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Abstract
Anthropogenic pollution has widespread impacts on development of marine organisms as myriad
chemicals make their way into coastal ecosystems. The present study examined effects of the
commonly used herbicide atrazine. Potential effects of atrazine were observed using
scyphozoans, model organisms useful as indicators of unbalanced ecosystems, specifically
testing effects on cultured planula larvae of the medusa Aurelia labiata. Planula settlement was
observed over 14 days in seawater with 3 dilutions of atrazine (1 ppb, 10 ppb, and 30 ppb)
Turnover time, when population dominance shifted from free-swimming larvae to sessile polyps,
was longest in the highest atrazine dilution relative to the control treatment (6.27 days versus
1.96 days, respectively), indicating a significant treatment effect of atrazine (P-0.0015),
Similarly, cumulative mortality was higher with increased atrazine concentration. These data
suggest atrazine negatively affects development of cnidarians. Scyphozoans are typically more
tolerant of poor ocean conditions, therefore these results raise concern for effects on other marine
organisms more sensitive to environmental conditions.
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Introduction
Coastal ecosystems bear the brunt of anthropogenic pollution as various chemicals that
are utilized on land meander their way into the marine environment from diverse sources.
Everyday activities such as laundry and sewage produce large amounts of chemical runoff, but
typically in a more diffuse manner; of more significance is the runoff produced by agricultural
operations. Large amounts of pesticides, herbicides, and fertilizers wash off crops and into the
waterways leading to the ocean, undoubtedly, having some impact on marine habitats. Runoff
from agriculture has already been found to produce eutrophication in coastal waters, as the
nitrates and phosphates from fertilizers fuel large-scale algal blooms. However, the effect of
chemical pesticides and herbicides in the marine realm has only recently come to light.
Atrazine (2-chloro-4-ethytlamino-6-isopropylamine-1,3,5-triazine) is the most commonly
used herbicide in the United States (30,000 tons annually), and is currently used in at least 80
other countries around the globe (U.S. Dept. Agriculture 1994; Hayes et al. 2002). Within the
state of California, atrazine use is relatively small, with approximately 24.1 tons of atrazine
applied in 2002 (Orme and Kegley 2004). Atrazine was assumed to have low environmental
impacts, but recently Hayes et al. (2002) reported that atrazine induced hermaphroditism in frogs
at concentrations as low as 0.1 ppb, and at higher concentrations, the larynges demasculinized
and testosterone levels dropped.
Scyphozoans are typically considered resilient to poor ocean conditions, thus they may
serve as indicators of poor environmental conditions (especially degraded ecosystems) (Mills
2001; Arai 2001) and may prove suitable for studies of pollutant effects on development. Recent
research suggests that adult scyphozoans may be affected by pollution. Trace metals (arsenic,
cadmium, lead, and mercury) have been found accumulating in tentacles of the scyphozoan
Pelagia noctiluca (Cimino et al. 1983). Researchers examining populations of medusae in the
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western Pacific Ocean found intriguing morphological variants in heavily polluted areas, where
the hydromedusae Gonionemus vertens and the scyphozoan Aurelia aurita exhibited abnormal
numbers of radial canals and gonads, respectively (Pogodin et al. 1997). To the contrary,
Gershwin (1999) suggests that variation in gonad number in Aurelia aurita (specifically the
occurrence of nontetramerous individuals with less or more than 4 gonad pouches) may be due to
natural genetic variation, with little influence by environmental factors.
Scyphozoans utilize both sexual and asexual reproductive strategies during their life
cycle. In Aurelia labiata studied in the present experiment, male medusae release strands of
sperm, which are taken up by the female medusae; the female broods developing embryos within
her bell, until the embryos become competent ciliated planula larvae and are released. The
negatively buoyant larvae sink until they come in contact with a desirable substrate, typically one
covered with organic/bacterial biofilms (F. Sommer, pers. comm.). Upon contact with a
substrate, the planula metamorphoses into a polyp (such settlement typically occurs within 3 to 4
days at 13 °C (C. Widmer, pers. comm.)). Unknown environmental factors trigger strobilation,
by which the polyp divides along its length into a set of identical clones (ephyrae), which bud off
of the polyp once fully developed and then mature into the adult medusa, completing the life
cycle.
