and mutagenic effects of PAH on
mammals. In comparison, relatively little information has
been collected on the acute and chronic toxicities of PAH
to aquatic organisms. Anthracene, a.3-ring PAH, has been
previously considered not to be toxic to aquatic organisms
even in supersaturated solutions (Herbes et al., 1976;
Applegate et al., 1957); however, the present study has
demonstrated that chronic exposure to anthracene in the
absence of UV-radiation adversely affects the fecundity of
Q. m. TOTL6BRD was reduced by 14% when 9. magna were
exposed to 8.2 ug anthracene/1.(or approximately 25% of the
aqueous solubilityJ

Solar ultraviolet radiation can have considerable
impact on the distribution, survivorship and reproduction
of certain aquatic organisms such.as bacteria, protozoa,
algae, zooplankton and fish (Damkaer, 1980; Calkins and
Thordardottir, 1980; Barcelo and Calkins, 1979; Bullock,
1982). UV-B, the most deleterious component of sunlight,
is readily absorbed by proteins and nucleic acids and is

effective in inducing photochemical reactions in plants and

48

I. .

 

49

animals (Damkaer et al., 1980). Prolonged exposure to UV-B
radiation can cause lesions in the epidermis of fish
(Allison, 1960) and can reduce the survival of algae
(Calkins and Thordardottir, 1980) and zooplankton (Damkaer
et al., 1980). The ecologically-relevant intensities of
UV-A and UV-B radiation used in this study, which were
comparable to UVHradiation intensities that routinely
penetrate to depths of 10 and 12 meters in Lake Michigan
(Gala and Giesy, 1987), did not affect the production of
neonates. TOTL6BRD by Q. magna exposed to UV-radiation in
the absence of anthracene were not significantly (p>0.05)
different from one another at any UV-radiation intensity.

Recent studies, which have incorporated
environmentally realistic lighting regimes with PAH
exposure, have demonstrated that anthracene and other PAH
are extremely toxic to fish (Oris and Giesy, 1986a, 1986b)
and zooplankton (Kagan et al., 1985 :1Newsted and Giesy,
1987; Allred and Giesy, 1985) at concentrations less than
aqueous solubility. Results from the study reported here
demonstrated that exposure of Q. m to low intensities
of UV-radiation and concentrations of anthracene between 6
and 14% of aqueous solubility reduced production of
neonates by Q. m up to 69%. If production by p_. m
which died during the 21 d is added, the observed maximum
decrease in production of neonates was 84%.

As expected, no observable adverse effect

concentrations (NOEC) were less when exposure to anthracene

50

was long-term (chronic) rather than short-term (acute). At
UV-A intensities of 29 or 116 uW/cmz, the acute NOEC's for
Q. magna exposed to anthracene for 48 h were between 7.66
and 21.4 and between 2.11 and 6.10 ug/L, respectively
(Newsted, 1986). Corresponding chronic NOEC's for Q.
mam exposed to either 31 or 117 uW/cm2 UV-A were between
0 and 1.9 and between 0 and 2.2 ug/L, respectively. The
chronic NOEC for D. M exposed to 60 uW/cm2 was between
2.2 and 4.1 ug anthracene/L.

Although no other studies have examined the effects of
chronic exposure of PAH and UV-radiation on aquatic
organisms, chronic effects have been estimated from
laboratory-derived acute dose-response data (Oris and
Giesy, 1986a). At a UV-A intensity of 100 uW/cmz, the
concentration of anthracene necessary to cause lethality in
1% of bluegill sunfish populations during exposure for an
infinitely long period of time (LCl) was predicted to be
3.8 ug/L, which is within the range of the NOEC values
which were determined for Q. m in the present study.
Although Q. m were more sensitive to acute exposure of
anthracene and UV-radiation than bluegill sunfish (Newsted,
1986), sensitivity to chronic exposure may not differ.
However, reproductive studies which involve chronic
exposure of bluegill sunfish to anthracene and UV-radiation
have not been conducted to verify previous predictions of

chronic effects to bluegill sunfish.

