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Bioaccumulation of atrazine: effects on population growth and reproduction of the rotifer Brachionus caly ciflorus fed atrazine exposed microalgae Dorian J. Feistel, Arthur B. Rudolph, Colette St. Mary Key Words : atrazine, bioaccumulation, ecotoxicology, rotifers, Brachionus calyciflorus
Abstract Little information is known about bioaccumulation of atrazine or the effects it has on consumers that ingest atrazine contaminated prey. For that reason, freshwater microalgae Chlorella vulgaris cultures were exposed to different concentrations of atrazine for twenty four hours and were then used as an exclusiv e food source for Brachionus calyciflorus for three days with the intent to evaluate the effects on rotifer population growth, reproduction, and survival. Considering a multivariate response, there was a deleterious effect of feeding on atrazine exposed algae. Specifically, t he rate of population growth, total ovigerous females and egg ratio (i.e. the total num ber of parthenogenic eggs divided by the total number of females in a population) in the rotifer population s significantly decreased as a trazine exposure concentrations of C. vulgaris increased The rate of mixis in rotifer population s fed atrazine exposed microalgal cells also decreased as herbicide concentration increased. The results of this study suggest that herbicides absorbed by microalgal cells can adversely affect higher trophic level organisms.
1. Introduction Increased use of herbicides in agribusinesses ha s raised a general concern about how they are affecting aquatic environment s Herbicides pollute rivers, streams, and lakes either indirectly by leaching into groundwater or directly through runoff (Graymore et al., 2 001; He et al., 2012). Originally introduced in the 1950's, atrazine a class of tri a zine herbicide is now one of the most heavily employed herbicides on the planet due to its relatively inexpensive cost (Graymore et al., 2001). Atrazine works by inhibiting the electron transport chain of photosystem II in plants, resulting in a disruption of photosynthesis (Corbett et al., 1984). It's primarily been used for pre and post emergence broadleaf weed control in both agricultural and nonagricultural areas (Girling et al., 2000). In 2012, t he USDA reported that 23 million kg s (~50.7 million lbs.) of atrazine was employed in corn production alone. In Florida, roughly 566,900 kg s (1.25 million lbs.) of atrazine were used between citrus, sugarcane, and vegetable crops, and other agricultural commodities such as field crops, o rnamental plants, and turf grass from 1999 to 2002 (Schuler & Rand, 2008). As a result of its high use, atrazine is one of the most frequently detected herbicide s i n contaminated ground soils and aquatic ecosystems (Vecchia et al., 2009; Chalifor & Juneau, 2011) Ecosystems polluted by atrazine have been shown to impact non target species such as copepods ( Forget Leray et al. 2005 ) fish ( Bringolf et al., 2002; Tillitt et al., 2010 ) and amphibians ( Hayes et al., 2002; Hayes et al. 2003; Hayes et al. 2010) Studies have found evidence that an accumulation of atrazine in aquatic systems generate s harmful effects on growth
and reproduction (Dornelles & Oliveira, 2014) and induce s alterations in behavior (McCallum et al., 2013) of non target species inhabiting contaminated areas. Hayes et al (2003 ) showed that exposure to atrazine polluted waters with a s little a 0.1 ppb caused gonadal dysgenesis (retarded gonadal development) and testicular oogenesis ( hermaphroditism ) in American Leopard Frogs, Rana pipien s These findings held not only for laborator y experiments but also for animals collected from atrazine polluted waters around the US The literature on atrazine exposure has extensively demonstrated the effects that direct exposure has on non target species However, little information is known about bioaccumulation of atrazine or the effects it has on consumers that ingest atrazine contaminated prey Zooplankton are among the most susceptible organisms to alterations within aquatic eco system s (Chang et al., 2005 ). Since zooplankton are a basal part of aquatic food webs, changes in their population size reproduction, b ehavior or physiology can potentially lead to significant ecosystem level impacts (Dodson and Hanazato, 1995 ) for instance via effects on higher trophic level taxa that are directly or indirectly associated with these basal trophic species To investigat e the toxicological effects of ingesting atrazine contaminated prey on consumers, I have chosen to study t he m onogonont rotifer Brachionus calyciflorus I ts short generation time, complex rep roductive biology ubiquity in aquatic systems, and ease to maintain in laboratory setting s (Janssen et al., 1993) make B. calyciflorus a prime candidate for this study. The primary goal of this experiment al study is to gain insight into the effects of ingestion of atrazine, specifi cally via atrazine exposed prey. This investigation will use Brachionus calyciflorus as the primary consumer and the microalgae, Chlorella vulgaris a commonly used species in toxicity tes ting as the primary food source. C. vulgaris cultures were exposed to different concentration s of atrazine and were then use d as an exclusive food source for B.
