The effect of whey on the growth and survival of Isthmohyla pseudopuma tadpoles Patricia L. Stamper Department of Ecology and Evolutionary Biology, University of Arizona Tucson, AZ 85711, USA ABSTRACT Water contamination and poor water quality in general have escalated in recent years. Streams and rivers located near dairy farms are commonly polluted b y a byproduct of cheese making, whey Due to factors such as water degradation, amphibian species have been experiencing rapid population declines wo rldwide The purpose of this study wa s to determi ne the effects of whey waste on Isthmohyla pseudopuma tadpole growth and survival. It was found that whey waste generally has a negative effect on I pseudopuma tadpole survivorship and little effect on the growth. Additional observations such as metamorphosis were observed towards the end of the experiment further research should look into rat e of metamorphosis and develop ment of deformities (if any) due to the presenc e of whey waste. Water is one of the most highly valued and necessary resources for all life forms. Globally approximately 50% of the fresh and accessible run off water is used by humanity. Due to the high demand for the limited supply of fresh water, humanity has extensively altered river systems through diversion and impoundments. Often rivers such as the Colorado, the Nile, and the Ganges are used so extensively that little w ater ever makes it to the ocean (Vitousek et al. 1997) Water contamination and poor water quality in general have escalated in recent years. This contamination of water sources is very common in areas of human population. Streams and rivers located near dairy farms are commonly polluted by a byproduct of cheese making, whey. Whe y has a high organic strength and a high chemical oxygen demand (COD), and therefore often causes disposal problems. The production of approximately 25.3x 10 9 kg of liquid whey was estimated (Mauro et al 1980) 37% was utilized for feeding livestock, 28% to produce dried whey and 9% to recover lactose. The remainder of the liquid whey was disposed of without any treatment, which causes a tremendous pollution problem, since the BOD of raw whey is about 30,000 50,000ppm ( Mauro et al 1980). Water quality needs of wil dlife have often been neglected and this neglect is particularly true for amphibians (Mauro et al 1980). Due to factors such as water degradation, a mphibian species have been experiencing rapid population declines worldwide (Blaustein et al. 2 003; Puschendorf et al 2006). Additionally, habitat alteration is increasing due to the growing human population and consequent land development. Habitat alteration leaves tadpoles and larvae at high risk due to their dependency on water for survival and inability to leave degraded areas. Tadpoles are also influenced by degradation of water quality by means of fertilizers and pesticides. An example of this influence is Rana pipiens whose hatching success is negatively correlated with water contamination (K arasov et al. 2007).
Founded by dairy farming Quakers, the economy of Monteverde, Costa Rica is largely based on the production and sales of dairy products. Facilities like the Cheese Factory in Monteverde have propertie s located near bodies of water Degradation of water quality, such as whey waste in riparian habitats, is a habitat modification that directly affects amphibian populations (Karasov et al. 2007). The purpose of this study is to determine the effects of degradation of water quality by mea ns of whey waste on Isthmohyla pseudopuma tadpole growth and survival. Due to the fact that amphibians are considered reliable indicators of environmental quality, i f growth and survival of I pseudopuma tadpoles is lower under the adverse conditions of degraded water quality, this result could shed light on other species surviving under these unfavorable conditions (Boyer & Grue 1995) METHODS Study Organism The organism that was tested in this experiment was H. pseudopuma (Anura) tadpoles. I pseudopuma are among the commonest Costa Rican treefrogs. This species is found primarily in the humid lower montane zones (1,120 2,340m) of Costa Rica. During the day these frogs hide in vegetation and are commonly found in ground a nd arboreal tank bromeliads The species is an explosive pond breeder which uses both temporary and permanent sources of water. Eggs hatch within 24 hours and in captivity tadpoles metamorphosed in fifty seven to eight one da ys. Two factors are especially important in the survival of the development states, desiccation and food limitation. Under simulated drying, development is accelerated and size at metamorphosis is significantly decreased. In the wild, tadpoles feed on plan t material and detritus (Savage 2002). Tadpole Capture 200 Isthmohyla pseudopuma ta d poles were collected from a temporary pond located on the property of Wil low Zuchowski, which is on the perimeter of the Cloud Forest Reserve in Monteverde, Costa Rica Tadpoles were collected using a fine hand held net. Tadpoles of varying size s were collected. After being captured the tadpoles were transferred to Ziploc bags Identification of the tadpoles as I pseudopuma was don e with the h elp of the staff from the Museo de Zoologia de la Universidad de Costa Rica. Experimental Set Up 40 plastic Glad containers (6cm x 10cm x 6cm) containing 200 milliliters of tap water were m ixed with one of four concentrations of whey (0%, 2%, 5% or 10%). The tap water come from the local river near the Estacion Biologica de Monteverde Whey waste was collected from the farm of Jesus Rojas in Monteverde, Costa Rica The concentrations of whey water were made volume to volume. Each of the concentration was represented wi th ten plastic Glad containers, with every individual container containing five tadpoles of varying size. The experimental set up is diagramed in Figure 1.
