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Sex, Drugs, and Rotifers: Endocrine Disrupting Water Contaminants and Rotifer Reproductive Cycling Charlotte Hovland 1 Advised by Kristin Gribble 2 1 The Biological Sciences Collegiate Division, University of Chicago, Chicago, IL 60637 USA 2 Josephine Bay Paul Center. Marine Biological Laboratory, Woods Hole, MA 02543 USA
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Page 1: Sex, Drugs, and Rotifers: Endocrine Disrupting Water ... · Sex, Drugs, and Rotifers: Endocrine Disrupting Water Contaminants and Rotifer Reproductive Cycling Charlotte Hovland1 Advised

Sex, Drugs, and Rotifers: Endocrine Disrupting Water Contaminants and Rotifer

Reproductive Cycling

Charlotte Hovland1

Advised by Kristin Gribble2

1The Biological Sciences Collegiate Division, University of Chicago,

Chicago, IL 60637 USA

2Josephine Bay Paul Center. Marine Biological Laboratory, Woods Hole, MA 02543 USA

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Abstract

Monogonot rotifers are near-ubiquitous invertebrate zooplankton, distinguished by their

cyclically parthanogenetic reproductive activity. This reproductive cycling is regulated by steroid

hormones, possibly including estrogen. In this study, I observed the effects of two estrogen

agonists and known environmental pollutants, 4-nonylphenol and 17α-ethynylestradiol, on rotifer

population growth rates, sex ratios, and egg quality. Neither nonylphenol or ethynylestradiol, nor

both in conjunction had any effect on population growth rates and they had only mixed effects on

sex ratios. However, the estrogen agonists did have a marked impact on egg quality. The

estrogen agonists’ effects on egg development were sex specific, with male eggs showing greater

susceptibility. Nonylphenol and ethynylestradiol influenced egg quality at far lower

concentrations in combination than were sufficient to produce results in tests of individual

compounds. This suggests interactions between the two endocrine-disrupting compounds that

may be cause for environmental concern.

Keywords: Rotifers, endocrine disruption, mixis

Introduction

Rotifers (phylum Rotifera) are zooplanktonic invertebrates. Rotifers are “ubiquitous,”

appearing globally in freshwater and marine systems, and even in moist soils (Fontaneto and De

Smet, 2015). Although they are small (generally < 1mm in length), rotifers are of great

ecological importance. As low-level consumers, rotifers take in energy and nutrients from

phytoplankton, detritus, and single-celled bacteria and protozoa (Fontaneto and De Smet, 2015).

In turn, rotifers are consumed by macro-organisms, including large zooplankton, benthic worms,

and larval fish. Rotifers package energy and nutrients from the smallest scale organisms in their

ecosystems and allow their export to larger animals, bridging the gap between the micro and

macroscopic worlds within aquatic ecosystems (Fontaneto and De Smet, 2015; Huang et al.,

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2012). Monogonota is the most specious rotifer sub-group, and includes my study organism,

Brachionus manjavacas.

Monogonot rotifers are cyclically parthenogenetic, and males and females are highly

sexually dimorphic, with females composing most of the population. Under ideal conditions,

asexual (amictic) females produce eggs by mitosis, resulting in the parthanogenetic development

of clonal, diploid daughters (Radix et al., 2001). However, at high populations densities and in

response to changes in photoperiod, sexual (mictic) reproduction is triggered, and a portion of

the eggs released by the amictic females develop into mictic females, which produce haploid ova

by meiosis (Snell, 2011). If these eggs go unfertilized, they develop into small, haploid males,

which are then available to mate with the mictic females. Once mating occurs, the zygote

develops into a ‘resting egg,’ an embryo surrounded by a thick shell. The resting eggs settle out

of the water column and enter a period of dormancy in the sediments below (Fontaneto and De

Smet, 2015). Once conditions are again favorable, the resting eggs emerge and complete their

development into amictic females.