calyciflorus We then evaluate d the consequence s of t hese exposed algae on rotifer population growth, reproduction and survival. 2. Methods 2.1. Study Species The heterogonic life cycle of B. calyciflorus (Figure 1) includes many generations of amictic (i.e. asexual) reproduction before mictic (i.e. sexual) reproduction takes place (Birky & Gilbert, 1971). Amictic females will mitotically reproduce diploid eg gs that develop into genetically identical amictic females by means of parthenogenesis. If environmental conditions become unfavorable, amictic reproduction is disrupted and triggers amictic females to reproduce diploid eggs that mature into mictic female s. Further amictic females excrete a chemical into their surrounding environment when they reach a high population density that induces mictic reproduction (Fussmann et al., 2007). Once mictic reproduction is triggered, mictic females will me i otically produce haploid eggs that develop into males capable of fertilizing mictic females. A t a bout one fourth the size of female rotifers, males do not possess a gut, are short lived, are fast swimmers Males only have roughly 30 sperm, of which only 2 to 3 are transferred during each copulation event ( Wallace & Snell, 2001 ). After a male successfully inseminates a mictic female, the female will produce ovoid, thick shelled embryos in diapause (called resting eggs or cys ts), which can lie dormant for extended periods of time and endure harsh environmental conditions. Once environmental conditions become favorable (e.g. the optimal conditions in light, temperature, pH, salinity and oxygen; Wallace & Snell, 2001 ) a resting egg hatches and releases a diploid ami ctic female, beginning the asexual life cycle anew.
2.2. Chlorella vulgar is twenty four hour exposu re The microalgal species, Chlorella vulgaris was obtained from Carolina Biological Supply Company (Burlington, North Carolina). For the experiment and routine cultures, C. vulgaris was cultured in 250 mL Erlenmeyer flask s containing 100 mL of Bristol medium ( Bold, 1949 ) Microalgae cultures were stored in a thermoregulated chamber at 25¡C 1¡C on top of a shaker table (100 10 rpm) and illuminated under cool white florescent lamps on a 16h:8h light:dark photoperiod The microalgae cultures were maintained u sing semi continuous condition s (i.e. preexisting algal cultures were partially harvested and topped with fresh media to the original volume, and harvested cells were added to a sterilized flask containing fresh media) with the intention to keep cells in log arithmic phas e a period during cell growth when essential nutrients are abundan t and growth is not limited driving cell populations to divide and proliferate exponentially. Algal cell culture densities were determined spectrophotometrically at a wavelength of 685nm ([OD 685 i.e. optical density at 685nm ]; Qian, 2008 ) The regression equation between the density of a cell culture (y[cells/ mL]) and OD 685 (x) was determined to be : ! ! !"#"$ !" ! ! !"!#$ !" ( R 2 = 99. 23 %) Chlorella vulgaris cells were exposed to atr azine concentrations at 2, 5 and 10 mg/ L for 24h prior to starting the experiment. ! ! ! !" cells/mL of C. vulgaris were harvested via pipette and added in to individual screw cap tubes and centrifuged at 3300 rpm for 1h r. The supernatant was decanted and 5 mL of atrazine contaminated EPA medium (96 mg NaHCO 3 60 mg CaSO4! 2H2O, 60 mg MgSO4! 7H2O, and 4 mg KCl per liter of distilled water) of 2, 5, or 10 mg/L was introduced into randomly selected tubes containing centrifuged algae (see atrazine solution description below) Tubes were sealed to prevent external co ntamination agitated and
resuspended algae, and stored in the same manner as describe above for the maintenance of algal cultures. Cell cultures not exposed to atrazine were used as a control C ultures expose d to acetone were carried out as a complementary control and showed no difference to the control without acetone The maximal amount of acetone in a n y treatment did not exceed 1.0% (v/v) which has previously been shown to not significantly affect results when comparing to the no acetone control (Tang et al, 1997; DeLorenzo et al., 2007; Qian et al., 2008, Snell and DesRosiers noac 2008; Lu et al., 2012) Prior to feeding rotifers exposed C. vulgaris cultures (atrazine and acetone only ) were washed with