Perhaps the most important transformation in the life cycle of a scyphozoan is the
settlement and metamorphosis of the planula larva into a polyp, a process known from most
scyphozoan species (with the exception of Pelagia noctiluca, which undergoes direct
development of the pelagic medusa from the swimming planula stage). Planula larvae are
lecithotrophic, with their own internal energy reserves that suffice until the planula settles and is
able to feed. Using calculations of respiration and waste production, the maximal survival time
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of Aurelia aurita planula was estimated around 7 days (Schneider and Weisse 1985). Obviously.
not all planulae will settle, given that scyphozoans are r-strategists that rely on quantity of larvae
rather than quality to produce the next generation. The ciliated planula attaches to a substrate
under very specific, but diverse, cues. Work by Brewer (1976a, 1976b, 1984) with the
scyphozoan Cyanea capillata suggests that qualities of the substrate may be critical in
determining settlement, given that C. capillata settles preferentially on rough substrates. The
orientation of the substrate also seems to be key, with planulae preferring to settle with the oral
end oriented downwards, a feature that also seems to be true for Aurelia aurita (Brewer 1978).
Observations of Aurelia labiata polyps in the field and laboratory culture support the notion that
this genus prefers to settle with the oral surface oriented downward, indeed some cultured polyps
often settling on water surface tension (F. Sommer, pers. comm.).
Unlike natural inducers of settlement and metamorphosis, some factors may delay or
prevent development. Goh (1991) found nickel significantly lowered settlement rates, but more
importantly induced higher levels of larval mortality in planulae of the coral Pocillopora
damicornis. With regard to crude oil pollution, coral planulae are similarly intolerant (Kushmaro
et al. 1997; Rinkevich and Loya 1977; Rinkevich and Loya 1979). Among the scyphozoans.
Spangenberg (1984a) found significant impacts of petroleum oil on established polyps of Aurelia
aurita, and suggested that A. aurita polyps may serve as an indicator for detecting subtle effects
of various chemical pollutants. Such effects were also noted in later stages of development (i.e.
ephyrae and juvenile medusae), due to abnormal strobilation in the presence of chemical
pollutants (Spangenberg 1980).
Atrazine was expected to have negative effects on scyphozoan development, delaying or
inhibiting settlement of planula in the Pacific scyphozoan Aurelia labiata. When observed over a
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14-day time series, settlement was indeed delayed, evidenced by the significant increase in
turnover time in high atrazine dilutions. In these treatments, it took longer for population
dominance to switch from free-swimming planulae to settled sessile polyps.
Materials and Methods
Atrazine Preparation
10 mg of solid powdered atrazine (98% purity, MW=215.6 gmol“) was diluted in 1 mL
(DMSO) organic solvent. The 1 mL of atrazine/DMSO solution was diluted into 1 L of unfiltered
seawater to make a stock solution at 4.6x10° M (1000 ppb). This stock solution was further
diluted for the individual treatments; treatments prepared included 1 ppb atrazine (4.6x10° M).
10 ppb atrazine (4.6x103 M), and 30 ppb atrazine (1.39x10" M).
For planula settlement studies, 1 L of each atrazine concentration (1, 10, 30 ppb) was
prepared. For the 1-ppb atrazine dilution, 100 ul of the stock solution was added to 1 L unfiltered
seawater. For the 10-ppb atrazine solution, 1000 ul of stock solution was diluted in 1 L unfiltered
seawater. The highest (30 ppb) atrazine dilution was prepared with 3000 ul of stock solution in 1
L'seawater.
Planula Settlement
New live-quality glass bowls (4-inch diameter) were prepared by rinsing and scrubbing in
freshwater (without any detergent or other cleaning chemicals) to remove any chemical residues
that might have confounded experimental results. The dishes were soaked in unfiltered seawater
for 12 hours to culture the organic biofilm preferred for planula settlement. Each dish was filled
with 150 mL of seawater with the appropriate dilution of the herbicide atrazine as prepared
above.
Planulae for study were pipetted off the manubrium of an adult female Aurelia labiata
brooding larvae (specimen held at the Monterey Bay Aquarium, Monterey, California).
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Approximately 50 planula larvae were pipetted into each dish. The dishes were placed in a
cooling water bath with continuous flow. For each treatment level (i.e. control, 1 ppb, 10 ppb.
and 30 ppb atrazine), there were 3 replicate dishes. Extraneous variation in dish conditions could
have confounded the study’s results. As such, light levels and water temperature were controlled