51

The study presented here has demonstrated that
exposure of Q. magna to anthracene causes a reduced
fecundity in the presence of UV-radiation in the
laboratory. The question remains, however, as to what are
the current and future impacts of the photo-enhanced
toxicity of PAH to zooplankton (and other organisms) in
aquatic systems? Concentrations of PAH in some aquatic
systems are currently at concentrations.(Table 7) which, in
this study, reduced production of Q. magna neonates by
nearly 40% at a Uv-A intensity of 117 uW/cmz. Therefore,
aquatic systems, which have sufficient‘UV light penetration
and concentrations of PAH, may be experiencing increased
mortality and reduced reproductive success of aquatic
organisms due to the photo-enhanced toxicity of PAH.
Concentrations of anthracene presently in the Great Lakes
do not appear to be at levels that would result in
significant photo-enhanced toxicity (Table 8).
Concentrations of anthracene would have to increase by
approximately 2 orders of magnitude to elicit a 10%
decrease in Daphnia reproduction (equation 2) in the Great
Lakes. However, pyrene and B[a]P, two PAH which are two
times more photo-toxic than anthracene (Newsted and Giesy,
1987), occur at greater concentrations in Lakes Michigan
and Erie than anthracene; therefore, increases in
concentrations of these PAH would probably have to occur at
lesser amounts to elicit adverse responses in zooplankton

reproduction.

52

Table 7: Concentrations of Total PAH in Various River
Waters (Neff, 1979) .

 

 

Source Total PAH (ug/L)
River Rhine, GFR 0.500 - 3.000
Thames River, England 0.800 - 2.350
Trent River, England 0.025 - 3.790
Delaware River, PA 0.352

Ohio River, WV 0.058

 

53

Table 8. Concentrations of PAH (ug/L) in Great Lakes Water
(Eadie et al., 1982) (n-6) .

 

 

PAH Mean Std. Dev.
Phenanthrene 0.024 0.025
Anthracene 0.006 0.006
Fluoranthene 0.015 0.009
Pyrene 0.014 0.006
Chrysene 0.014 0.010
Benzo[a]pyrene 0.012 0.008

 

1Six water samples from Lakes Michigan and
Erie.

CONCLUSIONS

This study has demonstrated that anthracene, in the
presence of UV-radiation, decreases the survival and
fecundity of Q. magna at concentrations well under aqueous
solubility limits. Exposure of Q. magna to 7.2 ug
anthracene/L and 117 uW/cm2 UV-A resulted in 70% mortality
or a 69% reduction in fecundity of the Q. magna which
survived. {Although anthracene has been demonstrated to
have adverse effects on the survival and fecundity of Q.
magna, further research needs to be conducted. Elucidation
of the mode of toxic action is the next logical step in
studying the photo-enhanced toxicity of PAH. In addition
to elucidation of the mode of toxic action, effort should
A be put into the l) examination of chronic effects of other
PAH besides anthracene on the survival and reproduction of
Q. magna and fish, 2) procurement of more accurate and
complete measurements of PAH concentrations in surface
waters, and 3) examination of possible synergistic

relationships among combinations of PAH.

54

APPENDICES

APPENDIX A

Neonates per Brood Produced in 21 Days per 9; magna Fed
Chlamydomonas reinhardti.

 

 

 

 

 

 

 

ALGAL cone REP N0* snooo TOTAL

(cells/ml)
1 2 3 4 5 6

1 x 105 1 6 17 26 29 13 19 110

2 8 16 20 22 17 20 103

3 11 18 27 17 10 20 103

4 10 13 19 27 21 -- 90

5 4 14 17 19 12 -- 66

i 7.8 15.6 21.8 22.8 14.6 19.7 94.4

s 2.9 2.1 4.4 5.1 4.4 0.6 17.4

2.5 x 105 1 7 17 19 27 7 17 94

2 7 13 24 29 14 13 100

3 10 18 21 29 13 16 107

4 8 11 20 30 10 10 89

5 6 12 19 26 5 18 86

i 7.6 14.2 20.6 28.2 9.8 14.8 95.2

s 1.5 3.1 2.1 1.6 3.8 3.3 8.5

_5 x 105 1 6 12 22 30 10 -- 80

2 5 13 28 28 6 -- 80

3 7 11 23 33 8 -- 82

4 6 11 24 28 7 -- 76

5 3 14 21 29 24 -- 91

i 5.4 12.2 23.6 29.6 11.0 -- 81.8

S 105 103 207 201 704 -- 506

 

