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Published in final edited form as: J Toxicol Environ Health A. 2013;76(20):1168–1181. doi: 10.1080/15287394.2013.845865

Burrowing in the freshwater mussel Elliptio complanata is sexually dimorphic and feminized by low levels of atrazine

Katherine Flynn †,‡,*,✉, Maria Belopolsky Wedin †,††, Josephine A Bonventre †, Marsha Dillon-White †, Jessica Hines †,††, Benjamin S Weeks †,‡, Chantale André ±, Martin P Schreibman ^, Francois Gagné ±
PMCID: PMC4369575  NIHMSID: NIHMS599673  PMID: 24279817

Abstract

The widely used herbicide atrazine (ATR) may have endocrine-associated adverse effects, including on behavior. In this study, 120 adult freshwater mussels, Elliptio complanata, were exposed to ATR at the environmentally-relevant concentrations of 1.5, 15, or 150 μg/L. Burrowing depth was evaluated hourly for 6 hr and at sacrifice animals were sexed by gonad smear. Female controls burrowed overall approximately 30% less than males, the first report of sexual dimorphism in this behavior. Atrazine at 15 μg/L feminized burrowing in both sexes in that exposed animals burrowed 20% less than their same sex controls. Males treated with 1.5 μg /L ATR displayed approximately 20-fold higher vitellogenin (VTG) levels than same sex controls. Higher concentrations of ATR were not associated with increasing effects. A scatterplot showed a weak binomial curve associating low burrowing with high VTG levels. Taken together, these data suggest a non-linear dose-response in behavioral and physiological feminization produced by ATR and support the need to reconsider the widespread use of this compound.

Introduction

Atrazine (ATR) is the most commonly used pesticide in the United States, with over 36 million kg applied to more than 25 million hectares annually (Graymore et al., 2001). The U.S. Environmental Protection Agency (EPA) has re-affirmed that the “level of concern” for this herbicide is 10–20 ppb (10–20 μg/L) in aquatic ecosystems (U.S. EPA, 2003). Freshwater bodies throughout the United States were found to be contaminated with ATR at concentrations ranging from less than one to over 100 μg/L and most freshwater not tested is believed to be contaminated at the lower end of this scale (Huber, 1993). Atrazine is a relatively hydrophilic chloro-s-triazine with water solubility of 70 ppm (70 mg/L) but its methanol solubility is 18,000 ppm (18 g/L), suggesting that it has the potential to settle in fatty tissues of exposed aquatic organisms (Gluth et al., 1985; Phyu et al., 2005).

Estrogenic effects of low dose ATR including hermaphroditism, demasculinized male larynges, and decreased plasma testosterone levels, were first reported in metamorphic African clawed frogs (Xenopus laevis) exposed as larvae to 0.01–200 μg/L ATR (Hayes et al., 2002). Other studies, however, reported no or only mild estrogenic effects under similar conditions: for example, no changes in gonad morphology or histology despite slightly lengthened larval periods in green frogs (Coady et al., 2004) and no marked impact on gonadal development nor sexual differentiation in zebrafish (Corvi et al., 2012). In addition, conditions may impact estrogenic actions of ATR, as predator stress was shown to amplify ATR-induced effects on gonadal development in anurans (LaFiandra et al., 2009). There are also conflicting reports regarding estrogenic effects of ATR in adults: Mature minnows (Gobiocypris rarus) showed increased expression of androgen and estrogen receptors (Yang et al., 2010) yet mature male X. laevis exposed to similar nanogram concentrations displayed no significant changes in circulating sex steroids (Hecker et al., 2005). A recent meta-analysis concluded that ATR exerted sufficient consistent indirect and sublethal effects on aquatic organisms, including on behavioral and reproductive systems, to be considered an endocrine disrupting chemical, while at the same time acknowledging that there is still controversy regarding certain endpoints and still much to learn about this compound (Rohr and McCoy, 2010). Behavioral assays are widely recognized as reliable indicators of low level exposure to endocrine disrupters (Clotfelter et al., 2004). In guppies, estrogen and xenoestrogens were reported to decrease sexual display behaviors by males (Bayley et al., 1999) and in E. complanata, 15 μg /L ATR reduced spatial aggregation (Flynn and Spellman, 2009). Measurements of reproductive behavior may be confounded by changes due to naturally occurring reproductive cycles, thus non-reproductive behaviors may be more reliable indicators of exposure to sex steroid-like compounds. Indeed, hyperactive swimming was shown in red drum (Sciaenops ocellatus) larvae following 4 days of exposure to 40 μg /L ATR (del Carmen Alvarez and Fuiman, 2005). However, despite the sexually dimorphic effects of natural sex steroids, and a recent call for more research in this area, little attention has been given in any animal model to the possibility of sexually dimorphic effects from exposure to endocrine disrupters (Orlando and Guillette, 2007).