EPA media to remove any un absorbed atrazine and centrifuged for 0.5hr The amount of atrazine absorbed by exposed C. vulgaris cultures was not measured. However, published evidence suggests that C. vulgaris cells exposed to trizine herbicides for 24h r removed > 90% of the surrounding toxicant from the media, and that viability of cells remained around 100% f or concentrations as high as 5 00n M (Rioboo et al., 2007) which is approximately 0.120 mg/L. 2.3. Brachionus calyciflorus three day test The rotifer species Brachionus calyciflorus Pallus w as obtained as resting eggs from Pentair Aquatic Eco Systems (Apopka, Florida) R esting eggs were hatched by hydrating eggs in a petri dish containing 10 mL of EPA medium adjusted to a pH of 7.5 0.02 Hydrating resting eggs were stored in a thermoregulated chamber at 25¡C 1¡C under continuous light of cool white florescent lamps 24 hr prior to the experimen t All the experiments were conducted by introducing 4 neonates in to sterilized 8 mL screw top glass chambers containing 2 mL of EPA medium with ! ! !" ! cells/mL of non exposed, acetone exposed, or atrazine exposed (2, 5 or 10 mg/mL) microalgal cultures with four replicates
of each treatment Rotifers were cultured under static nonrenewal conditions and stored in a thermoregulated chamber at 25¡C 1¡C on top of an shaker table (100 10 rpm) and illuminated under cool white florescent lamps on a 16h:8h light:dark photoperiod for 3 d Algae deposited at the bottom of a glass chamber was res u spended once every 12 hr us ing a micropipette. After 3 days the number of living and dead rotifers were counted per chamber and classified by the morphology and type of their eggs, which are different for females, males, and resting eggs (Wallace and Snell 2001 ). Rotifers were categorized as amictic female (AF), unfertilized mictic female (UMF), fertilized mictic female (FMF ), no novigerous female (NO F ), or male (M) From these counts, the intrinsic rate of population increase (r) was calculated for each glass chamber using the following equation : ! !" ! !" ! ! where N t and N 0 are the final and initial population densities respectively and t is the time in days. The ratio of ovigerous female s to nonovigerous (OF/NOF) females was calculated for each popula tion The sex ratio (M/F) was determined as the total number males divided by the total number of females within a population The mictic rate (MR) was calculated by the number of fertilized and unfertilized females divided by the total number of females in each glass chamber. The egg ratio (ER) was calculated as the total number of parthenogenic eggs divided by the total number of females in a population 2. 4. Atrazine Exposure Atrazine ( 1 Chloro 3 ethylamino 5 isopropylamino 2,4,6 triazine ; purity > 98% ) was obtained from Chem. Service, Inc (West Chester, PA). Atrazine stock solutions (1mg/mL) were
prepared by dissolving atrazine in acetone (99.5% purity). Test solutions were diluted to the appropriate concentrations by adding the atrazine stock solution to 100 mL EPA media prior to exposing algal cul tures. Previous experimental results suggest that B. calyciflorus has an LD 50 value of 39.2 mg/L of atrazine (Lu et al., 2012). Based on this result, three nominal atrazine concentrations (2, 5, and 10 mg/L) were chosen as treatments for the experiment. 2.5. Data analysis A multi variate an alysis of variance ( M ANOVA) with the concentration as the explanatory value and the intrinsic rate of population increase (r) OF/NOF, M/F and MR as the response variables was conducted to assess the effect of the test compound on asexual and sexual reproduction. 3. Results During the 3d exposure period some of the treatment replicates (three acetone and one 2mg/L) were found with a profusion of unidentified organism s that contaminated the algae These replicates were not used in the statistic al analysis. Overall, there was a significant effect of atrazine exposure on the response variables considered, (r), OF/NOF, MR, M/F, and EG (Wilk's Lambda: F= 9.815; df= 20, 24; P < 0.001). All of the response variables show a consistent response, with the exception of M/F (Figure 6 ). There were no significant differences among the exposure treatments observed for the M/F The experimental results for the control and treatments are listed in Table 1 and the specifics are discussed below.
