Effects of ultraviolet radiation and degraded water quality on growth and survival of Bufo marinus tadpoles Brenna Levine Department of Fish, Wildlife, and Conservation Biology, Colorado State University ABSTRACT Amphib ian species worldwide are curre ntly experiencing population declines and decreased ranges. This decrease in amphibian populations is due in part to habitat degradation such as canopy cover loss and decreased water quality. Other amphibian species, however, such as Bufo marinus , a re experiencing population growth and global expansion. In this study, I tested the effects of loss of canopy cover and degraded water quality on growth and survival of B. marinus tadpoles by exposing them to condition s of elevated levels of ultraviolet radi ation, degraded water quality , and a combination of the se two factors . I found that B. marinus tadpoles have high survivorship and low rates of mortality when exposed to ultraviolet radiation and degraded water quality separ ately. When these two environmental stresses are combined, however, B. marinus tadpoles experience low survivor ship and high rates of mortality . These results provide insight into why B. marinus is experiencing population expansions, population increases, and why it has become an invasive species in many countries while native anurans are going exti n ct. RESUMEN Alrededor del mundo las especies de anfibios est Ã¡n ex perimentando actualmente una disminuciÃ³n en las poblaciones y en el rango de distribuciÃ³n. Esta disminuciÃ³n es e n parte debido a la degradaciÃ³n del hÃ¡bitat como perdida de cobertura de dosel y disminuciÃ³n en la calidad del agua. Otras especies de anfibios, sin embargo, como Bufo marinus , estÃ¡n experimentando crecimiento en las poblaciones y expansiÃ³n global. En es te estudio, probÃ© el efecto que ejerce la pÃ©rdida de cobertura del dosel y la degradaciÃ³n de la calidad del agua en el crecimiento y sobrevivencia de renacuajos de B. marinus exponiÃ©ndolos a condiciones de elevada radiaciÃ³n ultravioleta, degradaciÃ³n de la calidad del agua y la combinaciÃ³n de estos factores. EncontrÃ© que los renacuajos de B. marinus tienen una mayor sobrevivencia y bajas tasas de mortalidad cuando estÃ¡n expuestos a radiaciÃ³n ultravioleta y agua de baja calidad separadamente. Cuando estos dos factores son mezclados, sin embargo, los renacuajos de B. marinus experimentan baja sobrevivencia y altas tasas de mortalidad. Estos resultados dan una idea de que las poblaciones de B. marinus estÃ¡n aumentando y el porque estÃ¡ especie se ha convertido una especie invasiva mientras otras especies nativas se estÃ¡n extinguiendo. INTRODUCTION Amphibian species worldwide are currently experiencing population declines Blaustein et al. 2003; Pusche ndorf et al. ; 2006 Nystrom et al. 2007 . Simultaneously , due to the rapidly growing human population and consequent land development, habitat alteration is increasing. Habitat altera tion include s such drastic effects as loss of canopy cover in riparian h abitats and degradation of water quality which can directly affect amphibian populations . Although habitat alteration a ffects adult amphibians, tadpoles and larvae are specifically at risk due to their dependency on water and consequential inability to l eave degraded areas. Loss of canopy cover may expose anuran tadpoles to increased levels of ultraviolet radiation and higher water temperatures. Thus, loss of canopy cover and increas ed exposure to ultraviolet radiation can cause both sub lethal and letha l effects in amphibians Crotaeu et al. 2008. For example, development and growth in Leptodactylus is related to temperature and exposure of sunlight to aquatic habitats, and Leptodactylus tadpoles experience decreased growth under exposed conditions free of canopy cover Ernst et al . 2007. Additionally, Rana cascadae larvae showed decreased growth and increased presence of physical abnormalities when exposed to hi gher levels of ultraviol et radiation Romansic et