Mictic reproduction is density dependent and regulates rotifer population dynamics. The

production of resting eggs removes rotifers from active circulation in preparation for winter,

during population booms, or when their aquatic habitat shrinks, concentrating the rotifers in the

remaining water and increasing their population densities. This allows rotifer populations to

endure dramatic fluctuations in habitat quality. Mixis is thought to be under the control of a

pheromone, released into water by female rotifers (Snell, 2006). As in bacterial quorum sensing,

the strength of the signal is related to the density of rotifers releasing the signaling molecule, so

that the rotifers will only transition to mixis when certain density thresholds are surpassed (Snell,

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2011; Stout et al., 2010). Once the mixis signal has been triggered, the rotifers’ reproductive

response is under hormonal control (Snell, 2011; Stout, 2010).

Steroid hormones in particular are increasingly well-supported candidates for the primary

regulators of rotifer reproduction. Snell et al. (2006) observed similarities between fragments of

a purported mixis signaling protein and a protein that induces steroidogenesis in humans, while

Stout et al. (2010) revealed the presence of progesterone receptors in male and female rotifers.

Recently, Jones et al. (2017) discovered an estrogen-like receptor in Brachionus rotifers. This

estrogen-like receptor is so highly conserved with respect to mammalian estrogen receptors that

it is able to bind human estradiol as a ligand. Several teams of researchers have shown that

rotifers respond when their environment is dosed with human steroid hormones, including

progesterone, estrogen, and testosterone (Snell and DesRosiers, 2008; Gardallo et al., 1997;

Preston et al., 2000). This has led to concerns that rotifer reproductive cycling may be disrupted

by water contaminants that are androgen and estrogen mimics or antagonists. These

contaminants include β –estradiol, ethynylestradiol, nonylphenol and nonylphenol-ethoxylate,

dioxin, and endosulfan (Depledge and Billinghurst, 1999; Roefer, 2000; Swartz et al., 2006; and

Zhang, 2015). In isolation, ethynylestradiol and nonylphenol have been shown to reduce the

number of females in rotifer populations as well as the proportion of mictic females (Radix,

2001). Endocrine agonists and antagonists have previously been shown to cause changes to the

sex ratios of fish populations and to hinder sexual development and reproduction in several

vertebrate groups (Depledge and Billinghurst, 1999). Diverse marine invertebrates, including

mollusks and arthropods, have been found to be sensitive to endocrine disruption (Depledge and

Billinghurst, 1999). Endocrine disruption is of particular concern in the effort to monitor and

regulate water pollution, as pollutants may produce reproduction-disrupting endocrine effects

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even at low concentrations that do not result in outright toxicity (Depledge and Billinghurst,

1999).

Little is yet known about the potential synergistic, additive, or antagonistic effects of

multiple endocrine-disrupting contaminants—despite the fact that most contaminated water

sources contain multiple contaminants, and contaminants cannot be expected to appear in

isolation in ecologically relevant environments (Depledge and Billinghurst, 1999; Standley et al.,

2008). When Thorpe et al. studied the combined effects of nonylphenol and ethynylestradiol in

developing rainbow trout (Oncorhynchus mykiss), they found an additive response on increasing

vitellogenin concentrations in the trouts’ blood plasma (2001). Given that steroid hormone

receptors appear to be widely conserved in bilatarians, it is of interest to see if additive effects of

endocrine-disrupting contamination are also observed in invertebrate members of impacted

ecosystems.

In this study, I investigated the effects of ethynylestradiol, an artificial estrogen

manufactured as an oral contraceptive and present in wastewater, and nonylphenol, an estrogen-

mimicking contaminant released from plastics and used as an industrial surfactant (Swartz et al.,

2006; Soares, 2008), on the growth rate and mictic/amictic ratios of rotifer populations, and on

the size of their eggs. I examined the effects of these contaminants in and above environmental

concentrations, as well as their potential effects in combination.

Given that monogonot rotifers appear to have active estrogen-like receptors localized to

their reproductive structures (Jones et al., 2017), I expected that their reproductive cycles would

be influenced by exposure to the estrogen agonists ethynylestradiol and nonylphenol. Based on

the work of Preston et al. (2000) and Radix et al., (2001), if the compounds were to have any

effect, I would have expected them to lower population growth rates, however, I was uncertain as

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to whether any effect would be seen at environmental-level concentrations. As the estrogen

hormone signaling pathway is deeply conserved between rotifers and vertebrates (Jones et al.,

2017), I expected that, as in the case of the rainbow trout, a combination of contaminants would

have a greater effect than would each contaminant alone (Thorpe et al., 2001).