*Each rep. no. corresponds to 1 Q. magna that was fed the
respective concentration of algal cells daily for 21 days.

55

56

APPENDIX Bl

Neonates per Brood Produced per 9. magna in 21 Days During
Experiment 1.

 

UV-A ANTH REP* BROOD TOTAL
couc NO

 

0 0 1 5 13 24 18 23 22 105
2 8 9 20 19 24 21 101

3 7 12 21 18 23 22 103

4 5 13 24 19 25 23 109

5 5 11 19 18 24 24 101

6 6 11 22 24 27 24 35 149

7 7 10 19 17 23 22 98

8 8 12 18 20 19 28 36 20 161

9 6 11 22 18 20 26 32 135

10 6 12 18 16 21 22 26 121

2.2 1 6 13 20 18 24 23 104
2 5 11 21 17 19 25 29 127

3 5 10 19 16 18 21 89

4 6 11 17 18 25 22 99

5 8 10 17 17 21 19 92

6 6 13 23 18 23 21 104

7 4 12 17 17 18 22 30 120

8 6 11 23 17 20 21 26 124

9 6 10 16 20 17 22 31 122

10 5 10 16 16 21 22 28 118

4.5 1 3 12 18 17 23 23 96
2 3 8 19 19 25 22 96

3 4 7 17 19 26 23 29 125

4 6 7 17 18 23 20 91

5 10 7 18 18 26 21 100

6 7 9 16 18 17 19 86

7 4 7 16 17 18 26 36 124

8 7 10 16 22 23 21 28 127

9 5 9 19 20 22 19 94

10 6 10 21 17 28 22 104

8.8 1 4 6 15 19 20 19 83
2 4 10 15 18 19 20 86

3 7 5 11 16 21 21 81

4 3 6 15 16 17 18 75

5 6 7 14 15 20 21 83

6 7 8 14 15 20 19 83

7 6 6 13 17 20 25 87

8 3 6 11 15 18 22 26 101

9 6 5 12 15 19 18 75

10 2 4 14 16 18 19 28 101

57

 

 

 

UV-A ANTH REP BROOD TOTAL
CONC N0
1 2 3 4 5 6 7
31 0 1 7 11 22 17 23 22 28 130
2 6 14 21 24 25 26 25 141
3 7 17 19 21 25 24 28 141
4 8 13 20 20 26 24 111
5 5 12 24 19 23 21 104
6 7 17 18 23 28 22 30 145
7 5 12 22 17 23 22 27 128
8 5 12 21 15 22 21 26 122
9 5 11 18 17 22 25 22 120
10 4 10 17 16 23 22 28 120
2.2 1 7 6 17 15 20 .18 83
2 6 7 19 17 22 19 90
3 5 10 20 16 22 23 96
4 5 12 18 15 20 19 28 117
5 6 10 18 16 19 19 29 117
6 7 14 21 15 20 20 97
7 7 6 20 16 21 23 93
8 6 9 14 16 22 19 29 115
9 6 8 17 20 24 20 27 122
10 8 10 16 19 18 18 89
4.5 l 3 6 14 15 17 17 72
2 2 8 12 11 16 17 23 89
3 2 7 14 12 18 20 73
4 6 11 12 12 17 18 25 101
5 7 11 11 20 18 25 92
6 9 9 10 15 16 18 77
7 5 7 10 14 19 18 73
8 2 7 13 14 21 18 75
9 6 7 14 12 18 16 73
10 6 7 11 13 17 20 74
8.8 1 3 10 9 13 10 14 59
2 3 7 8 13 17 16 64
3 2 6 10 12 16 18 64
4 2 9 12 14 17 54
5 3 7 9 12 14 17 22 84
6 2 5 8 13 14 17 59
7 3 5 11 9 14 16 20 78
8 4 8 9 7 16 14 58
9 3 6 7 10 17 15 58
10 4 7 11 8 15 16 21 82
*Each rep. no. corresponds to 1 9. ma na that was efposed
to the respective intensity of UV-rad ation.(uW/cm ) and