al. 2009. Tadpoles are also susceptible to changes in water quality due to application of fertilizers and pesticides to nearby land . For example, h atching success of Rana pipiens is negatively correlated with the level of water contamination Ka rasov et al. 2007. Additionally, the combination of nitrite water contamination and elevated levels of ultraviolet light is seven times more lethal to Bufo bufo tadpoles than nitrite or ultraviolet light separately Macia et al. 2007. While many amphibian species are experiencing population declines due to loss of canopy cover and degraded water quality, others, such as the invasive Cane Toad Bufo marinus , have been rapidly colonizing areas where they were introduced by humans an d are currently experiencing population growth and increased distribution Phillips et al. 2003; Phillips et al . 2007. The purpose of this study was to determine the effects of loss of canopy cover and degradation of water quality on B. marinus tadpole g rowth and survival. If growth and survival of B. marinus tadpoles is higher under the adverse conditions of degraded water quality and loss of canopy cover, this result could shed light on why this species is so invasive. MATERIALS AND METHODS Study Organisms The organism that was tested in this experiment was the Bufo marinus tadpole . B. marinus is Costa RicaÂ€s largest amphibian and the largest lowland toad that occurs between Texas and Central Brazil Zug 1983 . This species is widespread in Costa Rica and is common to disturbed areas near human development Savage 2002. It is an invasive species that has been introduced to over 40 countries including Guam and Australia , where it has sin ce spread throughout the continent Christy et al. 2007 ; Phi llips et al. 2007 . Adults and especially larvae of B. marinus are dependent upon water pools for growth and survival as they are highly susceptible to desiccation Zug and Zug 1979. Tadpole Capture I collected 160 B . marinus tadpoles from a man made pond located approximately 160 meters south of the road leading to the EstaciÃ³n BiolÃ³gica de Monteverde and approximately 40 meters east of Alan MastersÂ€ house in Monteverde, Costa Rica . Tadpoles were collected using a hand held net with fine mesh. Upon capture, tadpoles were immediately transferred to a bucket filled with water from this pond. I only collected tadpoles that were of approximately the same size and developmental stage. Tadpoles were only collected if they had no back legs or partial back legs, and no sign of front leg development. I dentification of the tadpoles as B. marinus was made using a dichotomous key Savage 2002 . Tadpole Husbandry Tadpoles were housed in plastic Glad brand con tainers until trials began . They were fed 1 gram of fish food in their holding containers on the day of capture. The tadpoles were kept in 400 milliliters of tap water until trials commenced . During the course of the experimental trials, tadpoles were fed 1 gram of fish food two times per week. Experimental Set Up Each of four treatments were represented in ten plastic G lad containers, the treatments being clean water with no ultraviolet radiation , contaminated water with no ultraviolet radiation , cl ean water with ultraviolet radiation , and contamin ated water with ultraviolet radiation . These containers were then thoroughly rinsed with tap water to remove any dust particles. 400 milliliters of tap water was then used to fill each plastic container. All containers within the c lean water/no ultraviolet radiation treatment and the contami nated water/no ultraviolet radiation treatment were separated from the contain ers within the ultraviolet
radiation treatments via a large black plastic garbage bag that acted as a wall. The twen ty containers on each sid e of the wall were arranged in three vertical columns. To test the condition of decreased water quality on tadpole growth, a soap solution was created by mixing 25 milliliters of tap water with 0.5 gram s of Axion brand detergent. Ten drops of this solution were then added using an eye dropper to each container within the contaminated water treatments . To determine the soap concentration needed to contaminate the water, two tadpoles were placed in each of five containers filled wi th 400 mill iliters of tap water and varying numbers of drops of