The intrinsic rate of population increase (r) was adversely affected by the presence of atrazine in the microalgal cells used as food. Ro tifer cultures fed with atrazine contaminated C. vulgaris showed significant difference s among the control and the treatments ( ! !" !"# !" ! !! ! ! ! !!" ; Figure 2 ). The average (r) was lower for all treatments relative to the control. As the concentration increased from 2 to 5 mg/L and from 5 to 10 mg/L, there was steady decrease and increase, respectively, in the intrinsic rate of population growth af ter three days The 10 mg/L treatment averaged higher than the other treatments but was still lower than the control. Atrazine exposed microalgal cultures fed to rotifer cultures altered the ratio of ovigerous to nonovigerous females (OF/NOF) after three days. Rotifer cultures fed with atrazine exposed C. vulgaris cultures showed signif icant difference s among the control and the treatments ( ! !" !"" !" ! !! ! ! !!" Figure 3 ). Compared to the control, the average OF/NOF was lower for all concentrations. The OF/NOF decreased and increased between 2 to 5 mg/L and 5 to 10 mg/L, respectively C. vulgaris cultures contaminate d with atrazine and then fed to rotifer cultures for three days had an impact on the m ictic rate (MR) Cultures fed with atrazine exposed microalgal cells showed statistical differences among the control and the treatments ( ! ! !"# !" ! !! ! ! ! !"# Figure 4 ). The mean value was lower for all treatments when compared to the control The MR decreased between 2 and 5 mg/L and moderately increase d between 5 and 10 mg/L Finally, atrazine contaminated algae cells fed to rotifer cultures for three days affected the egg ratio (ER). Cultures fed with atrazine exposed C. vulgaris cells showed statistically significant differences when comparing the control to the treatments ( ! !"# !"# ! !"
! !! ! ! ! !!" ; Figure 5 ) Compared to the control, the ER decrease d between 2 and 5mg/L and increase d between 5 and 10mg/L. Rotifers fed with atrazine contaminated microalgal cells show no significant differences in sex ratio (M/F) after three days when comparing the control to the treatments ( ! ! !"# ! !" ! !! ! ! ! !"# Figure 6 ) The 5 mg/L of atraz ine had the highest average M/F ratio, while the control, 2 mg/L and 10 mg/L had decreased averages It should be noted that one of the 5 mg/L replicates has an abnormally low number of total females when compared to all replicates, suggesting an explanation for the elevated average sex ratio compared to the other treatments 4. Discussion The primary goal of this study was to determine the toxic effects that atrazine bioaccumulated in algae have on consumer levels of the food chain. Utilizing zooplankton to assess the effects of atrazine bioaccumulation is important for a number of reasons (Hanazato T, 2001). Zooplankton are found in nearly all aquatic systems and are highly susceptible to a wide variety of toxicants (Snell & Janssen, 1995; Marcial et al., 2005) Studies can be performed using large population densities in a small volume of media and data can be collected quickly. Our results suggest that C. vulgaris cells have a propensity to absorb atrazine via water (bioconcentration) in as little as twenty four hours and that atrazine exposed microalgae consumed by Brachionus calyci florus alters a variety of biological parameters. The effect a toxicant has depends on the duration of exposure and the concentration absorbed by the microalgae. Although the effects direct exposure to tox icants like atrazine have been studied for several aquatic species the current results suggest that the effects of herbicides ingested via food