Mollusks are increasingly seen as reliable models in testing for effects of environmental toxicants as they are filter feeders with a sedentary adult phase, making them uniquely susceptible to water pollution. In addition, mollusks are widespread in freshwater systems and easily maintained in a lab environment. In the species used here, the freshwater bivalve Elliptio complanata, the gonads of an individual animal may range from 100% female to 100% male allowing for every quantitative combination (Downing et al., 1989). Nonetheless, E. complanata and most Unionid bivalves are predominantly dioecious, and this is particularly so for larger animals (Kat, 1983). E. complanata, has long been used for in situ testing of estrogenic pollutants in studies focusing on physiological endpoints, such as shell growth, tissue weight and expression of the egg yolk protein precursor, vitellogenin (VTG) in males (Gagné et al., 2001; Gagné and Blaise, 2003; Won et al., 2005). Behavioral endpoints have been tested in other bivalve species: inhibition of valve gaping in swan mussels (Anodonta cygnea) and burrowing in little neck clams (Protothaca staminea) following metal pollution (Kadar et al., 2001; Phelps et al., 1983). In the Asian clam Corbicula fluminea, a body of literature shows altered valve closure patterns as a way to detect aquatic metal contamination (Tran et al., 2003; Chen et al., 2012). In the marine bivalve (Macoma liliana), the pesticide chlordane was found to inhibit horizontal movement (Roper and Hickey, 1994).

Bivalve burrowing is postulated to aid in avoiding predation (Saarinen and Taskinen, 2003) but also to contribute to the overall health of the freshwater ecosystem by re-suspending deposited material and oxygenating the substrate (Vaughn and Hakenkamp, 2001; Vaughn et al., 2008). The aim of this study was to examine the effects of ecologically-relevant concentrations of ATR on burrowing behavior in the freshwater mussel, E. complanata. It was postulated that exposure to the endocrine disrupter would have a sexually dimorphic effect on burrowing behavior. Unpublished data from our lab suggested that females burrow less than males, thus the inherent sexual dimorphism of burrowing behavior, the effect of estrogen and the relationship between ATR effects on burrowing and on the estrogen-regulated protein, VTG were also examined.

Materials and Methods

I. Animals

Between 50 and 150 E. complanata were gathered by hand from approximately 0.6 m depth at Wildwood Lake in Suffolk County, NY (40°89′N / 72°67′W) on three separate occasions, once each in the autumn of 2004, 2005 and 2006. The lake is in the Central Pine Barrens Core Preservation Area of eastern Long Island, where there is limited agriculture and recreation and no development has been permitted since 1993 (Central Pine Barrens Joint Planning and Policy Commission, 2013). The mussels were transported to the lab by car in buckets of lake water and acclimated to lab conditions following previously established protocol (Flynn and Spellman, 2009). Animals were maintained for 2 to 5 months in 120-L tanks containing aged tap water at 18 ± 2 °C, standard aquarium gravel (Estes Ultra Stone, Clifford W. Estes Co. Inc., Totowa, NJ) and a common freshwater plant, Sagittaria subulata. The tap water was tested by the town of Garden City and no detection of synthetic organic contaminants, including pesticides and herbicides, was reported (Garden City Park Water District, 2006). Tank water was re-circulated through a carbon filter (AquaClear, Mansfield, MA), and 20% of the water was changed approximately every two weeks. The room was on a 12 hr light:dark cycle to mimic winter conditions and minimize animal activity. Mussels were fed a crushed fish food (TetraColor Tropical Flakes, Tetra GMBH, Germany) or a phytoplankton suspension (Zoomax, Kent Marine Inc., Franklin WI) approximately twice per week. All animals were treated in accordance with U.S. laws.