via dish covering and continuous water bath, respectively.
Each dish was observed under a dissecting scope daily to monitor planula metamorphosis
for comparison among treatments for any effect of atrazine concentration. Following
metamorphosis, the polyp exhibits clear morphological changes over time. The incipient polyp is
often globular in appearance, soon budding 4, then 8 tentacles buds. These buds continue to
elongate into feeding tentacles and the mouth develops. The polyp then produces another set of
feeding tentacles as it matures (a total of 16 tentacles). Numbers of planulae and life history
stages of developing polyps (i.e. new settlers, 4 rudimentary tentacles buds, 8 rudimentary
tentacle buds, 8 incomplete tentacles [tentacle length less than central widthl, 8 complete
tentacles [tentacle length equal to central 1-2x central width], 8 elongate tentacles I [tentacle
length »3x central width), 8 elongate tentacles II [tentacle length »4x central width), 16 tentacle
buds forming lelongating tentacles + new buds»8), and 16 fully formed tentacles) were counted
daily for a 14-day time series.
Data and Statistical Analysis
Plots of planula settlement were prepared over the full time series from raw counts. The
time at which the populations of planula and polyps were equal (each at 50%, Tso) was recorded
for each replicate across treatments. Trendlines (extrapolated via regression analysis) for rates of
planula disappearance and settled polyp appearance were used to determine the turnover point,
the time when the population dominance shifted from planulae to sessile polyps. Tso points,
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turnover points, rate of planula disappearance/polyp appearance, and cumulative mortality from
each replicate across the 4 treatments were each compared with a single-factor analysis of
variance (ANÖVA) (Underwood 1997).
Results
Planula Settlement
Observations of larval population in glass dishes over the 14-day time series were
converted to percentages for each life stage daily. When plotted, the data indicated a decrease in
the percentage of the population in the swimming planula stage and an increase in the percentage
of settled individuals (polyps). In controls with no atrazine, exemplified the clear shift in
population dominance from free-swimming planula larvae to settled polyps, with clear peaks of
consecutive life stages (fig. 1). At 2.83 days, the population of planulae in the sample was equal
to the population of settled polyps (T5o) (table 1).
In the 1 ppb atrazine treatment, there was also a clear shift in the population from
planulae to sessile polyps, however, there was no obvious trend in peaks of life stages over time
(fig. 2). Additionally, the amount of time that planulae dominated the population was longer in
the 1 ppb atrazine treatment than the control treatment (Tsy was 4 days for the 1 ppb atrazine
treatment versus 23 days in the control sample). Furthermore, the percentages of the various
polyp life stages were lower than those in control populations. There was no clear pattern in life
history stages, unlike the consecutive peaking life stages observed in the control. The 10 ppb
atrazine treatment remained in the planula stage for longer than the control and 1 ppb atrazine
treatments (fig. 3). This was evidenced by the Tso, which was higher in the 10 ppb atrazine
treatment (5.17 days, table 1). Unlike the control treatment dishes, there were no clear peaks in
consecutive life stages over time. Interestingly, some individuals reached latter life stages (e.g.
16 tentacle buds), more so than in the control sample. The highest atrazine treatment (30 ppb)
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had the longest duration of larvae in the planula stage (fig. 4). Planulae comprised a large (».50)
percentage of the experimental population for nearly 6 days (T5o-5.83). Again, there was no clear
pattern in peaks of life stages with time, with similar abundances of all life history stages.
Comparison of Ts times with a single-factor analysis of variance found a significant treatment
effect of atrazine (P=0.0192).
Analysis of Turnover Time
The plots of planulae and polyp life history stages for each treatment were summed to
reflect solely free-swimming planula larvae and all polyp stages grouped into one category; these
were re-plotted to identify the turnover point, when population dominance shifted from planula
to polyp. Calculation of the linear trendline determined the rate of planula disappearance (as
percent per day) and appearance of settled polyps. For the control treatment, planula
disappearance and polyp appearance was found to be .0582 percent per day (fig. 5); the 1 ppb, 10
ppb, and 30 ppb atrazine treatments were calculated at .0726, .0784, and .0865 percent per day
respectively (fig. 6, 7, 8). The intersection point of the trendlines provided an estimate for the