Methods

I reared Brachionus manjavacas rotifers under eleven chemical treatments: a control

without added contaminants; a control to which only ethanol was added; three treatments to

which 4-nonylphenol (Standard grade, Sigma-Aldrich, Milwaukee USA) was added dissolved in

ethanol to concentrations of 1 ug/L (0.0045 uM), 5 ug/L (0.0227 uM), and 50 ug/L (0.2273 uM);

three treatments to which 17α-ethynylestradiol (> 98%, Sigma-Aldrich, Milwaukee USA) was

added to concentrations of 1 ng/L (3.378x10-6

uM), 5 ng/L (1.689x10-5

uM), and 50 ng/L

(1.689x10-4

uM); and three additional treatments with both 4-nonylphenol and 17α-

ethynylestradiol in combination. In the treatments with both 4-nonylphenol and 17α-

ethynylestradiol, one treatment contained 4-nonylphenol at a concentration of 1 ug/L and 17α-

ethynylestradiol at a concentration of 1 ng/L, while the next contained 5 ug/L 4-nonylphenol and

5 ng/L 17α-ethynylestradiol, and the final treatment contained 50 ug/L 4-nonylphenol and 50

ng/L 17α-ethynylestradiol. Each treatment was carried out in three replicates.

I prepared each replicate in a 50 mL glass vial. Initially, I filled each replicate with 20

mL from a single, mixed solution of Tetraselmis suecica, Instant Ocean, and B. manjavacas. The

initial concentration of T. suecica was ~6x105

cells per millilitre, while the initial concentration

of rotifers was ~5 individuals per millilitre. The rotifers were sourced from mixed-age laboratory

cultures maintained by Kristin Gribble at the MBL. I spiked each vial individually with the

appropriate volume of contaminant solution. Given that the greatest added volume of

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contaminant was 200 uL, only 1% of the 20 mL volume of the total solution, it was not necessary

to add any additional volume to the control vials.

I incubated the control vials and the vials treated with 17α-ethynylestradiol for 7 days,

but the ethanol controls and the vials treated with 4-nonylphenol and with both contaminants

were only incubated for 6 days. After the control vials and 17α-ethynylestradiol replicates had

incubated for 5 days, and the ethanol controls, 4-nonylphenol replicates, and combined replicates

had incubated for 4 days, I replenished each vial with 6 additional millilitres of algae solution. I

also added contaminant solution to the vials so that the increased volume of algae solution would

not result in more dilute contaminants. All incubation took place at 21°C and on a 12 hour light-

dark cycle.

I took 1 mL subsamples from each replicate during each day of their incubation period. I

observed these live subsamples using a dissecting microscope, noting the number of active male

rotifers. I then fixed each subsample with 20 uL of Lugol’s solution. Using a dissecting

microscope, I counted the rotifers and characterized them as mictic females, amictic females,

nonovigerous females, or males. I also counted the free eggs in each subsample, and determined

whether they were diploid female eggs, haploid male eggs, or resting cysts.

After the seventh day of incubation for the control and ethynylestradiol vials and the sixth

day of incubation for the ethanol control, nonylphenol, and combined treatments, I transferred 2

mL subsamples from each replicate into fresh vials, each containing 18 mL of algae solution and

the appropriate concentration of contaminants. I allowed these vials to incubate undisturbed for 5

days. On the fifth day, I took 1 mL subsamples from each vial, fixed them with Lugol’s solution,

and counted and categorized the mictic, amictic, and nonovigerous females, males, and eggs. I

then replenished the vials with 6 mL of algae solution, added the appropriate amounts of

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contaminants to maintain their concentrations, and allowed the vials to incubate overnight. The

next day, I took 7 mL subsamples from each vial, vortexed each subsample for a minute to

detach as many eggs as possible from the female rotifers, and treated the subsamples with

Lugol’s solution. I let the treated subsamples settle overnight and then removed 5 mL of the

supernatant. I prepared microscope slides using 30 uL of the material remaining at the bottom of

each subsample. I viewed these slides using a light microscope under a 40x objective lens. I

photographed the first 8-12 female and male eggs I saw from each sample. If there were not at

least 8 eggs of each type present on a single slide, I prepared a second slide from the same

sample. Resting cysts were rare, and I took photographs of each that I observed.