For each ATR-exposure trial, 5 35-L tanks were prepared with 7 ± 0.5 cm of the same gravel as above and 30 L of aged tap water only. Animals were not fed during the exposure period. Water was maintained at 22 ± 4 °C and the room on a 16:8 hr light cycle to simulate summer conditions and increase animal activity. For each trial, 40 mussels, each between 7.5 and 10 cm long, were randomly assigned to one of 5 experimental tanks (8 per treatment) and acclimated for 2 weeks. One week prior to exposure, all animals were numbered (1–8) with a white marker on a colored tape glued to their shell, and measured (longest tip to tip) to the nearest 0.1mm with a standard vernier caliper (Carolina Biological Supply Co., Burlington, NC).

Treatment

Atrazine (ChemService, Inc., West Chester, PA) was prepared from a stock solution of 5 mg/ml in 70% ethanol, and dilutions were made such that an addition of less than 1 ml produced concentrations of 1.5, 15, or 150 μg/L in the tanks. Less than 1 ml of estradiol (Sigma-Aldrich, Inc., St. Louis, MO) in 70% ethanol resulted in a final tank concentration of 100 μg/L and served as the positive control, and 1 ml 70% ethanol alone was the negative control. At time = 0, either ATR, estradiol or vehicle was added to the tanks and carbon filters were removed, but water continued to circulate and all other conditions were maintained as above. Atrazine degradation in aquaria was shown to be minimal (Mazzanti et al., 2003), and other studies used a single ATR water contamination for exposures lasting 72 and 96 hr (Griggs and Belden, 2008; Pandey et al., 2011), suggesting that the desired concentrations were maintained for the 6-hr experimental period although ATR concentration in the tank water was not independently verified.

At t=0, immediately before the addition of the test substances, animals were equidistantly distributed on the surface of the gravel and “fraction burrowed” was recorded as zero. Burrowing measurements were then taken once every hr using calipers to record each mussel’s exposed length without disturbing the mussel’s position. All measurements were taken by research personnel blind to the experimental treatments. At the end of each 6-hr exposure, mussels were sacrificed by flash freezing and dissected on ice. A gonad smear was made on a glass slide and allowed to air dry for at least 48 hr. Smears were analyzed by two independent reviewers using a Zeiss light microscope at 40–200X magnification and animals were classified as per Gagné et al. (2001): female if eggs were present. While the absence of eggs might indicate a sexually undifferentiated and immature animal, all mussels were more than 8 cm long suggesting that they were mature, thus the absence of eggs was taken as indicative of a male. If reviewers disagreed on the gonad classification, those animals were not used in the analysis.

The ATR exposure described above was repeated thrice, on three separate occasions, using new animals each time. Experimental tanks were emptied, cleaned with a mild detergent and tap water and air-dried between trials. Tanks were re-filled with fresh aged tap water and clean, autoclaved gravel. All potentially ATR-contaminated water and animal tissue was disposed of in accordance with local regulations.

II. Burrowing Analysis

Fraction burrowed was calculated for each animal at each time period by subtracting the mussel’s exposed length from its full length to get the burrowed length, and dividing that by the full length. Those animals that did not burrow at all were not used in statistical analyses. SPSS 15.0 statistical software was used for all analyses with p<0.05 always required for significance. Average fraction burrowed was calculated independently for male and female controls, and analyzed with the paired samples t-test using fraction burrowed at each time as the dependent variable and sex as the between subject factor.