turnover time. The turnover time was longest in the highest atrazine dilution, at 6.27 days, and
lowest in the control treatment (1.96 days) (table II). Analysis with a single-factor ANOVA,
revealed a statistically significant effect of atrazine on turnover time (P-0.0015). Plotting all
treatments for the entire 14-day time series, there was a visibly obvious shift in the turnover
point (when planula and polyp plots intersected) with atrazine treatments (fig. 9).
Further examination of the rates of planula disappearance and polyp appearance also
demonstrated a significant treatment effect of atrazine. Comparing the rates of planula
disappearance and polyp formation (via trendline extrapolation from plotted time series) among
replicates across treatments with an ANÖVA, atrazine appeared to have a significant effect
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(P-0.0017). Interestingly, the rate of planula disappearance and the rate of polyp appearance
were higher in the atrazine treatments than the control (table III).
Mortality Analysis
Data on mortality in each treatment was also collected for the entire time series. Mortality
data was rather confounded due to the counting methodology used to analyze planula and polyp
populations within each dish (see discussion), however, some general trends were evident.
Analyzing cumulative mortality (i.e. the total number of dead individuals by day, evidenced by
daily differences from the initial population), all treatments showed an increase in deaths over
time, though the degree of difference varied by treatments (table IV). The control group
increased mortality by 0.29 deaths per day per day (i.e. 0.29 individuals died daily, fig. 10). The
increase in mortality for the 1 ppb, 10 ppb, and 30 ppb atrazine treatments were 1.34, 1.91, and
1.23 new deaths per day per day, respectively (fig. 11, 12, and 13). With an ANOVA, there was
a statistically significant difference in cumulative mortality with atrazine concentration
(P=0.0354).
Discussion
Analysis of the data gathered during the 14-day observation of A. labiata planula
settlement indicates that atrazine indeed impedes the transition from planula to polyp. Atrazine
clearly prolongs settlement time in A. labiata, given the large disparity in turnover time between
atrazine treatments and the control. For each part per billion of atrazine, settlement time
(evidenced by turnover time) seemed to increase by 0.12 days (fig. 14); such finding calls to
attention the danger of large atrazine concentrations (230 ppb) that may prolong settlement
beyond the natural 7-day maximal survival period for Aurelia planulae.
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Ephyra production via polyp strobilation is a key determinant in scyphozoan populations,
thus increased production and survival of polyps is needed to boost medusa numbers (as is
hypothesized to occur with seasonal jelly blooms). However, recruitment of the pelagic larvae to
à substrate is a necessary prerequisite for subsequent asexual propagation of a species. Like
many marine invertebrates, scyphozoan medusae are r-selected, relying on large numbers of
offspring (with a high mortality rate) to further the species, however, prolonging settlement
likely artificially increases mortality among planulae. Studies on respiration and metabolism on
the lecithotrophic planulae estimate maximal survival period at one week for A. aurita
(Schneider and Weisse 1985); should chemicals (like atrazine) delay settlement for a sufficient
amount of time beyond this maximal survival time, individual larvae may use up their energy
reserves and die. Any factors that influence settlement and thereby increase larval mortality
likely have lasting effects on the population, should they decrease abundance of polyps on a
sufficiently large scale.
There were various factors which may have confounded some aspects of the present
study, most importantly simple human error during observation contributing to inaccurate
counts. Motile planulae were small, but placed in a relatively large dish; active individuals may
have moved around within the dish and thereby been counted multiple times. To rectify this
problem, the lowest count was used in determining planulae numbers in each dish, as this
counting methodology theoretically accounted for double-counting motile planulae. However,
this method confounded the mortality data, given planula numbers may have been
underestimated by taking the lowest number, thus subsequent days may have had a higher
planula count. This created a negative mortality among planulae, seen in the cumulative and
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daily mortality plots. Despite this fact, mortality data are still valid given that they follow a clear
trend.
The availability of food may have been the largest source of variation among dishes.