this solution were added to each container. The s oap concentrations tested were six drops, eight drops, ten drops, twelve drops, and fourteen drops of the solution . Ten drops of the soap sol ution was chosen as it was the highest concentration tested that did not kill the tadpoles. To test the condition of loss of canopy cover and conseque nt increase in ultraviolet radiation exposure, a lighting system consisting of a linear circuit of five u ltraviolet lights was constructed and h ung twenty centimeters above the center column of contain ers within the ultraviolet radiation treatments. The lighting syst em produced an ultraviolet radiation intensity of 3 compared to 0 without the lighting system . Pebbles were placed in one corner of each conta iner to allow tadpoles that metamorphosized during the experiment to be able to crawl partially out of the water. The experimental set up is d iagramed in Fig . 1. Fig . 1. Experimental design. Tadpoles were randomly assigned to one of the four following treatments: clean water with no ultraviolet radiation , contaminated water with no ultraviolet radiation , clean water with ultraviolet radiation , and contaminated water wi th ultraviolet radiation . Experi mental Trials Experimental trials were conducted in the lower lab of the EstaciÃ³n BiolÃ³gica de Monteverde in Monteverde, Costa Rica from April 23, 2009 to May 4, 2009. Tadpoles were randomly assigned to a treatment and a container wi thin a treatment so th at four tadpoles were assigned to each container. Each tadpole was then removed from its home container and weighed by blotting the tadpole off on a paper towel to remove water and then placing the tadpole on a scale. Tadpole weights were then
summed fo r each container as the identities of individual tadpoles were unknown. Tadpoles were placed in their assigned containers. After all of the tadpoles were placed in their respective containers, all containers were covered with mosquito nets to prevent inse cts that were attracted to the ultraviolet light from entering the water. The mosquito nets also kept tadpoles that metamorphosized during the experiment from escaping. At this time, the ultraviolet lighting system was turned on and trials began. Each d ay, tadpole mortality within each container was assessed . If mortalities occurred within a container, the death was recorded and the dead tadpole was immediately removed and disposed of. Every other day, surviving tadpoles were removed from their containe rs and weighed using the previously described method. Each day that the tadpoles were weighed, the water within each container was replaced with 400 mill iliters of new tap water, and ten drops of the soap solution were added to the containers within the c ontaminated water treatments. Statistical Analyses A Kruskal Wallis test was used to determine if there was a significant difference between the average numbers of tadpoles alive per container on the final day of measurements. A Kolmogorov Smirnov test w as used to determine if there was a significant differenc e between the mortality rates for each treatment. Tadpole weights were analyzed using a Kruskal Wallis test. The Kruskal Wallis test was used to determine if the total average weight loss of an individual tadpole differed between treatments. Weight loss was analyzed, rather than weight gain, because tadpoles lose mass as they grow and metamorphosize. RESULTS Tadpole Growth N o significant difference between the total average weight loss of tadpoles in the four treatments was detected K W: Â 2 = 3.4929; df = 3; p = 0.3217 ; Fig. 2 . The average total weight lost by an individual in each of the treatments was approximately equal regardless of the environmental conditions in which they were exposed to. Average weight lost by an individual in the four treatments ranged was 0.0696 + 0.0406 grams in the clean water/no ultraviolet radiation treatment , 0.0748 + 0.02 56 grams in the contaminated water/no ultraviolet radiation treatment, 0.0938 + 0.0240 grams in the clean water/ultraviolet radiation treatment, and 0.0968 + 0.0077 grams in the contaminated wa ter/ultraviolet r adiation treatment.