Fraction burrowed was expressed for each animal at each time period as % of same sex control burrowing. Two-way repeated measures analysis of variance (RM-ANOVA) used tank (treatment) as the unit of analysis and time and sex as the factors. Mauchly’s test of sphericity determined homogeneity of variance, and if equal variance could not be assured the Greenhouse-Geisser correction was applied before identifying any within-subjects effects i.e., time, time by sex, time by treatment and time by sex by treatment. For any significant effects, all times were compared to time 0 using the Student’s t-test with the Bonferroni correction for multiple samples. If the RM-ANOVA revealed a significant between-subjects effect (treatment) all groups were compared to control using the Dunnett’s t, 2-sided post-hoc.

III. Vitellogenin (VTG) Analysis

VTG levels in gonadal tissue were measured by the alkali-labile phosphate (ALP) assay following the method of Gagné and Blaise (2003). Won et al. (2005) showed that levels of gonadal ALP are significantly correlated with the sex-specific proteins characteristic of VTG, and the phosphate measured by the ALP assay was shown to originate from the highly phosphorylated VTG molecules (Hallgren et al., 2009). Following the 2005 exposure, gonadal tissue was dissected on ice, weighed and added at 5 g /ml to ice-cold 10 mM HEPES-NaOH buffer at pH 7.4, containing 1.15% KCl, 1 mM EDTA and 1 mM dithiothreitol. The mixture was homogenized on ice for 30 sec and centrifuged at 15,000× g for 20 min at 2 °C. The supernatant was saved as two separate aliquots per animal and stored at −80 °C for less than 6 months.

Samples were transported on dry ice to the Environment Canada Laboratory where the ALP assay was performed. Briefly, samples were subjected to acetone fractionation followed by centrifugation at 1000× g for 5 min, NaOH was added to liberate phosphates and the phosphomolybdenum method was used to determine ALP concentration. Molybdate, the reducer ANSA (1,2,4-aminoaphtholsulfonic acid) and distilled water were added to samples in a 96 well plate and read on a spectrophotometer. Data were normalized to a KH2PO4 (potassium phosphate) standard curve.

Independent ALP assays were performed on two separate days, each assay contained gonad samples in duplicate from 4 of 8 animals in each tank. If duplicate values differed by more than 100%, those values were not used in the analyses. For each day’s trial, average VTG concentrations (μg ALP/g gonadal tissue) were calculated for control males and females separately and the Student’s t-test for independent variables was used to compare the means on each day. Levene’s Test for Equality of Variance was performed and if p>0.05, equal variance was not assumed and the appropriate correction was applied. The average VTG concentration for each treated animal was then converted to % of same sex same day control and a 2-way ANOVA using treatment and sex as dependent variables compared VTG concentrations. If the ANOVA revealed a significant between-subjects effect (treatment), all groups were compared to the control using the Dunnett’s t, 2-sided post-hoc and if an interaction between sex and treatment was revealed, a 1-way ANOVA for each sex compared treatment groups to controls. All statistical analyses were performed with SPSS 15.0 statistical software and p<0.05 was always required for significance.

IV. Correlation

Average % of same sex control burrowing and average % of same sex same day control ALP values for those animals used in both analyses were diagrammed as a scatter plot and regression analysis used to find the best possible curve with correlation expressed using Pearson’s correlation coefficient (r).

Results

Of the original 120 animals tested, 44 were excluded from the burrowing analysis because they did not burrow at all and 2 were excluded because researchers disagreed on their sex. Overall approximately half of the males and one third of the females did not burrow (Table 1). The distribution of excluded animals among the 5 treatment groups did not appear to follow any pattern although this was not specifically tested for.

Table 1.

Summary of animals used in each of the three experimental repetitions.

MALES FEMALES
Non-Burrowers Burrowers Non-Burrowers Burrowers
2004 Control 0 4 0 4
Estradiol 0 1 0 7
Low ATR 0 1 1 6
Mid ATR 1 2 1 4
High ATR 0 3 1 4
2005 Control 3 1 2 2
Estradiol 0 1 2 5
Low ATR 6 0 1 1
Mid ATR 2 1 4 1
High ATR 3 2 2 1
2006 Control 2 1 2 3
Estradiol 2* 2 3* 0
Low ATR 2* 1 0* 4
Mid ATR 0 3 4 1
High ATR 2 1 0 5
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*

animal from this group not included in analyses because researchers disagreed on sex.