I processed the resulting photos (Figure 1) using Fiji (Schindelin et al., 2012), and used a

known scale to measure the area, perimeter, feret diameter (maximum caliper diameter), and

roundness of each egg, where:

𝑅𝑜𝑢𝑛𝑑𝑛𝑒𝑠𝑠 = 4 ∗ 𝐴𝑟𝑒𝑎

𝜋 (𝑀𝑎𝑗𝑜𝑟 𝑎𝑥𝑖𝑠)2

To test whether the contaminants resulted in significant effects, I used a Student’s t-test

to compare the mean proportions of mictic, amictic, nonovigerous, and male rotifers in treated

populations to those in control populations and to compare the average dimensions of the eggs

between the treated populations and control populations. I compared the populations that had

been treated with ethynylestradiol only to the control populations. The nonylphenol treated

populations and the combined contaminant treated populations all contained ethanol as a solvent,

and thus were compared to the ethanol control populations. Growth rates were determined by

plotting the numbers of adult female rotifers and fitting an exponential curve to the data.

Results

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Time Series

No general toxicity seems to have been caused by any of the compounds used at any of

the test concentrations. All of the rotifer populations increased exponentially over time. Their

rate of growth, in terms of the number of adult females, was not impacted by any of the

contaminant treatments (Figure 2), nor was the time it took for the female population to double

(Figure 3).

End Point Population Mixis Ratios

No significant differences were observed between the total numbers of either rotifers or

eggs in between any treatments and their respective controls. However, the proportions of mictic

(Figure 4), and eggless females did vary somewhat between treatments, as did the ratios of male

and female eggs (Figures 5-6).

Here, it should be noted that the proportions of male and female eggs differed

significantly between the control replicates made with only Instant Ocean and the control

replicates to which ethanol was added. In the following analyses, all treatments containing

nonylphenol dissolved in ethanol have been compared only to the ethanol control, while

treatments containing only ethynylestradiol have been compared only to the control without

ethanol. Nevertheless, the presence of ethanol may represent a confounding factor in my

examination of estrogen-mimicking compounds.

The 1 ug/L nonylphenol treatment resulted in a lower proportion of mictic females and a

higher proportion of nonovigerous females, with the proportion of mictic females out of total

females dropping from an average of 5.3% (S.E. = 0.41%) to an average of 2.3% (S.E. = 0.63%)

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(P = 0.0155) (Figure 4). The rotifers reared in 1 ug/L nonylphenol also produced a lower

proportion of male eggs and a correspondingly greater proportion of female eggs (Figures 5-6).

In the ethanol control, male eggs averaged 43% of the total egg yield (S.E. = 4.1%), while female

eggs made up 57% of the total eggs (S.E. = 4.1 %). In the 1 ug/L nonylphenol treatment, male

eggs composed only 27% of total eggs and female eggs made up the remaining 73% (S.E. =

3.2%). However, no significant deviations from the control were observed for the 5 ug/L or 50

ug/L nonylphenol treatments (Figure 5).

Mictic females made up a smaller proportion of total adult females in the 1 ng/L

ethynylestradiol and 1 ug/L nonylphenol combined treatment than in the ethanol control (5.3%

vs. 3.3%) (P = 0.0156). A similar, but more dramatic effect was observed when both compounds

were present at 50 ng/L and 50 ug/L, respectively (P = 0.0067). When both compounds were

present at their highest concentration, on average mictic females only made up 1.2% of the

female population (S.E. = 0.64%). In this treatment, the proportion of female eggs out of total

eggs also increased, from an average of 57% to an average of 81% (S.E. = 4.9%) (P = 0.0184).

Ethynylestradiol alone did not appear to have any significant effects on the sex ratios of

either adult rotifer populations or their eggs.

Resting cysts appeared so infrequently that it was impractical to subject their counts to

statistical testing.