by herbivores should be taken into account. The concentration of a toxicant can decrease the size of a population without changing the survivability of individuals within an affected population (Snell and Janssen, 1995). Toxicity studies for instance, have corroborated that a decrease in the rate of populat ion growth caused by toxicity exposure is usually due to a decrease in egg production ( Rioboo et al, 2007 ) In the current study, the intrinsic rate of population increase, OF/NOF and ER in B. calyciflorus fed with atrazine contaminated microalgae was significantly altered in all treatments when compared to the control. Even t he lowest concentration reduced the population growth rate and decreased the OF/NOF and ER in rotifers fed with at razine contaminated microalgae. Sexual reproduction is a necessary component in the life cycle of m onogonont rotifers, which is triggered by environmental stimuli such as crowding, perennial photoperiods, and diet (Gilbert, 1975 ). Amictic reproduction rapidly increases a population of rotifers to a considerable size; however it is the production of resting eggs via mictic reproduction that ensures the survival of rotifers from year to year (Snell & Serra, 2000 ) and promot es genetic variability via recombination A population of rotifers affected by ingesting herbicide contaminated microalgae will reduce the probability of resting egg produc tion This will consequently lower the number of resting eggs added to the egg bank and therefore decrease the chance for a population to recover after an environmental disturbance or even predictable season variation In the current study, rotifers fed atrazine bioaccumulated C. vulgaris cells showed a significant decreased in the MR when comparing the treatments and the control ; however, no significant difference s were observed for the M/F A drop in the rate of mixis (MR) leads to a decreased number of mictic females in a population (Lu et al., 2012), and therefore results in a decreased number of males capable of fertilizing mictic females. Brachionus sp males have a copulation
success rate between 10 and 75% (Snell and Hawkinson, 1983; Watson and Snell, 2001) A lower number of males with in a mictic population might negatively shift this percentage Moreover t his would reduce the overall rate of recombination, and thus decrease the frequency of novel genotypes within the population The current study demonstrates the importance of evaluating the bioaccumulation of atrazine and the effects it has on consumers that ingest atrazine contaminated prey Increasing the sample size and number of replicates per treatment, including more intermediate concentration levels, and extending the duration rotifers are exposed to atrazine polluted algae allowing female rotifers to developmentally mature could strengt hen future studies on this subject. Additionally, incorporating further response variables such as resting egg production and the hatchability of resting eggs would enhance bioaccumulation studies
Figure 1. The bisexual life cycle of the monogonont rotifer Brachionus calyciflorus ( taken from Birky & Gilbert, 1971 ) Read section 2.1 for information about the life cycle. Figure 2 The effects of atrazine exposed microalgae on the intrinsic rate of population growth (r) of B. calyciflorus after three d ays (mean SE; n=4 for C, 5, and 10mg/L; n=3 for 2mg/L). !"# !"$# !"%# !" !"'# ("# ("$# Atrazine Concentration (mg/L) Population growth (r) C 2 5 10