Figure 1. Experimental design. Isthmohyla pseudopuma t adpoles of varying sizes were assigned to one of the four following treatments (0%, 2%, 5%, 10% whey waste). Each container contained 5 tadpoles each. Experimental Trials The experiment was performed in the lower lab of the Estacion Biologica de Monteverde in Monteverde, Costa Rica from July 19 th 2010 to August 2nd, 2010, a two week period. To test the condi tion of decreased water quality, I psuedopuma tadpoles were exposed to different concentration of whey within their habitat (Glad container). Each container of tadpol es was fed 1 gram of fish food daily. In addition to being fed, fresh wate r and whey replaced the old mixture dai ly. To record the growth rate of the tadpoles, they were weighed using a handheld scale. F ive tadpoles were removed from their home container, blotted with a paper towel to remove any excess water and then placed in a container that was already torn on the scale. Five tadpoles within a container were weighed toge ther for one combine weight. It was also reco rd ed if there were any deaths If mortalities occurred within a container, the death was recorded and the dead tadpole was immediately removed and disposed of. Tadpoles were then placed back in their assigned container with fresh water and whey. After all of the tadpoles were placed in their respective containers, all containers were covered with mosquito nets to prevent insects from entering their habitat. The mosquito nets also kept tadpoles that had metamorphosized during the experiment from escaping.
Statistical Analyses The Kaplan Meier (also known as the product limit estimator) test was used to compare the survival rate of the tadpoles within varying concentrations. Mantel Haenszel test for posthoc comparison of groups was also used to determine if the difference in survivorship was significant between the individual groups. One way ANOVAS s were used for day one, day three, and day five to determine differences in weight by treatment of whey. By this time the other two treatments had no tadpoles remaining. RESULTS Tadpole Survivorship Survivorship of Isthmohyla pseudopuma tadpoles was negatively impacted by the presence of whey waste. After day two all of the tadpoles in the presence of 10% whey waste had died. The percentage of tadpoles alive in the presence of 5% whey waste decreased greatly (six of fifty individuals alive) after day six and no ne of these tadpoles survived af ter day thirteen T he whey waste had no effect on the tadpoles in the presence of 2% whey waste during the expe rimental timeframe. It was shown though that there is a significant difference between the four treatment The Mantel H aenszel test for posthoc comparison of groups showed that there was a significant difference between each of th e treatments; expect the 0% and 2%, which had 100% alive for the duration of the experiment. Figure 2 Kaplan Meier survival curves for I psuedopuma tadpoles It was shown that there is a
Tadpole Growth On day zero all of the tadpole groups had the same weight even though they were of varying size within a container (ANOVA, F (3,31) =1.16, p<.34, Fig.2). On day three the growth between the three remaining treatments was not significant (ANOVA, F (2,27) = 2.82, Fig. 2), but a strong positive trend was visible. Beginn ing on day three fatalities began to occur in the 5%. Figure 2 is calculated out of how many containers, not individuals still living. For example, on day three there were 9 containers of tadpoles alive, so the weight was averaged for those nine containers This is represented by floating numbers in Figure 2. It was shown on day thirteen that the weight of the control gr oup and 2% whey waste was not different (t=0.00 0 n=20, p=0.99 8 ). The final day of the experiment the control group grew .03 g more than the group exposed to 2% whey waste, but the growth was not significant (t=2.484, n=20, p=0.132). Additionally, it was observed on the fifteen th day that many of the tadpoles in both the control and the 2% whey treatment had developed legs. Figure 3 Average (SD) weight (g) for Isthmohyla pseudopuma tadpoles being exposed to four different concentrations of whey over a two week period (N=10). The line that represents the 5% treatment has f loating numbers below each point to represent the amount of containers with living tadpoles
DISCUSSION The results indic ate that the presence of whey waste has a negative effect on Isthmohyla pseudopuma tadpoles. This was clearly shown by the treatment group which habitat was exposed to 10% whey waste. Within this treatment, al l tadpoles within the containers had died just two days after the experiment had commenced A potential cause of death f or the tadpoles could have been thei r inability to breath. When drying off the excess water from the tadpoles before weighing them every day, I found that there was a film of whey waste developing on their skin. Since the skin of a frog is permeable to oxygen and carbon dioxide, as well as w ater this may have blocked their ability to breathe successfully. The 5% treatment tadpoles were also negatively impacted by the presence of whey waste. The amount of tadpoles alive approximately half way through the experiment was greatly decreased, six of the fifty individuals were still living. After day thirteen all of the individuals in the treatm ent of 5% whey waste were dead. It is likely the cause of death for this treatment group was also the inability to breathe. I suspect that the 5% died more s lowly than the 10% simply because they had less whey present in their habitat. The survivorship of the 2% treatment was not a ffect ed by the presence of whey waste This is clearly observed in the results seeing as the 2% treatment sustained their population for the duration of the experiment. Additionally, it was observed on day fifteen that four of the individuals within the 2% treatment had died. results would be mor e conclusive if the experiment had been run for a longer amount of time. If the experiment were to be repeated I would suggest allowing enough time for the tadpoles to become fully mature adults. If this time frame were allowed you would be able to obs erve if there were any long term effects on the frogs In the case of growth rate, t he results suggest that the presence of whey waste has little effect on the tadpoles but a trend is obvious On day three the growth between the four treatments was proven n ot to be significant but there was a strong positive trend that showed the growth differences. On day thirteen it was proven that whey does not affect the growth rate of H. pseudopuma tadpoles. The results indicated that the growth rate of these two groups growth was nearly identical (t=0.000, n=20, p=0.998 ). This result illustrates that the control group could had potentially grown fast after the two week period then the remaining treatment group (2 %). Additionally, it was observed on the fifteenth day that many of the tadpoles in both the control group and the group exposed to 2% whey waste had started to metamorphosize This only exemplifies further that the experiment may not have run long enough to truly see the e ffect that whey waste has on the growth rate of I pseudopuma tadpoles. Further investigation should be done to observe rate of metamorphosis and if adults develop deformities due to the presence of whey waste. Overall, it was observed that whey has a neg ative effect on the survivorship of Isthmohyla pseudopuma tadpoles. Because amphibians are such good indicators of response to degradation, I believe this results shows evidence that other riparian animals may also be affected by whey waste negatively. The presence of whey waste seemed to have little to no effect on the growth of Isthmohyla pseudopuma tadpoles. This may be a positive results for the people category.