Egg Qualities

Area: After 5 days of undisturbed incubation (making 11/12 days of total contaminant

exposure), all treatments had plentiful male and female eggs, but there were not enough resting

cysts to produce sample sizes sufficient for further analysis.

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The area of female eggs ranged from 8069.456 um2 to 20014.049 um

2 with an overall

average of 14813.35 um2 (S.E. = 128.08 um

2). A significant difference between the mean area of

contaminant-exposed eggs and that of the control (P = 0.0051) was found only when the rotifers

were incubated with both ethynylestradiol and nonylphenol at the highest tested concentrations

(Figure 7). The mean area of the eggs from the ethanol control group was 15253.97 um2 with a

standard error of 443.04 um2, while the mean area of female eggs developed in 50 ng/L

ethynylestradiol and 50 ug/L nonylphenol was 13483.55 um2 with a standard error of 415.83

um2. On average, the affected eggs were 11% smaller by area than the control eggs.

Male eggs were smaller than female eggs, with an overall average area of only 5909.15

um2 (S.E. = 42.71 um

2). Significant differences were observed between the mean areas of the

controls, the 50 ug/L nonylphenol treatment, the 50 ng/L ethynylestradiol treatment, and all

treatments using both contaminants (Figure 8). On average, the eggs developed in 50 ng/L

ethynylestradiol were 4.4% larger by area than those of the control, while the male eggs

developed in 50 ug/L nonylphenol and in both nonylphenol and ethynylestradiol were smaller,

on average, than those in the ethanol control treatment (Table 1). Although the combination of

compounds produced statistically significant effects at lower concentrations than nonylphenol

alone, at the concentration when both produced an effect (50 ug/L nonylphenol and a

combination of 50 ug/L nonylphenol and 50 ng/L ethynylestradiol), there was not a significant

difference in the effect’s magnitude. The mean area of male eggs treated with both

ethynylestradiol and nonylphenol was not significantly different from those treated with

nonylphenol alone (P = 0.9028).

Perimeter: Neither ethylestradiol or nonylphenol had a significant effect on female egg

perimeter as individual compounds. When the compounds were applied in combination, results

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were mixed. The mean perimeter of the female control eggs was significantly greater (P < 0.05)

that that of the female eggs produced in 1 ng/L ethynylestradiol and 1 ug/L nonylphenol or that

of the female eggs produced in the highest concentration of both compounds (Figure 9).

However, the intermediate treatment, with 5 ng/L ethynylestradiol and 5 ug/L nonylphenol,

showed no significant deviation from the control (P = 0.1004).

The mean perimeters of male eggs (Figure 10) roughly corresponded to the mean areas.

Whether measured by area or perimeter, male egg size was affected by the combined

contaminants at lower concentrations than affected the female eggs. All cultures treated with

both compounds had significantly smaller mean male egg perimeters compared to controls. Male

eggs from the 50 ug/L nonylphenol treatment had, on average, 4.5% smaller perimeters than

male eggs from the ethanol control, and the difference between the 50 ug/L nonylphenol and

ethanol control treatments was found to be significant (P = 0.003). None of the ethynylestradiol

treatments were found to have any effect on male egg perimeter.

Feret Diameter: Under no circumstances did the average feret diameters of the female

eggs differ significantly from that of their relevant controls (Figure 11).

In contrast, the mean diameter of male eggs from the 50 ug/L nonylphenol treatment was

significantly smaller than that of the ethanol control (P = 0.0020). All treatments using both

contaminants also resulted in male eggs with small mean feret diameters compared to the ethanol

control (Figure 12).

Roundness: Out of a maximum of 1, the average female overall had a roundness of

0.8081 (S.E. = 0.0037). The average egg developed in 50 ng/L ethynylestradiol was significantly

rounder than the average control (0.8669 as compared to 0.7872; P = 0.00004). The average

female egg developed in 1 ng/L ethynylestradiol and 1 ug nonylphenol was less round than the

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average control (P = 0.0258), however no other treatments had any significant effects on female

egg roundness, including higher concentrations of both contaminants (Figure 13).

