Effects of low-dose ionizing
radiation on
eggs and larvae of
Bufo
terrestris (southern toad)
Karolina Stark--Research Projects--SREL
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Effects of low-dose ionizing
radiation on |
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Experimental design
Hypothesis: Current regulatory guidance on acceptable dose rates to aquatic organisms (10mGy/d) established by IAEA (1992) sufficiently protects frogs under chronic exposure conditions.
Facility: LoDIF with 137Cs-sources at Par Pond Laboratory and SREL molecular lab.
The LoDIF consists of 40 tanks divided into eight blocks with five tanks in each block. Four blocks will be used during the exposure period in this experiment, with four tanks per block used for the different treatments.
Frog species: Bufo terrestris (Southern toad), inhabitant of SRS. Typical toad and life-cycle (2-3 years). Egg development, 4-10 days; larval development period 1-2 months (30-60 or 45-50 days). Good representative of toads because there is ~ 250 Bufo species in the world.
Prior to this experiment, toad eggs and tadpoles (B. terrestris) will be collected in the field and brought to lab.
In this experiment we hypothesize that 10 mGy/d sufficiently protect frogs under chronic exposure conditions. We test our hypothesis by exposing toad tadpoles to dose rates of 0, 1.8, 18, and 180 mGy/d during their development period to metamorphosis. The metamorphosis is considered to be a radiosensitive period during the development of amphibians. Three buckets (size: 16 L) will be placed in the center of every tank. In total 48 buckets will be used. Every bucket has 3 holes in them covered with nets that allow a flow-through system of water in the tanks. An exposure period of 20 days would result in a total dose of 0, 0.036, 0.36, and 3.6 Gy, respectively.
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Freshly oviposited eggs (< 1 day old) will be exposed to the four radiation treatments (approximately 100 eggs per tank). At hatching survivors will be counted, and larvae will be sub-sampled and partitioned into two density treatments. In bucket A in every tank 30 tadpoles will be placed.
Set-up for each tank |
Bucket A in every tank will be exposed until metamorphosis of the tadpoles. In bucket B 10 tadpoles will be placed and exposed until metamorphosis. Bucket C is empty. The different densities of tadpoles in bucket A and bucket B are expected to promote differences in the length of the larval period. The tadpoles in bucket A will likely need more time until they start to metamorphose. This will result in a longer exposure period for tadpoles in bucket A and a higher total dose to these tadpoles. A comparison of radiation effects can therefore be made between bucket A and B regarding the importance of dose rate vs. total dose received. Both bucket A and B will be exposed to the same dose rates but the tadpoles in bucket A will most likely receive a higher total dose prior to metamorphosis than the tadpoles in bucket B due to the density effect. The exposure will be interrupted two times to record the stage of development and mortality. The first interruption will be after 9 days and the second interruption will be after 18 days or prior to the expected start of metamorphosis. The tadpoles will be fed with ground TetraMin 1-2 times/week during the whole exposure period by using an extended feeding device. The first week the flow-through system will be turned off to mimic natural conditions for tadpoles and to raise the water temperature. If the ammonium levels rise the flow-through system will be turned on. The exposure period will be ended after metamorphosis. The water temperature will be measured throughout the exposure period. |
Ecological endpoint
Egg mortality will be measured during the egg exposure period. Larval mortality, morphological damage and time of metamorphosis will be recorded in bucket A & B and results from irradiated tadpoles will be compared to controls. After the exposure the toadlets will be moved into the lab and body size and body mass will be recorded of all animals. A sample of metamorphs will be collected from each replicate to measure total lipid content as an indicator of body condition. We will Ultra freeze samples until the analysis can be conducted, and will pool samples if necessary due to small metamorph body size.
Molecular endpoint
Blood samples (0.5 µL) will be taken from all toadlets and analyzed for DNA damage (double strand breaks) with the Comet Assay. Irradiated toads will be compared to unirradiated toads. Also, the results will be compared to the results from the ecological endpoint. The Comet Assay will have to be conducted right away after the samples are taken to minimize post-exposure DNA repair. We plan to collect a minimum of 6 samples per treatment group, which will require two separate runs (i.e., 4 days) for the Comet Assay. It might be difficult to take blood samples because they are so small.
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