______________________ ______________________________________________________________ Fig. 2. Average + SE total change in weight g for individual B. marinus tadpoles within four treatments. Tadpoles were weighed every other day from April 23, 2009 to May 4, 2009 in Monteverde, Costa Rica. Tadpole Survivorship Average Tadpole Survivorship There was no significant difference between the total survivorsh ip of tadpoles within all of the treatments K W: Â 2 = 7.1752; df = 3; p = 0.0665 ; Fig. 3 , but a strong trend wa s visible. The average number of tadpoles that survived per container was much larger in the contami nated water/no ultraviolet radiation and the clean water/ultraviolet radiation treatment s. Clearly, even though a significant difference in survivorship be tween treatments was not detected, survivorship in the contami nated water/no ultraviolet radiation treatment was noticeably higher than in the other treatments . 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 Clean/ No UV Contaminated/ No UV Clean/ UV Contaminated/ UV Treatment Average Change in Weight g
Fig. 3. Average + SE B. marinus tadpole survivorship across four treatments. Mortality was assessed daily from April 23, 2009 to May 4, 2009 in Monteverde, Costa Rica. Rate of Tadpole Mortality There was a significantly higher rate of tadpole mortality in the clean water/no ultraviolet radiation treatment than in the contaminated water/no ultraviolet radiation treatment Dmax = 0.4412; n1 = 34; n2 = 20; 0.01 < p < 0.05 ; fig.4 . T he rate of mortality in the contaminated water/ultraviolet radiation treatment was much greater than the rate of mortality in the clean water/no ultraviol et radiation treatment Dmax = 0.44; n1 = 34; n2 = 34; p < 0.01 ; fig.4 The rate of mortality in the contaminated water/ultraviolet radiation treatment was higher than in the contaminated water/no ultraviolet radiation treatment. Dmax = 0.7029; n1 = 20; n2 = 34; p < 0.01 ; fig.4 . 0 0.5 1 1.5 2 2.5 3 Clean/ No UV Contaminated/ No UV Clean/ UV Contaminated/ UV Avg. # Alive Per Container
Fi g. 4. B. marinus tadpole survivorship across 4 tr eatments over a 12 day period. This experiment was conducted in the lower lab of the EstaciÃ³n BiolÃ³gica in Monteverde, Costa Rica. Exposure to ultraviolet represents loss of canopy cover and subsequent exposure of tadpoles to elevated UV levels. DISCUSSION There was no difference detected betw een the average tadpole weight losses within the four treatments b ecause neither ultraviolet radiation , degraded water quality, nor t he interaction of these two environmental factors slowed tadpole growth. Tadpoles within these treatments continued to devel op in the same way that the tad poles in the clean water/no ultraviolet radiation treatment developed. Previous studies have shown that degraded water quality, ultraviolet radiation , and their interaction can cause decreased growth in anurans. Rana cascadae larvae exhibited decreased growth when exposed to elevated levels of ultraviolet radiation Romansic et al . 2009. Additionally , Leptodactylus tadpoles experienced decreased growth when exposed to elevated levels of ultraviolet radiation Ernst et al. 2007. Because Bufo marinus tadpoles that were exposed to elev ated levels of ultraviolet radiation and degraded water quality expe rienced the same average growth as those tadpoles not exposed to these adverse conditions, I believe that B. marinus tadpoles have a higher tolerance for adverse environmental conditions than other anuran species. This higher tolerance to environmental stress could be a factor that allows B. marinus to be an invasive species. In the face of climate change and habitat destruction in which water quality in aquatic systems is often times degraded and canopy cover over aquatic systems is often times lost, B . marinus may be able to continue to rapidly colonize new systems and to extend its natural range by being less affected or unaffected by these conditions. Additionally, since the growth of tadpoles in other anuran species is negati vely affected by these environmental stresses , B. marinus may be able to outcompete native anurans for resources. Because there were significant differences between the rates of mortality of tadpoles within the four treatments, it is clear that ultraviolet radiation and degra ded water quality have an effect on the rate of tadpole mortality. Additionally, because there was almost a significant difference between the overall 0 5 10 15 20 25 30 35 40 45 4/23/2009 4/24/2009 4/25/2009 4/26/2009 4/27/2009 4/28/2009 4/29/2009 4/30/2009 5/1/2009 5/2/2009 5/3/2009 5/4/2009 Number of Tadpoles Clean/No UV Dirty/No UV Clean/UV Dirty/UV