Male eggs that developed in 50 ng/L ethynylestradiol were, on average, significantly

rounder than control male eggs (0.8371 as compared to 0.7997, P = 0.0348). Male eggs that

developed in 50 ug/L nonylphenol were significantly rounder than control eggs (P = 0.0046).

Male eggs from the low concentration treatment with both compounds were rounder than the

control, as were male eggs from the highest concentration treatment, but the intermediate

concentration did not show significant effects on male egg shape (P = 0.0778) (Figure 14).

Discussion

Neither nonylphenol nor ethynyestradiol in isolation, nor both in conjunction, had a

significant effect on the growth rate or doubling time of B. plicatilis populations. These findings

are consistent with those of Radix et al. (2001), who found that ethynylestradiol only decreased

rates of population increase at concentrations of 1.72 uM or higher, while nonylphenol only

decreased the rate of population increase at concentrations higher than 0.59 uM. The maximum

concentrations used in the present study, selected to approximate the concentrations found in

Cape Cod wastewater (Swartz et al., 2006; Rudel et al., 1998; Bhandari, 2015) were 0.2273 uM

nonylphenol and 1.689x10-4

uM ethynylestradiol. The concentrations of nonylphenol and

ethynylestradiol typically seen in coastal waters (4.5x10-4

uM to 0.381 uM nonylphenol; 5x10-6

uM ethynylestradiol) are not sufficient to significantly impact the growth rates of local rotifer

populations (Swartz et al., 2006; Bhandari, 2015). However, nonylphenol and ethynylestradiol

are only two of many estrogen agonists that have been detected entering the ocean, and the

effects of many compounds in conjunction, although present at low concentrations, remain

worthy of study and concern.

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Exposure to estrogen agonists did not have any significant effect on the total number of

females or on the proportion of amictic females present. Treatment with nonylphenol or with

both nonylphenol and ethynylestradiol did result in a significantly lowered ratio of mictic

females to total females in three cases, but these effects did not follow a clear pattern with

respect to concentration. While somewhat perplexing, this is consistent with the 2001 findings of

Radix et al., who report, “it was difficult to establish a clear dose-response relationship” between

exposure to nonylphenol and changing ratios of mictic and amictic females. Previous researchers

have speculated that sexual reproductive processes in rotifers are more likely to be affected by

endocrine disruption than are asexual reproductive processes, perhaps because of the greater

complexity of endocrine signaling involved in carrying out sexual reproduction (Preston et al.,

2000). This study, which saw some effect of estrogen agonists on mictic female proportions but

none on the proportions of amictic females, could lend support to this hypothesis.

Preston et al. found alterations to the proportion of fertilized mictic females (mictic

females bearing resting cysts) to be a reliable indicator of endocrine disruption, with the

proportion of fertilized mictic females declining when exposed to 50 ug/L nonylphenol (2000).

However, in my experiment, which tested 50 ug/L nonylphenol, fertilized mictic females were so

rarely seen in any of the treatments that no useful comparisons between treatments could be

made. This scarcity of fertilized mictic females is likely a result of the short time period over

which the experiment was run. Each population was allowed to develop for only two

generations. It’s possible that a longer incubation time would have allowed more males to

accumulate, resulting in higher rates of fertilization and a greater accumulation of resting eggs.

While the total numbers of eggs did not vary between treatments, and the proportions of

male and female eggs did not vary with any clear pattern, several trends emerged when I

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compared the shapes and sizes of eggs extracted from the different treatments. In general, male

eggs were much more susceptible to effects of the contaminants than were female eggs. Male

eggs responded to single contaminants when female eggs did not, and consistently responded to

lower concentrations of the combined contaminants. Preston et al. (2000) hypothesized that

certain “aspects of the life history of male rotifers” could cause them to be more vulnerable to

endocrine disruption. Although the mechanisms that would cause such a disparity are not well

characterized, they could stem from differences in the biochemical pathways used in the

development of males and females. This sex specific susceptibility to estrogen agonists appears

to extend to eggs, and indicates that nonylphenol and ethynylestradiol are causing endocrine

effects, not merely general toxicity, which would be expected to impact all egg morphologies

equally.