During the 14-day time series, the developing polyps were not fed. However, sand-filtered
seawater was used to prepare the atrazine and control dilutions and this seawater may have
contained various microorganisms, upon which the polyps may have fed. During observation,
several dishes were observed with ciliate infestations and polyps were often observed eating
ciliates. Differences in food availability would have affected growth and development in
established polyps, but would have had negligible effects on observations of planula settlement.
Predation on unmetamorphosed planulae by settled polyps may also have confounded mortality
data.
A variety of potential mechanisms potentially account for the negative effect of atrazine
on A. labiata planula settlement. Atrazine is an herbicide designed to kill plants, thus it may have
reduced (via interference with the photosynthetic electron transport system) the amount of
organic biofilm coating the experimental dishes, making the substrates were less desirable to the
planula larvae, accounting for decreased settlement (DeLorenzo et al. 2001). Various chemicals
have been shown to induce metamorphosis of the planula larva into a polyp, especially in
tropical rhizostome jellies (such as Cassiopeia xamachana and C. andromeda). Planulae of
Cassiopeia seem to favor “dirty" substrates with a bio-organic film present, such as that
produced by bacteria whilst decomposing mangrove leaves, thereby releasing proline-rich
peptides, indicating the presence of mangroves, ideal habitat for Cassiopeia (Müller and Leitz
2002; Fitt and Hoffmann 1985; Fleck et al. 1999; Fleck and Fitt 1999). Induction of settlement
via chemical cues suggests a chemosensory mechanism in planula larvae. Given A. labiata’s
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preferred settlement on substrates with a biofilm, perhaps atrazine somehow interfered with the
chemosensory mechanism(s) of the planula, accounting for decreased settlement in higher
atrazine concentrations.
Atrazine has been shown to induce aneuploidy in oysters (Bouilly et al. 2003); such
chromosome mutations significantly affect growth and development. Any occurrence of
aneuploidy in planulae may potentially account for decreased settlement with higher atrazine
concentrations. Given the necessity of intact, functional chromosomes in developmental
processes, alteration via chemical pollutants may significantly delay settlement if genes involved
in settlement are somehow tainted. Similarly, any interruptions of other chemical or protein
systems within the planula may also explain the negative effects of atrazine. In salmon, atrazine
has been shown to induce elevated levels of thyroxine, a chemical Spangenberg (1984b) reported
to decrease statolith formation in Aurelia polyps and ephyrae (Waring and Moore 2004). If
similar processes are occurring in planula and affecting their ability to determine orientation
during settlement, this may explain decreased settlement at high levels of the herbicide.
Such a finding of negative impacts on key phases in scyphozoan development merits
further investigation due to its significance on a broader scale. Scyphozoans are typically tolerant
of poor ocean conditions, able to thrive in conditions few other animals would be able to survive.
The scyphozoan Aurelia aurita is a generalist, able to persist in a variety of habitats (including
polluted waters) by relying on its benthic polyp stage for persistence in such diverse
environmental conditions; the polyps of A. aurita are quite hardy, able to survive for long
periods of time without food and exhibit little specificity of diet (Lucas 2001). Recent research
has suggested polyps of the medusa Chrysaora quinquecirrha are able to tolerate hypoxic
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conditions (Condon et al. 2001). However, it is clear that scyphozoans may not be adapted to the
chemical alterations in water chemistry associated with chemical pollutants.
Given the widespread use of atrazine, there are undoubtedly large amounts of the
chemical in runoff from land (especially in areas with developed agriculture industries). With
such large quantities of atrazine potentially entering the ocean, there is cause for concern as
scyphozoan development is susceptible to atrazine, since this implies the chemical may likely be
affecting less resilient species as well. Further study is clearly needed as to the effects of atrazine
in development in other marine organisms. Planula settlement in other scyphozoans and indeed
other cnidarians would be of key interest. Öther scyphozoan species (such as Aurelia aurita and
Chrysaora quinquecirrha) may be more resilient than the Aurelia labiata species used in this
study. Both A. aurita and C. quinquecirrha are found in the eutrophic "dead" waters of the Gulf