anthracene concentration (ug/L) for 21 days.

58

APPENDIX 82

Neonates per Brood Produced per 2. magna in 21 Days During
Experiment 2.

 

UV-A ANTH REP* BROOD TOTAL
CONC NO

 

0 0 1 11 15 23 34 26 33 142
2 8 17 24 31 25 37 142

3 8 15 22 31 28 30 134

4 9 14 24 36 29 28 140

5 10 16 25 32 28 29 140

6 9 16 26 31 26 32 140

7 10 15 22 33 25 33 138

8 8 14 26 36 32 29 145

9 8 14 23 31 28 28 24 156

10 10 15 23 34 29 34 145

2.2 1 9 14 22 30 28 36 139
2 12 14 24 30 27 107

3 9 15 21 30 27 34 136

4 9 17 26 34 30 34 150

5 8 16 22 29 24 30 129

6 10 15 24 32 20 29 130

7 10 14 24 33 30 36 147

8 9 14 20 29 24 27 123

9 11 19 25 31 26 112

10 8 15 22 29 30 32 136

4.1 1 8 13 20 30 29 32 132
2 11 12 20 28 27 32 130

3 8 12 20 29 27 29 125

4 8 13 21 31 25 35 133

5 8 13 19 34 30 31 135

6 7 12 19 30 28 36 132

7 10 14 19 29 29 29 130

8 10 13 20 30 24 97

9 9 12 22 32 29 34 138

10 7 12 21 32 22 32 126

8.5 1 6 10 20 27 26 29 118
2 8 11 19 35 28 28 129

3 9 10 19 28 27 29 122

4 10 12 21 27 26 96

5 9 10 18 31 31 29 128

6 9 13 22 34 31 30 139

7 9 11 22 26 27 28 123

8 8 12 19 26 29 34 128

9 7 14 23 27 28 31 130

10 9 14 22 26 27 30 128

59

 

UV-A ANTH REP BROOD TOTAL

 

60 0 1 11 16 25 32 29 33 146
2 12 16 23 36 32 27 37 183

3 8 14 23 34 27 29 135

4 8 15 24 30 28 36 141

5 8 16 20 29 29 31 133

6 10 17 25 34 27 33 146

7 10 15 24 32 25 32 138

8 9 16 22 32 29 34 142

9 9 19 23 31 27 29 138

10 10 14 26 28 26 32 136

2.2 1 8 16 24 31 27 27 133
2 10 14 23 33 25 32 137

3 9 13 23 30 26 28 129

4 9 15 22 28 25 29 128

5 7 14 23 31 24 31 130

6 8 18 24 34 24 26 134

7 9 18 23 31 28 36 145

8 8 15 25 34 29 34 145

9 8 14 24 33 30 31 140

10 7 14 26 31 26 30 134

4.1 1 8 10 19 25 25 26 113
2 6 9 18 26 24 26 109

3 9 12 18 28 19 24 110

4 8 11 18 26 23 24 110

5 7 8 15 26 25 20 101

6 7 11 18 28 24 25 113

7 8 11 18 28 19 27 111

8 6 11 17 25 20 26 105

9 8 11 19 28 23 26 115

10 8 14 17 28 22 23 112

8.5 1 4 5 18 17 14 22 80
2 4 4 15 19 17 16 24 99

3 6 7 16 23 15 20 87

4 6 4 14 26 13 19 82

5 5 5 17 18 15 23 83

6 5 8 18 20 12 22 85

7 4 5 4 13

8 7 9 18 23 11 23 91

9 7 4 17 22 16 21 87

10 4 _ 9 15 21 15 19 83

 

fEach rep. no. corresponds to 1 Q. magna that was ex§>osed
to the respective intensity of UV-rad ation (uW/cm ) and
anthracene concentration (ug/L) for 21 days.