ACKNOWLEDGEMENTS Great thanks to my advisor, Pablo Allen for guiding me with my project concept and assistance during the experiment. Thank you to Jesus Ro jas for the use of his whey waste. Great thanks to Willow Zuchowski for allowing me on her property to collect tadpoles. Thank you to Calli Thompson for helping me to work though my original ideas and her constructive peer review of my paper. Thank you to Moncho Calderon for providing me with all the necessary supplies. I am very grateful to Alan and Karen Masters for their exceptional guidance during the CIEE Costa Rica summer 2010 Ecology program. Finally, thank you to the E stacion Biologica de Monteverde staff for their outstanding hospitality during our stay LITERATURE CITED Blaustein, A.R., J.M. Romansic, J.M. Kiesecker, and A.C. Hatch. 2003. Ultraviolet radiation, toxic chemicals, and amphibian population declines. Diversity & Distributions 9: 123 140. Boyer, R. and C.E. Grue, 1995. The Need for Water Quality Criteria for Frogs. Environme ntal Health Issues 103: 353 354. 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 Aby ecosystem in Wisconson, USA. Environmental Toxicology and Chemistry 24: 943 953. Mauro, M., A. Colicchio,F. Sansovini and E. Sebastiani. 1980. Chemical Oxygen Demand Reduction in a Whey Fermentation. Applied Microbiology and Biotechnology 9: 261. 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. Savage, J.M. 2002. The Amphibians and Reptiles of Costa Rica, p. 328 330. The U niversity of Chicago press, Chicago. Ecosystems. Science 277: 496 497.
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Stamper, Patricia, L.
El efecto del suero sobre el crecimiento y la supervivencia de los renacuajos Isthmohyla pseudopuma
The effect of whey on the growth and survival of Isthmohyla pseudopuma tadpoles
Water contamination and poor water quality in general have escalated in recent years. Streams and rivers located near dairy farms are commonly polluted by a byproduct of cheese making,whey. Due to factors such as water degradation, amphibian species have been experiencing rapid population declines worldwide. The purpose of this study was to determine the effects of whey waste on Isthmohyla pseudopuma tadpole growth and survival. It was found that whey waste
generally has a negative effect on I. pseudopuma tadpole survival and little effect on the growth. Additional observations such as metamorphosis were observed towards the end of the
experiment, further research should look into rate of metamorphosis and development of
deformities (if any) due to the presence of whey waste.
La contaminacin del agua y la mala calidad del agua, en general se han incrementado en los ltimos aos. Las quebradas y ros ubicados cerca de las fincas lecheras son comnmente contaminados por un subproducto de la fabricacin del queso, el suero. Debido a los factores como la degradacin del agua, especies de anfibios han experimentado un rpido decrecimiento de poblacin mundialmente. El propsito de este estudio fue determinar los efectos de los residuos de suero de leche en el crecimiento y supervivencia de los renacuajos Isthmohyla pseudopuma. Se encontr que los residuos de suero de leche tienen generalmente un efecto negativo sobre la sobrevivencia en el renacuajo I. pseudopuma y poco efecto sobre el crecimiento. Observaciones adicionales, tales como la metamorfosis fue observado hacia el final del experimento, la investigacin adicional debe mirar la tasa de la metamorfosis y el desarrollo de las deformidades (si los hay) debido a la presencia de residuos de suero de leche.
Text in English.
Aquatic organisms--Effect of water pollution on
Costa Rica--Puntarenas--Monteverde Zone
Organismos acuticos--Efecto de la contaminacin del agua en
Costa Rica--Puntarenas--Zona de Monteverde
Tropical Ecology Summer 2010
Rana (Isthmohyla pseudopuma)
Ecologa Tropical Verano 2010
Rana (Isthmohyla pseudopuma)
t Monteverde Institute : Tropical Ecology