I. Sexually dimorphic burrowing

The repeated measures t-test with the Greenhouse-Geisser correction compared burrowing in the 14 control animals and returned a significant overall sex effect in which females burrowed 30% less than males (Figure 1). There was no time by sex interaction on fraction burrowed, although females did appear to burrow less in the first hr than at the other time points (Figure 1 insert). A significant overall time effect indicated that all animals were significantly burrowed at all times compared to time 0 and no other time differences.

Fig. 1.

Fig. 1

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Average fraction burrowed for male and female controls. Each bar represents the average + SEM of data collected once per hr for 6 hrs; these numbers are shown above the error bars. Female burrowing is significantly less than male, p=0.05. Insert. Average fraction burrowed at each hour of the 6-hrtrial, + SD in controls. Burrowing was significantly greater at each time point in each sex compared to burrowing at time 0, p<0.05.

II. Atrazine effect on burrowing

For analysis of control versus treated groups, the 2-way RM-ANOVA with the Greenhouse-Geisser correction revealed a significant main effect of treatment on burrowing and the Dunnett post-hoc indicated that the mid-ATR treatment group of 15 μg /L and the estradiol-treatment group both burrowed significantly less than same-sex controls, 28 and 30% less, respectively (Figure 2). The low and high ATR groups appeared to burrow approximately 20% less than controls, but these differences did not reach significance. The same ANOVA revealed a significant main effect of time, with burrowing at all times significantly different from time 0 and no differences in burrowing among times 1 through 6. Figure 2, insert illustrates the burrowing by time relationship for the mid-ATR group for each of the three years that the experiment was repeated. There were significant interactions between time and treatment and among time, sex and treatment, but post-hocs demonstrated no marked differences between any treatment group and control nor between the sexes at any specific time nor between the sexes in any of the treatment groups (data not shown).

Fig. 2.

Fig. 2

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% same-sex control burrowing - SEM in treated E. complanata. The mid ATR- (15 μg /ml) and estradiol-treated groups burrowed 28 and 30% less than their same sex controls, * indicates significantly different from control burrowing when 2 sexes are combined, p<0.05. Insert: % same-sex control burrowing at each hr of the 6-hr trial in each year of the three repetitions, - SD, in the mid ATR-treated groups. Combining the 3 repetitions reveals a significant main effect of time, p<0.05, with all times different from time 0.

III. Vitellogenin analysis

Of the original 40 animals prepared for VTG analysis, 5 were not tested because the tissue sample was too small and one was not included in the analysis because the difference between duplicate assays was more than 100%. The Student’s t-test for independent samples compared ALP/g of gonadal tissue in control males and females on each of the 2 days of testing and a significant sex difference was seen on both days indicating that females had at least twice as much VTG as males (Figure 3 insert).

Fig. 3.

Fig. 3

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% same-sex control VTG + SEM in treated E. complanata. The low ATR- (1.5 μg /ml) treated males had approximately 180 fold more VTG than same-sex controls, p<0.05. Estradiol and other ATR-treated males appear to have higher than control VTG, but none reach significance. Insert: The absolute values of alkali-labile phosphate (ALP) in controls in μg/g gonad on the two independent days of testing, indicating that females had more than twice as much ALP as males, p<0.05.

In treated groups, the two-way ANOVA combined data from both day’s trials – expressed as % of same sex, same day control VTG, and showed a significant interaction between sex and treatment and main effects of each. The 1 way ANOVA post-hoc for males demonstrated a significant treatment effect indicating that animals exposed to 1.5 μg /L ATR displayed 180 fold higher VTG than their same sex controls, the same test applied to females showed no marked differences among treatment groups (Figure 3).