of Mexico; A. aurita is also an invasive species that has established itself quite successfully in
various habitats, and thus may be more tolerant of stressors (like chemicals).
Conversely, some rhizostome species (Cassiopeia sp. and Mastigias sp.) may be more
sensitive, given their natural habitat of pristine mangroves and tropical lagoons, respectively.
Studies on hydrozoan medusae would be invaluable, given that the current decrease in
hydromedusae populations in the Adriatic Sea and elsewhere (Benovic et al. 1987, Benovic and
Lucic 1996). These decreases may be due to pollution and eutrophication (Benovic et al. 2000);
examination of the effect of atrazine and other chemical pollutants on hydromedusa development
would support or refute this hypothesis. On a large scale, coral reefs are critical ecological and
economic resources in many areas of the south Pacific Ocean. The Great Barrier Reef is a major
source of income for Australia through ecotourism, fishing, etc., but is located directly offshore
from major agriculture operations in northern Queensland. Undoubtedly agricultural runoff is
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significant in areas of the Great Barrier Reef, but exactly how much chemical pollution from
agricultural pesticides and herbicides is being released remains unknown. Analysis of
agricultural chemicals’ effects on coral reproduction (specifically planula settlement) is indeed
urgent given the potential for large impacts over a broad scale.
Basic studies of atrazine on other marine invertebrate embryos and larvae during
development is key to understanding the full scale of this chemical’s potential environmental
impact. This study was initiated well after the establishment of atrazine as a common
commercial herbicide; despite the fact that it has been widely used for a considerable amount of
time, only recently have the negative effects of this chemical been elucidated. Such findings
suggest a major revision in chemical testing criteria prior to release for large-scale application is
necessary. The ecological implications of atrazine in marine communities may be widespread
and merit a critical overhaul of current pesticide usage in agriculture to better account for
inevitable leakage of these toxic chemicals into the ocean.
Conclusions
In high atrazine concentrations, there was a clear increase in the time planulae dominated
the experimental population, consistent with a treatment effect of the herbicide, suggesting it
prolongs settlement time. Atrazine also induced higher mortality over the full time series,
potentially due to delaying of settlement beyond maximal survival period of the larva. Together,
these data present strong evidence for the negative effects of atrazine, the most commonly used
herbicide in the United States and as such, there is sufficient need for a critical re-analysis of
herbicide usage and potential ecological effects of agricultural runoff.
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Acknowledgements
The author wishes to thank David Epel for his expertise and guidance over the course of the
project. Freya Sommer provided invaluable advice on the culture of Aurelia despite the
constraints of available resources. Chad Widmer graciously provided specimens (courtesy of the
Monterey Bay Aquarium) and his expertise in culture of Aurelia labiata. James Watanabe
provided support, both in experimental design/logistics and statistical analysis. The Epel Lab at
Hopkins Marine Station (Pacific Grove, California) provided supplies; the Monterey Bay
Aquarium’s Drifters Lab provided specimens.
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Spangenberg, D. B. 1980. Strobilation aberrations in Aurelia induced by petroleum-related
aniline and phenol. In Development and Cellular Biology of Coelenterates. Ed. P. Tardent and R.
Tardent. Elsevier/North Holland Biomedical Press, Amsterdam.
Spangenberg, D. B. 1984a. Üse of the Aurelia metamorphosis test system to detect subtle effects
of selected hydrocarbons and petroleum oil. Marine Environmental Research. 14: 281-303.
Spangenberg, D. B. 1984b. Effects of exogenous thyroxine on statolith synthesis and resorption
in Aurelia. American Zoologist. 24: 917-923.
Underwood, A. J. 1997. Experiments in ecology: their logical design and interpretation using
analysis of variance. Cambridge University Press, New York, NY.
U.S. Department of Agriculture. 1994. Pesticides Industry Sales and Usage: 1992 and 1993
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Nava 19
Tables
Table 1: Estimated Tyo for planula to polyp population shift in Aurelia labiata across treatments
Atrazine
5090 Time Tag (days,
Concentration
Replicate 1 Replicate II
Replicate III
Mean
2.8340.76
Control (Oppb)
2.0
4.0040.50
1 ppb
4.0
45
5.1741.15
10 ppb
4.5
45
30 ppb
6.5
5.8341.15
6.5
Table II: Turnover time in Aurelia labiata planulae by replicate and treatment
Atrazine
Turnover Time (days)