Figure 3 The effects of atrazine exposed microalgae on the OF/NOF of of B. calyciflorus after three days (mean SE; n=4 for C, 5, and 10mg/L; n=3 for 2mg/L). Figure 4 The effects of atrazine exposed microalgae on the mictic rate (MR) of B. calyciflorus after three days (mean SE; n=4 for C, 5, and 10mg/L; n=3 for 2mg/L). !"# !"$# !"%# !" !"'# ("# ("$# Atrazine Concentration (mg/L) OF/NOF C 2 5 10 !"# !"!$# !"!%# !"! !"!'# !"(# !"($# !"(%# !"( !"('# Atrazine Concentration (mg/L) Mictc rate (MR) C 2 5 10
Figure 5 The effects of atrazine exposed microalgae on the egg ratio (ER) of B. calyciflorus after three days (mean SE; n=4 for C, 5, and 10mg/L; n=3 for 2mg/L). Figure 6 The effects of atrazine exposed microalgae on the sex rat i o (M/F) of B. calyciflorus after three days (mean SE; n=4 for C, 5, and 10mg/L; n=3 for 2mg/L). !"# !"!)# !"(# !"()# !"$# !"$)# !"*# !"*)# !"%# Atrazine Concentration (mg/L) Egg Ratio (ER) C 2 5 10 !"# !"!)# !"(# !"()# !"$# !"$)# !"*# !"*)# !"%# Atrazine Concentration (mg/mL) Sex ratio (M/F) C 2 5 10
Table 1. Intrinsic rate of increase (r), ovigerous to non ovigerous females ratio (OF/NOF), mictic rate (MR), and sex ratio (M/F) of Brachionus calyciflorus fed with atrazine exposed microalgae to different concentrations calculated after the three day exposure pe riod. Treatments (r) OF/NOF MR ER M/F Control 0.9405 0.057 0.8241 0.179 0.1764 0.089 0.3322 0.017 0.1050 0.053 2 mg/L 0.7152 0.022 0.6328 0.076 0.1549 0.081 0.3123 0.021 0.0726 0.048 5 mg/L 0.6272 0.107 0.4584 0.027 0.2094 0.165 0.2853 0.019 0.0283 0.018 10 mg/L 0.7488 0.092 0.5571 0.093 0.0789 0.048 0.3057 0.036 0.0458 0.017 Data are given as mean values the standard errors of the mean
Literature Cites Birky, C. W. and J. J. Gilbert. 1971. PARTHENOGENESIS IN ROTIFERS CONTROL OF SEXUAL AND ASEXUAL REPRODUCTION. American Zoologist 11:245 &. Bringolf, R. B., J. B. Belden, and R. C. Summerfelt. 2004. Effects of atrazine on fathead minnow in a short term reproduction assay. Environmental Toxicology and Chemistry 23:1019 1025. Chalifour, A. and P. Juneau. 2011. Temperature dependent sensitivity of growth and photosynthesis of Scenedesmus obliquus, Navicula pelliculosa and two strains of Microcystis aeruginosa to the herbicide atrazine. Aquatic Toxicology 103:9 17. Chang, K. H., M. Sakamoto, and T Hanazato. 2005. Impact of pesticide application on zooplankton communities with different densities of invertebrate predators: An experimental analysis using small scale mesocosms. Aquatic Toxicology 72:373 382. Corbett, J. R. 1974. THE BIOCHEMICAL MODE OF ACTION OF PESTICIDES. The Biochemical Mode of Action of Pesticides:330. DeLorenzo, M. E., M. Leatherbury, J. A. Weiner, A. J. Lewitus, and M. H. Fulton. 2004. Physiological factors contributing to the species specific sensitivity of four estuarine micro algal species exposed to the herbicide atrazine. Aquatic Ecosystem Health & Management 7:137 146. Dodson, S. I. and T. Hanazato. 1995. COMMENTARY ON EFFECTS OF ANTHROPOGENIC AND NATURAL ORGANIC CHEMICALS ON DEVELOPMENT, SWIMMING BEHAVIOR, AND REPRODUCTION OF DAPHNIA, A KEY MEMBER OF AQUATIC ECOSYSTEMS. Environmental Health Perspectives 103:7 11. Dornelles, M. F. and G. T. Oliveira. 2014. Effect of Atrazine, Glyphosate and Quinclorac on Biochemical Parameters, Lipid Peroxidation and Survival in Bullfrog Tadp oles (Lithobates catesbeianus). Archives of Environmental Contamination and Toxicology 66:415 429. Forget Leray, J., I. Landriau, C. Minier, and F. Leboulenger. 2005. Impact of endocrine toxicants on survival, development, and reproduction of the estuarine copepod Eurytemora affinis (Poppe). Ecotoxicology and Environmental Safety 60:288 294. Fussmann, G. F., G. Kramer, and M. Labib. 2007. Incomplete induction of mixis in Brachionus calyciflorus: patterns of reproduction at the individual level. Hydrobiologi a 593:111 119.