final survivorship per container withi n a treatment, ultraviolet radiation and degraded water quality ma y have an effect on survivorship as well. The lowest rate of tadpole mortality and highest survivorship occurred in tadpoles within the contami nated water/no ultraviolet radiation treatment. Addition ally, the second lowest rate of tadpole mortality and t he second highest sur vivorship occurred in the clean water/ultraviolet radiation treatment. Interestingly, the highest rate of tadpole mortality occurred in the contaminated w ater/ultraviolet radiation treatment . It seems to me that individually, degrade d wat er quality and ultraviolet radiation have positive effects on tadpole survival. Tadpoles may fare better in degraded water quality because they prefer higher phosphorous levels. Additionally, ultraviolet radiation causes B. marinus tadpoles to have higher survival than when tadpoles of the same species are not exposed to ultraviolet radiation . Because the lowest survival and highest rate of mortality occurred in the cont aminated water/ultraviolet radiation treatment, it seems that the combination of two factors that produce positive effects when separate produce an extremely negative effect when combined. Other studies have been conducted on the interaction of t wo positive factors producing negative results. For example, in a study of Rana clamitans , it w as discovered that the chemical compounds carbaryl and nitr ate had positive effects on R. clamitans survival when they were present separately, yet negative effects when they were present together Boone et al. 2005. There may be severa l reasons wh y ultraviolet radiation and degraded water quality have positive effects on tadpole survival when separate and combine to produce a negative effect. First, the ultraviolet radiation may alter a chemical present in the soap solution that was added to contaminate the water. There may be a compound present within the soap solution that is harmless until altered by ultraviolet radiation. Additionally, ultraviolet radiation may alter the tadpolesÂ€ ability to cope w ith degraded water quality conditions. It may be that tadpoles are able to survive in degraded water quality conditions only as long as no other environmental stresses are present. In the same way, the degraded water quality may alter the tadpolesÂ€ abili ty to cope with elevated levels of ultraviolet radiation. Finally, it is interesting to note that overall average tadpole survival was equal between the clean water/no ultraviolet radiation treatment and the cont aminated water/ultraviolet radiation treatm ent. Overall, it seems th at the absence of either factor or the pre sence of both factors creates lethal conditions for B. marinus tadpoles. Although the rate of mortality was significantly higher in the cont aminated water/ultraviolet radiation treatment than in the clean water/no ultraviolet radiation treatment, the overall survivorship in both treatments was low. These results lead me to wonder, however, the frequenc y at which habitats are disrupted without both los ing canopy cover and creating degraded water quality. Often times, habitat loss and urban development produce both conditions. In order for B. marinus tadpoles to be so successful under the se adverse conditions, most habita t degradation must either only a ffect canopy cover or water quality, and not both. I would expect that in areas where water quality has been degraded and canopy cover has been lost, that B. marinus tadpoles would have low survivorship and high rates of mortality compared to areas where only one of these factors is present. Clearly, this research has created many more questions which require further research. ACKNOWLEDGEMENTS Thank you to my advisor Pablo Allen for helping me to formulate my ideas, for providing guidance throughout my project, and for much needed statistics help. Thank you to Yi men Araya as well for teaching me about excel and statistics. I would also like to thank Anjali Kumar for helping me to work through my original ideas. Thank you to Yi men Araya and Moncho CalderÃ³ n for buying me a ll of the equipment that was needed for m y project. Many thanks to Rolando MarÃn for building my lighting system without which my project would not have been possi ble. Thank you to Laura Heil for her constructive peer review of my paper. Finally, thank y ou to CIEE and the EstaciÃ³n BiolÃ³gica de Monteverde for providing me the equipment and lab space to conduct my experiment.