Concentration
Replicate l
Replicate II
Replicate III
2.5
Control (Opp
0.73
4.52
4.47
3.85
1pp
10 pp
4.62
5.61
705
30 ppb
503
6.72
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Mean
1.9641.07
4.2840.37
4.9340.50
6.2741.09
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Table III: Rate of Aurelia labiata planula disappearance and polyp appearance across treatments
Atrazine
Rate of Change (percentage per day)
Concentration
Replicate 1
Replicate Il
Replicate III
Mean
0.04
Control (Oppb)
0.062
0.064
0.05840.008
0.074
1 ppb
007
0.070
0.07340.003
10 ppb
0.076
0.080
0.080
0.07840.002
0.082
30 ppb
0.084
0.094
0.08740.007
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Table IV: Cumulative mortality of Aurelia labiata planulae by treatment over 14-day time series
Atrazine
Rate of Change in Cumulative Mortality (new deaths per day)
Concentration
Replicate l
Replicate Il
Replicate III
Mean
P
Control (Oppb)
0.60
0.66
-0.40
0.2940.60
1.3440.06
135
1.39
127
1 ppb
140
10 ppb
2.49
1.85
1.9140.55
30 ppb
1.87
— 048
1.2340.70
1.34
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Figure Legend
Fig. 1. Aurelia labiata planula settlement: control treatment. Successive life-stages (from planula
to developing polyps) plotted over 14-day time series. Clear, rapid decrease in percent of
planulae in population after 2 days. Error bars omitted for clarity.
Fig. 2. Aurelia labiata planula settlement in 1 ppb atrazine. Successive life-stages plotted over 14
days; decline in planula abundance gradual. Error bars omitted for clarity,
Fig. 3. Aurelia labiata planula settlement in 10 ppb atrazine. Life-stages plotted over full time
series. Planula dominance clearly prolonged, with planula still present until 27 days from start,
Error bars omitted for clarity.
Fig. 4. Aurelia labiata planula settlement in 30 ppb atrazine. Plots of life stages yield obvious
planula dominance (250% population) for nearly 6 days. Error bars omitted for clarity.
Fig. 5. Aurelia labiata turnover point. Trendlines for planulae and total settled polyps intersect to
give time (1.96 days) when dominance shifted from larvae to polyp within all control treatments,
Error bars represent standard deviation from the mean.
Fig. 6. Aurelia labiata turnover point in 1 ppb atrazine. Planulae and settled polyp populations
used to extrapolate trendline; intersection gives turnover time (4.28 days) for population
dominance shift. Error bars represent standard deviation from the mean.
Fig. 7. Aurelia labiata turnover point in 10 ppb atrazine. Trendlines extrapolated from plots of
planulae and sessile polyps over full time series. Intersection used to determine time (4.93 days)
when population shifted from predominantly free-swimming larvae to settled polyps. Error bars
represent standard deviation from the mean.
Fig. 8. Aurelia labiata turnover point in 30 ppb atrazine. Plotted trendline from planulae and
polyp counts gave intersection point, where population dominance shifts. Turnover time (6.27
days) was the highest of all experimental treatments. Error bars represent standard deviation
from the mean.
Fig. 9. Aurelia labiata turnover for all experimental treatments and control. Plot with all
treatments shows clear shift in intersection point of planula and polyp percentages with increased
atrazine concentration. Intersection shifted right (i.e. turnover time longer) with greater atrazine
concentrations. Error bars omitted for clarity.
Fig. 10. Aurelia labiata planula cumulative mortality. Total number of dead individuals after
successive days following start of time series increases over time. Average increase of 0.29
deaths daily. Error bars represent standard deviation from the mean.
Fig. 11. Aurelia labiata planula cumulative mortality in 1 ppb atrazine treatment. Total number
of deaths summed after each day. Average increase of 1.34 deaths per day. Error bars represent
standard deviation from the mean.
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Fig. 12. Aurelia labiata planula cumulative mortality in 10 ppb atrazine. Total number of deaths
increased daily by a rate of 1.91 new deaths per day. Highest cumulative mortality rate of all
treatments observed in 10 ppb atrazine dilution. Error bars represent standard deviation from the
mean.
Fig. 13. Aurelia labiata planula cumulative mortality in 30 ppb atrazine. Cumulative deaths of
planula since start of experimental run. Average increase of 1.23 new deaths per day. Error bars
represent standard deviation from the mean.
Fig. 14. Turnover time in Aurelia labiata planulae in relation to atrazine concentration. Mean
turnover times for each atrazine concentration plotted to determine any correlation with atrazine.
With increased atrazine concentration (ppb), turnover time lengthens by 0.12 days. Error bars
represent standard deviation from the treatment mean.
Fig. 1.
0.4
0.2
A. labiata Planula Settlement: Control
L