Gilbert, J.J., Litton, J.R. 1975. Dietary trocopherol and sexual reproduction in the rotifers Brachionus calyciflorus and Asplanchna sieboldi Journalof Experimental Zoology 194:485 493 Girling, A. E., D. Pascoe, C. R. Janssen, A. Peither, A. Wenzel, H. Schafer, B. Neumeier, G. C. Mitchell, E. J. Taylor, S. J. Maund, J. P. Lay, I. Juttner, N. O. Crossland, R. R. Stephenson, and G. Personne. 2000. Development of methods for evaluating toxicity to freshwater ecosystems. Ecotoxicology and Envir onmental Safety 45:148 176. Graymore, M., F. Stagnitti, and G. Allinson. 2001. Impacts of atrazine in aquatic ecosystems. Environment International 26:483 495. Hanazato, T. 2001. Pesticide effects on freshwater zooplankton: an ecological perspective. Envir onmental Pollution 112:1 10. Hayes, T., K. Haston, M. Tsui, A. Hoang, C. Haeffele, and A. Vonk. 2003. Atrazine induced hermaphroditism at 0.1 ppb in American leopard frogs (Rana pipiens): Laboratory and field evidence. Environmental Health Perspectives 111 :568 575. Hayes, T. B., A. Collins, M. Lee, M. Mendoza, N. Noriega, A. A. Stuart, and A. Vonk. 2002. Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proceedings of the National Academy of Sc iences of the United States of America 99:5476 5480. Hayes, T. B., V. Khoury, A. Narayan, M. Nazir, A. Park, T. Brown, L. Adame, E. Chan, D. Buchholz, T. Stueve, and S. Gallipeau. 2010. Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis). Proceedings of the National Academy of Sciences of the United States of America 107:4612 4617. He, H. Z., J. Yu, G. K. Chen, W. Y. Li, J. B. He, and H. S. Li. 2012. Acute toxicity of butachlor and atrazine to freshwa ter green alga Scenedesmus obliquus and cladoceran Daphnia carinata. Ecotoxicology and Environmental Safety 80:91 96. Janssen, C. R., M. D. F. Rodrigo, and G. Persoone. 1993. ECOTOXICOLOGICAL STUDIES WITH THE FRESH WATER ROTIFER BRACHIONUS CALYCIFLORUS .1. CONCEPTUAL FRAMEWORK AND APPLICATIONS. Hydrobiologia 255:21 32. Lu, Z., B. Zhao, J. Yang, and T. W. Snell. 2012. Effects of atrazine and carbaryl on growth and reproduction of the rotifer Brachionus calyciflorus Pallas. Journal of Freshwater Ecology 27:52 7 537. Marcial, H. S., A. Hagiwara, and T. W. Snell. 2005. Effect of some pesticides on reproduction of rotifer Brachionus plicatilis Muller. Hydrobiologia 546:569 575.
McCallum, M. L., M. Matlock, J. Treas, B. Safi, W. Sanson, and J. L. McCallum. 2013. E ndocrine disruption of sexual selection by an estrogenic herbicide in the mealworm beetle (Tenebrio molitor). Ecotoxicology 22:1461 1466. Qian, H. F., G. D. Sheng, W. P. Liu, Y. C. Lu, Z. G. Liu, and Z. W. Fu. 2008. Inhibitory effects of atrazine on Chlore lla vulgaris as assessed by real time polymerase chain reaction. Environmental Toxicology and Chemistry 27:182 187. Rioboo, C., R. Prado, C. Herrero, and A. Cid. 2007. Population growth study of the rotifer Brachionus sp fed with triazine exposed microalga e. Aquatic Toxicology (Amsterdam) 83:247 253. Schuler, L. J. and G. M. Rand. 2008. Aquatic risk assessment of herbicides in freshwater ecosystems of south Florida. Archives of Environmental Contamination and Toxicology 54:571 583. Snell, T. W. and C. A. Hawkinson. 1983. BEHAVIORAL REPRODUCTIVE ISOLATION AMONG POPULATIONS OF THE ROTIFER BRACHIONUS PLICATILIS. Evolution 37:1294 1305. Snell, T. W. and C. R. Janssen. 1995. Rotifers in ecotoxicology: A review. Hydrobiologia 313 314:231 247. Snell, T. W. and M. Serra. 2000. Using probability of extinction to evaluate the ecological significance of toxicant effects. Environmental Toxicology and Chemistry 19:2357 2363. Tang, J. X., K. D. Hoagland, and B. D. Siegfried. 1997. Differential toxicity of atrazine to sel ected freshwater algae. Bulletin of Environmental Contamination and Toxicology 59:631 637. Tillitt, D. E., D. M. Papoulias, J. J. Whyte, and C. A. Richter. 2010. Atrazine reduces reproduction in fathead minnow (Pimephales promelas). Aquatic Toxicology 99:1 49 159. Vecchia, A. V., R. J. Gilliom, D. J. Sullivan, D. L. Lorenz, and J. D. Martin. 2009. Trends in Concentrations and Use of Agricultural Herbicides for Corn Belt Rivers, 1996 2006. Environmental Science & Technology 43:9096 9102. Wallace, R.L., Snell, T.W. 2001. Rotifera. In: Throp JH, Covish AP, editors. Ecology and systematics of North American freshwater invertebrates. Second edition. New Your: Academic Press. p. 187 248.