IV. Correlation between burrowing and VTG

Of the 13 animals that were both burrowers and tested for ALP, the best-fit curve applied to the scatter plot demonstrated a very weak binomial relationship, r2=0.13 (Figure 4). For animals that burrowed less than control, a high ALP concentration appeared associated with decreased burrowing.

Fig. 4.

Fig. 4

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% same sex control burrowing plotted against % same sex ALP concentration in treated animals. The best fit curve shows a weak binomial relationship, R2 = 0.13. Each point represents one animal of which 3 were males and 9 females.

Discussion

These data suggest that in the freshwater mussel Elliptio complanata, depth of burrowing is sexually dimorphic with females burrowing less deeply, and that burrowing depth is decreased, and the sexual dimorphism augmented, following treatment with both estradiol and ATR. The lack of burrowing in 37% of animals seen here appears to follow a natural pattern; 62% of 157 E. complanata did not move at all during an 18-day early spring field study (Amyot and Downing, 1997). In some mussel species, burrowing has been linked to daylight and/or water temperature, with animals more likely to remain on the surface during the early summer spawning season (Watters et al., 2001). In other species, current velocity was reported as the dominant factor driving vertical movement (Schwalb and Pusch, 2007). Our conditions of 22 °C and 16 hr light with an absence of current may thus have contributed to a large number of non-burrowing animals.

Burrowing is a common behavior in bivalve species and was reported in E. complanata in the wild (Amyot and Downing, 1991, 1997; Balfour and Smock, 1995). To our knowledge, this is the first report of sexual dimorphism in depth of bivalve burrowing. In untreated animals, females on average burrowed 30% less than males (Figure 1). It appears from Figure 1 insert that the velocity of burrowing was less in females, as the largest difference between the sexes was after the first hr, 23%, and then fell to 5–7% and remained there through the end of the experiment. However, perhaps due to the relatively small number of animals, these time-based differences did not reach significance. Sexually dimorphic movement behavior is well known in other animals, from mammals (Smale et al., 1997), to insects (Martin, 2004), thus it is not terribly surprising to find the same in bivalves. Sexual dimorphisms in reproductive behavior were reported for other mollusks (Jordaens et al., 2009) and there are recent reports of sexually dimorphic shell shapes in freshwater mussels, including other Unionids (Widarto, 2009; Zieritz and Aldridge, 2011). Thus, this study of gender-specific movement behavior in E. complanata is also an extension of previous knowledge on bivalve sexual dimorphisms. With confirmation from other labs and from field studies, this investigation contributes to placing bivalves with other animals exhibiting sexually dimorphic non-reproductive behaviors, and thus may provide an alternate model system in which to study toxicant effects on sexual dimorphisms.

Both estradiol and ATR reduced burrowing depth in E. complanata. At the first measurement, 1 hr after treatment, ATR-exposed mussels burrowed an average of 31% less than controls (Figure 2 insert), and this pattern was the same each of the three times the experiment was repeated. In oysters (Crassostrea gigas), a similarly quick response in valve closure behavior was shown following exposure to toxic algae (Tran et al., 2010) and a 30 min exposure to the pesticide deltamethrin changed syphon opening behavior (Kontreczky et al., 1997), thus the rapidity of this response is within the range of other bivalve reports. Burrowing per time in the mid-ATR-treated group (Figure 2 insert) was similar to control burrowing per time in that the largest difference between treated and control groups was in the first hr in each of the 3 repetitions, between 28 and 50% less than control, with an apparent recovery by hr 6, when burrowing depth was between 9 and 31% of control. However, these time-based differences, again perhaps due to the relatively small number of animals, did not reach significance. While only the mid-ATR treatment group (15 μg /L) burrowed significantly less than controls (Figure 2), burrowing in the low- and high-ATR groups did not differ significantly from either mid or control; thus it is unclear if all ATR concentrations exerted the same inhibitory effect or if, at low and high concentrations, burrowing remained at control values. In either case, the burrowing response to ATR did not follow a classic linear dose-response curve in which rising exposure induces increasing effects and as such is similar to other non-linear dose-response curves seen following exposure to xenoestrogens (Myers et al., 2009). Additional ATR and burrowing studies, using more animals and intermediate concentrations, are necessary to determine the shape of this dose-response curve. In other aquatic testing paradigms and at concentrations similar to those used here, ATR exposure also resulted in non-linear dose-responses. These include reduced survival in the streamside salamander where the lowest exposure, 4 μg/L, produced the most morbidity (Storrs and Kiesecker, 2004), nuclear abnormalities in the freshwater fish Oreochromis niloticus where ATR concentrations between 6.25 and 25 μg/L all resulted in the same levels of damage (de Campos Ventura et al., 2008) and decreased spatial aggregation in E. complanata at 15 but not at 1.5 nor 150 μg /L ATR (Flynn and Spellman, 2009). These results contribute to and extend earlier reports by showing a non-linear burrowing response to ecologically-relevant concentrations of ATR in the freshwater mussel E. complanata.