9
2 3 4 5
Day
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10 11 12 13 14
Fig. 2.
0.4
0.2
A. labiata Planula Settlement: 1 ppb Atrazine


4 5 6
11
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12 13 14
Fig. 3.
0.4
0.2

A. labiata Planula Settlement: 10 ppb Atrazine


—


S


8 9 10 11 12 13 14
4 5
Day
Nava 27
Fig. 4.
0.6
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A. labiata Planula Settlement: 30 ppb Atrazine





8 9 10 11 12 13 14
1 2 3 4 5 6
Day
Fig. 5.
0.8 -
0.4
0.2
-0.2
-0.4
Average Settlement and Turnover ofA. labiata Planulae:
Control Treatment


y = 0.0582x + 0.3798

R2 = 0.606
45
2


y = -0.0582x + 0.6202

S
R°= 0.606
—
1 2 3 4 5 6 7 8
10 11
Day
Nava 29
Fig. 6.
0.6
-0.2
-0.4
Average Settlement and Turnover of A. labiata Planulae:
1 ppb Atrazine
L
= 0.0726x + 0.1889

-0.75

.
1 2 3 4 5
Nava 30
Fig. 7.
0.8
0.6
0.2
-0.2
-0.4
Nava 31
Average Settlement and Turnover of A. labiata Planulae:
10 ppb Atrazine



y- 0.0784x 4 0.1133
R? = 0.8495
-O.0784x + 0.8867

R? = 0.8495



10 11 2--98.
1 2 3 4 5 6 | 7 | 8 |
Day
Fig. 8.
0.6
0.2
-0.2
-0.4
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Average Settlement and Turnover of A. labiata Planulae:
30 ppb Atrazine

y = 0.0865x - 0.0432
R° = 0.8738


-0.0865x + 1.0432

R2 J0.8738


2..
1 2 3 4 5 6 7
11 12
Day
Fig. 9.
0.8
Average Settlement of A. labiata : All Treatments


2
EE
10 11 12 13 14
2 3 4 5 6
Day
Nava 33
Fig. 10.
1
10
Average Cumulative Mortality of A. labiata Planulae: Control
13.29
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Fig. 11.
20
315
Average Cumulative Mortality of A. labiata Planulae: 1 ppb Atrazine


1.3378 4 15.
R° = 0.7613
Days After Start
Nava 35
Fig. 12.
Average Cumulative Mortality of A. labiata Planulae: 10 ppb Atrazine
y - 1.9139

Nava 36
Fig. 13.
Average Cumulative Mortality of A. labiata Planulae: 30 ppb Atrazine
1.2308x + 14.53
R2 = 0.8654

Days Äfter Start
Nava 37
Fig. 14.
Atrazine Concentration vs. Turnover Time in A. labiata Planulae
V - 0.10827 4 3.2499

R° = 0.7003


Atrazine Concentration (ppb)
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