This AOP is licensed under a Creative Commons Attribution 4.0 International License.
Energy deposition from internalized Ra-226 decay leads to altered ventilation behavior and slower growth in Lymnaea stagnalis due to apparent hypoxia from decreased hemocyanin oxygen binding capacity
Point of Contact
- Danielle Beaton
|Author status||OECD status||OECD project||SAAOP status|
|Under development: Not open for comment. Do not cite|
This AOP was last modified on November 21, 2022 16:37
|Deposition of Energy||October 17, 2022 16:40|
|Increase in reactive oxygen and nitrogen species (RONS)||May 08, 2019 12:30|
|increase oxidation of the di-copper moiety of the hemocyanin active site||November 16, 2022 11:37|
|Decreased, oxygen binding capacity by hemocyanin||November 16, 2022 12:10|
|Cognitive, sensed as hypoxic/low oxygen environment||November 16, 2022 12:28|
|Increase, hemocyanin mRNA||November 16, 2022 12:32|
|Increase, pulmonate breathing||November 16, 2022 12:38|
|Decrease, Growth||July 06, 2022 07:36|
|Decreased, Reproductive Success||December 03, 2016 16:37|
|Energy Deposition leads to Increase in RONS||March 28, 2022 07:19|
|Increase in RONS leads to methemocyanin formation (decrease overall oxygen binding capacity)||November 16, 2022 12:49|
|methemocyanin formation (decrease overall oxygen binding capacity) leads to Decrease overall oxygen binding capacity (methemocyanin formation)||November 16, 2022 12:50|
|methemocyanin formation (decrease overall oxygen binding capacity) leads to Hemocyanin Bohr effect decrease||November 16, 2022 12:51|
|Hemocyanin Bohr effect decrease leads to behavioral change leading to possible reduced feeding opportunity||November 16, 2022 12:52|
|behavioral change leading to possible reduced feeding opportunity leads to Decrease, Growth||November 16, 2022 12:53|
|Decrease overall oxygen binding capacity (methemocyanin formation) leads to Increase, hemocyanin mRNA||November 16, 2022 12:54|
|Decrease, Growth leads to Decreased, Reproductive Success||November 16, 2022 12:59|
This AOP features the freshwater giant pond snail, Lymnaea stagnalis, as an example freshwater aquatic organism and one that is included as a test organism for setting water quality criteria. The Giant pond snail is an intermediary organism in freshwater ecosystems where it consumes vegetation, algae and decaying organic matter in the lower trophic levels and is a source of food to fish in the higher trophic levels. They are geographically widespread, air breathing (pulmonate), simultaneous hermaphroditic gastropods with simple nervous systems and well-characterized life cycles . This makes them useful research species in understanding facets of the neurobiology of learning and motivation and in toxicology research. Male maturation takes ~30 days and female maturation takes ~60 days. In the wild their lifespan is roughly one year  . The Lymnaea shell forms from the mantle where an organic matrix is secreted, forming a scaffold for calcium carbonate biomineralization akin to that found for the oyster  .
Calcium and other alklaine earth elements from the environment are taken up by the snail. Throughout its soft parts, calcium stores help to maintain a constant calcium concentration within the the snail's hemolymph. Uptake of the alkaline earth radionuclide radium-226 from the environment follows the dynamics of calcium distribution and storage, suggesting that the hemolymph may offer a way to test for possible early events that follow from the decay of internalized radium-226 related to growth, reproduction and survival. This AOP proposes that decay of internalized radium-226 produces radiolysis products that react with the snail's oxygen carrying protein, hemocyanin, with the effect of lowering its oxygen binding capacity. This change is interpreted as hypoxia motivating the snail to engage in longer and/or more frequent pulmonate breathing. This change, in turn, could lower feeding opportunity and thus lower growth.
AOP Development Strategy
Changes in Hemocyanin as an Early Event following Energy Deposition
The Giant pond snail uses the copper bearing protein, hemocyanin, for oxygen uptake and delivery to cells. The copper elements at the oxygen binding center of this protein display a characteristic UV-visible spectrum depending on whether the copper moiety is bound to oxygen or not. In transitioning from its deoxygenated form to its oxygenated form, the hemocyanin protein changes from colorless to blue with an UV-visible absorption peak at ~348 nm . For this reason, species with hemocyanin are referred to as “blue blooded”.