The ATR effect on burrowing was not sex-specific; yet the sexual dimorphism in burrowing depth seen in controls appeared augmented in ATR-treated animals. Female controls burrowed 30% less than males and ATR-treated females burrowed 54% less than males. This subtlety, a change in sexually dimorphic behavioral differences, was found in mice exposed to low doses of the xenoestrogens bisphenol-A and methoxychlor (Gioiosa et al., 2007) and these authors postulated that such changes (while not a traditional part of toxicological studies) may reflect a loss of adaptive differences which resulted from sexual selection, and thus may be important for survival. Further examination of effects of ATR exposure on sex differences in bivalve burrowing behavior, with particular attention to potential changes in sexual dimorphisms, are needed to elucidate the importance of this observation.

Despite ongoing discussion regarding the mechanism of action (Fan et al., 2007; Hayes et al., 2006), it is generally agreed that ATR exerts estrogenic effects (Holloway et al., 2008). Stimulation of VTG is a classic estrogenic endpoint as this protein is a precursor of egg yolk and normally found only in oviparous females. VTG production by males is a bioassay for estrogenic contamination in many aquatic species including other bivalves (Ciocan et al., 2010; Quinn et al., 2004). The ALP assay used here has been routinely used in E. complanata (Gagné et al., 2011; Bouchard et al., 2009; Gagné and Blaise, 2003) and was validated for this system by showing the expected gender difference in control animals: females had almost 10-fold higher VTG than males.

Atrazine-exposed males had up to 180-fold higher VTG than their same sex controls, yet only the low ATR-treatment group reached significance; this was perhaps due to the low number of animals - particularly only one male in the estradiol-treated group (Figure 3). The mid-, high- and estradiol-treatment groups were not different from controls nor from the low ATR group, making it unclear if all treatments produced the same VTG response or if only the low ATR-group displayed increased VTG. Indeed, there is no agreement regarding estradiol-induced VTG in bivalves, and data on sex-specificity of the VTG response to xenoestrogens is contradictory. In zebra mussels, Dreissena polymorpha, the amount of VTG-like protein was reported to be the same in controls of both sexes (Quinn et al., 2004). A 30-day exposure to 2 mM estradiol (over 500 mg/L) did not induce VTG in male nor female E. complanata (Won et al., 2005) while a 10-day exposure to 50 ng/L but not 200 ng/L elevated VTG levels in both sexes of M. edulis (Ciocan et al., 2010). In xenoestrogen-contaminated waters, male M. galloprovincialis, displayed elevated VTG while females did not (Pampanin et al., 2005) yet in D. polymorpha females showed a 40% higher rise in VTG than males (Quinn et al., 2004), and Gagné et al. (2001) reported increased VTG in both sexes of E. complanata. In addition, recent reports of estrogenic activity of commercial fish food (Beresford et al., 2011) suggest that feeding regimens may contribute to increases in VTG seen in lab settings; yet the low VTG values in control males in our study indicate a minimal effect from feeding. Thus, the short exposure of 6 hr and/or the 100 μg/L estradiol used here may not have been appropriate for inducing VTG in Elliptio complanata. Overall, the relationship between estradiol and VTG in bivalves may be time, concentration, gender, diet and/or species specific, and the ATR-VTG relationship may demonstrate similar intricacies. While more data are needed for this picture to become clear, our study shows an ATR-induced increase in VTG in male E. complanata.