60

APPENDIX 33

Neonates per Brood Produced per 2. magna in 21 Days During
Experiment 3.

 

UV-A ANTH REP* BROOD TOTAL
CONC N0

 

0 0 1 6 15 24 29 28 33 135
2 9 19 26 28 31 24 137

3 8 15 25 33 27 29 137

4 8 18 26 29 25 27 26 159

5 7 14 25 27 25 30 128

6 8 16 28 28 28 31 139

7 8 18 25 28 27 28 134

8 7 17 28 27 26 31 136

9 6 15 30 26 27 28 132

10 6 14 26 29 26 32 133

1.9 1 7 16 25 30 25 25 128
2 8 14 26 27 30 32 137

3 8 14 28 29 26 31 136

4 9 15 28 26 28 32 138

5 10 17 29 27 25 29 137

6 9 16 26 26 28 30 135

7 6 13 25 26 29 23 20 142

8 9 14 24 25 25 28 125

9 8 15 26 24 26 31 130

10 9 18 27 26 25 26 131

3.6 1 7 14 27 26 27 30 131
2 8 13 26 29 25 31 132

3 7 15 28 22 24 31 127

4 8 16 25 26 24 24 123

5 8 17 26 24 22 26 123

6 8 17 28 23 24 28 128

7 9 15 18 22 24 18 106

8 9 18 28 23 23 31 132

9 8 14 30 26 23 22 123

10 6 14 26 24 26 25 121

7.2 1 9 13 25 23 23 29 122
2 8 15 26 19 22 23 113

3 9 15 26 23 21 24 118

4 9 18 30 23 25 34 139

5 8 17 26 22 22 25 120

6 7 17 30 25 24 27 130

7 6 17 28 24 26 28 129

8 8 16 27 20 24 28 123

9 7 14 25 23 26 26 121

10 8 19 25 24 26 27 129

61

 

 

 

UV-A ANTH REP BROOD TOTAL
CONC NO
1 2 3 4 5 6 7
117 0 1 7 16 24 27 26 29 129
2 8 15 25 28 24 26 126
3 9 19 26 30 31 27 22 164
4 9 17 30 29 26 28 139
5 6 15 28 30 28 27 134
6 7 19 29 26 27 33 141
7 9 17 27 28 28 22 131
8 6 17 26 28 27 28 132
9 6 15 26 25 28 26 126
10 6 15 24 26 26 28 125
1.9 1 6 13 18 21 22 23 103
2 6 12 16 19 24 25 102
3 7 12 17 24 24 26 110
4 8 13 19 19 24 22 105
5 9 12 23 20 25 24 113
6 6 15 17 17 23 19 97
7 6 16 21 23 24 25 115
8 7 16 18 20 18 18 97
9 9 17 17 20 19 24 106
10 7 13 16 18 24 22 100
3.6 1 7 13 10 17 18 17 82
2 5 8 15 21 18 23 90
3 3 9 13 21 19 23 88
4 5 13 13 19 20 21 91
5 4 8 8 12 22 24 78
6 6 13 12 14 20 22 87
7 3 12 14 19 17 22 87
8 4 9 15 20 23 24 95
9 7 12 12 18 18 67
10 6 10 13 20 24 20 93
7.2 1 6 13 12 8 39
2 6 ' 6
3 6 9 9 24
4 7 14 21
5 6 8 6 13 10 14 57
6 4 6 10 20
7 3 3
8 4 8 12
9 3 3
10 5 7 4 14 30
*Each rep. no. corresponds to 1 Q. ma na that was easposed
to the respective intensity of UV-rad ation.(uW/cm ) and

anthracene concentration (ug/L) for 21 days.

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