Additionally, effects on egg size and shape occurred at concentrations of the combined

compounds that were insufficient to produce significant change when each compound was tested

individually. For all measures of egg size, nonylphenol alone had a significant effect on male

egg size only at its highest concentration. When both compounds were present, significant

effects were observed at all concentrations. The interactions between the two compounds do not

appear to be simply additive: in the case of male egg area, high concentrations of nonylphenol

alone resulted in a slightly smaller average egg, while high concentrations of ethynylestradiol

resulted in a slightly larger average egg. If the effects of the two compounds were simply added

together, one might expect to see the two compounds counteract each other. Instead, even the

lowest concentration of both contaminants resulted in male eggs that were, on average, smaller

than the control, an effect which was not achieved by similar concentrations of nonylphenol or

ethynylestradiol alone. Additionally, at concentrations where both an individual compound and

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the combination of compounds had an effect, the magnitude of the effect was not significantly

greater under the combined treatment. In order to more definitely characterize the interaction

between nonylphenol and ethynylestradiol, a wider range of concentrations should be tested in

order to establish curve by which responses to various doses of individual and combined

compounds could be compared. This question is of great interest going forward, as real

environments are usually polluted with trace amounts of many different contaminants, rather

than high concentrations of a only a few compounds. If the interaction of multiple endocrine

disruptors allows them to have greater effects at lowered concentrations, then even trace amounts

of endocrine agonists may be cause for grave environmental concern.

Acknowledgments

This work would not have been possible without Kristin Gribble’s mentorship, Emily

Corey’s aid and patience, and the generosity of the Gribble Lab in sharing their knowledge and

resources with me.

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Tables

Table 1: Statistically significant differences in haploid (male) egg area

Figures

Figure 1: Representative images of different egg morphologies

Figure 2: Comparison of population growth rate across treatments

Figure 3: Comparison of population doubling time across treatments

Figure 4: Percentage of mictic females out of adult females

Figure 5: Percentage of male eggs out of total eggs

Figure 6: Percentage of female eggs out of total eggs

Figure 7: Comparison of diploid (female) egg areas

Figure 8: Comparison of haploid (male) egg areas

Figure 9: Comparison of diploid (female) egg perimeters

Figure 10: Comparison of haploid (male) egg perimeters

Figure 11: Comparison of diploid (female) eggs – maximum diameter

Figure 12: Comparison of haploid (male) eggs – maximum diameter

Figure 13: Comparison of roundness among diploid (female) eggs

Figure 14: Comparison of roundness among haploid (male) eggs

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Table 1: Results of Student’s t-test comparing the mean areas of eggs between treatments. The

null hypothesis was that the mean areas would be the same between contaminant treatments and

the relevant controls.

A. Male Eggs

B. Female Eggs

Treatment Mean Area (um2) S.E. (um

2) P-value

Control 15544.63 365.33 N/A

Ethanol Control 15253.97 443.04 N/A

P-value relative to

Control

17EE 1 16018.22 277.61 0.3067

17EE 5 15111.66 434.60 0.4444

17EE 50 15626.45 348.13 0.8718

P-value relative to

Ethanol Control

4N 1 14363.94 443.63 0.1612

4N 5 15269.80 400.68 0.9788

4N 50 14269.99 496.94 0.1456

Both 1 14359.03 370.75 0.1271

Both 5 14188.79 390.61 0.0745

Both 50 13483.55 415.83 0.0051

Treatment Mean Area (um2) S.E. (um

2) P-value

Control 6158.16 100.55 N/A

Ethanol Control 6057.90 119.63 N/A

P-value relative to

Control

17EE 1 5854.38 147.66 0.0945

17EE 5 6076.66 248.60 0.7629

17EE 50 6428.84 83.19 0.0426

P-value relative to

Ethanol Control

4N 1 6022.17 115.23 0.8305

4N 5 6040.91 63.07 0.9006

4N 50 5575.70 104.95 0.0037

Both 1 5522.81 117.33 0.0023

Both 5 5638.03 139.32 0.0259

Both 50 5559.88 154.30 0.0136

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Figure 1: Typical female (A.) and male (B.) eggs, and resting cyst (C.) Stained with Lugol’s

solution and captured at 40x magnification.