LITERATURE CITED Blaustein, A. R., J. M. Romansic, J. M. Kiesecker, and A. C. Hatch. 2003. Ultraviolet radiation, toxic chemic als, and amphibian population declines. Diversity & Distributions 9: 123 140. Boone, M. D., C. M. Bridges , J. F. Fairchild, and E. E. Little. 2005. Multiple sublethal chemicals negatively affect tadpoles of the green frog, Rana clamitans . Toxicology & Chemistry 24: 26. Christy, M. T., J. A. Savidge, and G. H. Rodda. 2007. Multiple pathways for invasion of anurans on a Pacific island. Diversity & Distributions 13:598 607. Croteau, M. C., M. A. Davidson, D. R. S. Lean, and V. L. Trudeau. 2008. Global inc reases in ultraviolet B radiation: potential impacts on amphibian development and metamorphosis. Biochemical Zoology 81: 743 761. Ernst, R., T. Konrad, K. E. Linsenmair, and M. O. Roedel. 2007. The impacts of selective logging on three sympatric speci es of Leptodactylus in a Central Guyana rainforest. Amphibia Reptilia 28: 51 64. Karasov, W. H., R. E. Jung, S. Vanden Langenberg, and T. L. E. Bergeson. 2005. Field exposure of frog embryos and tadpoles along a pollution gradient in the Fox River and Green Bay ecosystem in Wisconsin, USA. Environmental Toxicology and Chemistry 24: 942 953. Macias, G., A. Marco, and A. R. Blaustein. 2007. Combined exposure to ambient UVB radiation and nitrite negatively affects survival of amphibian early life stages . Science of the Total Environment 385: 55 65. Nystrom, P., J. Hansson, J. Mansson, M. Sundstedt, C. Reslow, and A. Brostrom. 2007. A documented amphibian decline over 40 years: Possible causes and implications for species recovery. Biological Conserva tion 138: 399 411. Phillips, B. L, G. P. Brown, M. Greenlees, J. K. Webb, and R. Shine. 2007. Rapid expansion of the cane t oad Bufo marinus invasion front in tropical Australia . Austral Ecology 32 : 169 176 . Phillips, B. L., G. P. Brown, and R. Shine. 20 03. Assessing the potential impact of cane toads on Australian snakes. Conservation Biology 17: 1738 1747. Puschendorf, R. F. Bolanos, and G. Chaves. 2006. The amphibian chytrid fungus along an altitudinal transect before the first reported declines in Costa Rica. Biological Conservation 132: 136 142. Romansic, J. M., A. A. Waggener, B. A. Bancroft, and A. R. Blaustein. 2009. Influence of ultraviolet B on growth, prevalence of deformities, and susceptibility to predation in Cascades frog Rana cascadae larvae. HidrobiologÃa 624: 219 233. Savage, J. M. 2002. The Amphibians and Reptiles of Costa Rica , p. 199 202. The University of Chicago Press, Chicago. Zug, G. R. 1983 Bufo marinus In D.H Janzen. Costa Rican Natural History, p. 386 387. Unive rsity of Chicago Press, Chicago. Zug, G. R., and P. B. Zug. 1979. The marine toad, Bufo marinus : a natural history resume of native populations. Smithsonian Contrib. Zoo. 284: 1 58.
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Efectos de las radiaciones ultravioletas y degradacin del agua en el crecimiento y la sobrevivencia de los renacuajos Bufo marinus
Effects of ultraviolet radiation and degraded water quality on growth and survival of Bufo marinus tadpoles
Amphibian species worldwide are currently experiencing population declines and decreased ranges. This decrease in amphibian populations is due in part to habitat degradation such as canopy cover loss and decreased water quality. Other amphibian species, however, such as Bufo marinus, are experiencing population growth and global expansion. In this study, I tested the effects of loss of canopy cover and degraded water quality on growth and survival of B. marinus tadpoles by exposing them to conditions of elevated levels of ultraviolet radiation, degraded water quality, and a combination of these two factors. I found that B. marinus tadpoles have high survivorship and low rates of mortality when exposed to ultraviolet radiation and degraded water quality separately. When these two environmental stresses are combined, however, B. marinus tadpoles experience low survivorship and high rates of mortality. These results provide insight into why B. marinus is experiencing population expansions, population increases, and why it has become an invasive species in many countries while native anurans are going extinct.
Alrededor del mundo las especies de anfibios estn actualmente experimentando un descenso en las poblaciones y en el rango de distribucin. Esta disminucin en las poblaciones de anfibios se debe en parte a la degradacin del hbitat, tales como la prdida de la cobertura del dosel y la disminucin en la calidad del agua. Otras especies de anfibios, sin embargo, como Bufo marinus, estn experimentando un crecimiento en las poblaciones y expandindose globalmente. En este estudio, yo analice los efectos que ejerce la perdida de cobertura del dosel y la degradacin de la calidad del agua en el crecimiento y sobrevivencia de renacuajos de B. marinus exponindolos a condiciones de elevada radiacin ultravioleta, degradacin de la calidad del agua y la combinacin de estos factores.
Text in English.
Tropical Ecology 2009
Ecologia Tropical Verano 2009
Degradacin del hbitat
t Monteverde Institute : Tropical Ecology