The VTG data together with the burrowing findings suggest either no relationship or a weak binomial relationship between VTG and burrowing. The best fit curve for the scatterplot shows a weak negative correlation when burrowing is inhibited and a weaker positive correlation when burrowing is enhanced (Figure. 4). Such a relationship suggests that the more inhibited the burrowing, the more VTG the animal possesses i.e., a more feminized behavior goes with a more feminized physiological state. Since the elevated VTG and decreased burrowing are in response to the xenoestrogen ATR, this correlation suggests that decreased burrowing following ATR exposure may be an estrogenic response. A cell surface estrogen receptor might account for the rapidity of both the VTG and burrowing response, as in the mussel Mytilus edulis where estrogen inhibited nitric oxide release via such a mechanism (Stefano et al., 2003). The notion that burrowing is estrogen-modulated is also supported by the lesser amount of burrowing seen in control females. This hypothesis needs the support of many more data points and a stronger correlation than that shown here. The other possible hypothesis from our data is that there is no relationship at all between VTG and burrowing. In that case, one may postulate that estrogens are not inhibitors of burrowing in E. complanata and that females burrow less than males for a reason unrelated to sex steroids, such as e.g., a sexual dimorphism in their shell shape (as shown for other mollusks (Widarto, 2009; Zieritz and Aldridge, 2011)) or a lower overall level of locomotor activity (as shown for other aquatic animals (Herrel et al., 2012), and that the ATR-induced decrease in burrowing is not an estrogenic effect. Further supporting evidence for the hypothesis of no relationship between VTG, estrogen and burrowing, is that the best fit curve suggests that once control values are reached, animals burrowing more than control also possess more VTG.

The common and acceptable levels of ATR used here impaired burrowing, a behavior which has been linked to predator avoidance and survival in other mollusk species (Rochette et al., 1999, Saarinen and Taskinen, 2003). Bivalve burrowing is also important for bioturbation, the reworking of soils and/or sediments which increase sediment turnover, nutrient availability and water oxygenation thus contributing to the stability and function of the entire aquatic ecosystem (Vaughn and Hakenkamp, 2001). The impact of ATR on the bioturbation efficiency of bivalves may be an overlooked toxicological effect of this pesticide, and as such remains an important open question for future investigations. Whether or not the ATR-induced reduction in burrowing is an estrogenic response, decreased mussel burrowing in waters contaminated with low environmentally-relevant concentrations of ATR may exert a greater effect on freshwater ecosystems than is currently understood.

Acknowledgments

A special thank you to Peter R. Warny of WildMetro Inc. in New York for showing us where mussels live and teaching us to collect them and to Matt Sherman and Christopher Powers for maintaining the mussels in the laboratory.

Role of the Funding Source

This study was supported by an Adelphi University Faculty Research Grant to KF. The funders had no role in the details of the study design; data collection analysis and interpretation; writing of the report; nor decision to publish.

Footnotes

Disclosure Statement

None of the authors of this manuscript have or have had any financial, personal, or other relationships with any people or organizations that could inappropriately influence this work.

Contributor Information

Katherine Flynn, Email: katherine.flynn@safeconsortium.org.

Maria Belopolsky Wedin, Email: maria.belopolsky@gmail.com.

Josephine A. Bonventre, Email: josephine.bonventre@oregonstate.edu.

Jessica Hines, Email: jrae.hines@gmail.com.

Benjamin S. Weeks, Email: weeks@adelphi.edu.

Chantale André, Email: chantale.andre@ec.gc.ca.

Martin P. Schreibman, Email: martins@brooklyn.cuny.edu.

Francois Gagné, Email: francois.gagne@ec.gc.ca.

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