A. B.

C.

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Figure 2: Population growth, in terms of total adult females. Growth rates were unchanged

between controls and treatments. Error bars represent standard error.

A.

B.

C.

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Figure 3: Doubling time of female rotifer population. In no case did the average doubling time of

any treatment differ significantly from its relevant control. (Respective P values, 0.131, 0.977,

0.346, 0.256, 0.349, 0.434. All are > 0.05.)

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Figure 4: Mictic females made up 1.2 to 5.3 percent of adult females. Treatment with 1 ug/L

nonylphenol, with 1 ng/L ethynylestradiol and 1 ug/L nonylphenol, or with 50 ng/L

ethynylestradiol and 50 ug/L nonylphenol all depressed the proportion of mictic females. Error

bars show standard error. * denotes significance (P < 0.5)

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Figure 5: Male eggs made up 23-44% of the total eggs observed. The proportion of male eggs

differed from the relevant control only in the 1 ug/L nonylphenol treatment (4N 1). Error bars

represent standard error. * denotes significance (P < 0.5)

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Figure 6: Female eggs made up 55 to 88% of the total eggs observed. Error bars represent

standard error. * denotes significance (P < 0.5)

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Figure 7: The mean areas of diploid, female eggs collected after 11/12 total days of incubation.

Error bars show standard error. A significant difference from the control (P = 0.0051) was found

only when the rotifers were incubated with both 50 ng/L ethynylestradiol and 50 ug/L

nonylphenol (Both 50). * denotes significance (P < 0.5)

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Figure 8: The mean areas of haploid, male eggs collected after 11/12 total days of incubation.

Error bars show standard error. Significant deviations from the control (P < 0.05) were found in

the 50 ng/L ethynylestradiol treatment (17EE 50), the 50 ug/L nonylphenol treatment (4N 50),

and in all treatments with both contaminants (Both 1, Both 5, and Both 50). * denotes

significance (P < 0.5)

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Figure 9: The mean perimeters of diploid, female eggs. Error bars show standard error. The mean

perimeter of the control eggs was significantly greater than that of the eggs developed in 1 ng/L

ethynylestradiol and 1 ug/L nonylphenol (Both 1) or in 50 ng/L ethynylestradiol and 50 ug/L

nonylphenol (Both 50), but was not statistically significantly different from the intermediate

Both 5 treatment. * denotes significance (P < 0.5)

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Figure 10: Mean perimeters of haploid, male eggs. Error bars represent standard error. Eggs from

the 50 ug/L nonylphenol treatment (4N 50), and from all the cultures treated with both

compounds (Both 1, Both 5, Both 50), had significantly smaller mean perimeters compared to

the controls. * denotes significance (P < 0.5)

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Figure 11: Feret diameters (diameter at widest point) of diploid, female eggs. Error bars display

standard error. Under no circumstances did the diameters deviate significantly from the control

values.

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Figure 12: Feret diameters (diameter at widest point) of haploid, male eggs. Error bars show

standard error. The mean diameter of the eggs from the 50 ug/L nonylphenol treatment (4N 50)

was significantly smaller than that of the control. This was also the case for all treatments using

both contaminants (Both 1, Both 5, Both 50). * denotes significance (P < 0.5)

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Figure 13: Roundness of diploid, female eggs. Error bars show standard error. The average egg

developed in 50 ng/L ethynylestradiol (17EE 50) was significantly more circular than the

average control (P = 0.00004). The average egg developed in 1 ng/L ethynylestradiol and 1 ug

nonylphenol was less round than the average control. * denotes significance (P < 0.5)

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Figure 14: Roundness of haploid, male eggs. Error bars show standard error. The average egg

developed in 50 ng/L ethynylestradiol (17EE 50) was slightly more round than the average

control (P = 0.0348), as was the average male egg developed in 50 ug/L nonylphenol (4N 50) (P

= 0.0046). The average eggs developed in the lowest and highest concentrations of both

compounds (Both 1 and Both 50), were significantly rounder than the average control, but this

was not the case for the intermediate concentration (Both 5). * denotes significance (P < 0.5)


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