In its met‑hemocyanin form, the oxidized copper moiety cannot bind oxygen as measured by the disapearance of tthe UV-visible absorption peak at 348 nm and the appearance of a low broad peak around 380-410 nm . Observable changes in the hemocyanin UV-visible strectrum upon the oxidation of copper suggest it may be possibleto connect this change in hemocyanin properties to the oxidizing nature of radiolysis products formed following energy depositon (onto biological matter) from ionizing radiation.
Calcium uptake as a proxy for Ra-226 uptake: Justification to Study Proteins in Hemolymph
The snail acquires calcium from the environment where uptake follows saturable Michaelis Menten type kinetics with a half saturation coefficient of 0.3 mM in artificial tap water . After uptake, the calcium ion enters the hemolymph, where it is taken up by other organs and used in cellular ATP production. In this study on L. stagnalis physiology, it was found that the calcium ion activity in hemolymph was lower than what would be predicted by the Debye-Huckel theory , suggesting that the bicarbonate ion produced as part of cellular metabolism forms complexes with some of the internalized calcium ions. In addition to the hard parts of the body, calcium carbonate deposits distribute through the soft parts of the snail's body within ‘calcium cells’ (aka rhogocytes, pore cells) that line the blood vessels, connective tissue, the foot muscle and the digestive gland .
Uptake of Ra-226 would distribute thoughout the snail body in a similar manner to the distribution of calcium, suggesting that a proportion of the internalized Ra‑226 resides within the soft tissues where calcium cells/rhogocytes are located. This accumulation is akin to Ra-226 uptake and distribution into granules within a freshwater muscle .
An electrochemical potential difference between calcium in the medium and calcium in the hemolymph varies with the external calcium . The corresponding equilibrium potential suggests that there is a positive calcium balance within the snail hemolymph when the calcium concentration in the medium is 0.062 mM or higher. Uptake is likely an active process operating against an electrochemical gradient at external calcium concentrations of 0.062-0.5 mM, while uptake occurs with minimal free energy change in the absence of an electrochemical gradient at external calcium concentrations higher than 0.5 mM.
The accumulation half-life for calcim in hemolymph is around 20 to 24 hours, eventually achieving ~70% of calcium content of the medium  . Lymnaea stagnalis grown in a medium of both calcium and Ra-226 also accumulated ~70% of the Ra-226 activity , further suggesting that the distribution and dynamics of Ra-226 within the snail body corresponds to the distribution and dynamics of calcium. Studies using labeled calcium found that the rate of calcium accumulation into the shell was slower than its accumulation into the blood, but that eventually the shell, as the main location in the snail for biomineralization, accumulated most of the labeled calcium.
Notably, the dynamics of calcium uptake, storage and loss work to maintain a constant blood calcium concentration and calcium flux , even under conditions of variable calcium in the medium . This “calcium buffer” system is linked to how bicarbonate is handled in the hemolymph such that when calcium in the environment is present in excess, some of the calcium is stored as calcium carbonate within the calcium cells/rhogocytes and the shell . However, at times when calcium from the environment is lacking, stored calcium from these reserves is returned to the hemolymph, thus maintaining a stable circulating calcium concentration.
Possible Early Biological Events from Internalized Radium-226 in the Snail, Lymnaea Stagnalis
The MIE for ionizing radiation is energy deposition. This event is followed by tracks of ionization and excitation events as the ionizing particles traverse the biological medium, which is largely composed of water.
At the sites of ionizations and excitations along the particle track, the ensuing dissipation of energy via the abundant secondary electrons contributes to the radiolytic formation of hydrated electrons, hydroxyl radicals, hydride radicals and superoxide radicals. These in turn further react to form the final yields of reactive and molecule species, including hydrogen gas and hydrogen peroxide . The combination of direct physical events and indirect physicochemical events set the stage for biological events and possible radiation-induced adverse outcomes.
The snails's soft tissues are bathed in hemolymph [REF]. A major protein in hemolymph is hemocyanin.
Rhogocytes are the cells that express hemocyanin [REF] [REF] and they are the sites of soft tissue calcium storage [REF]. Increased hemocyanin RNA expression is observed via RNA in situ hybridization of rhogocyte histology slides ##########
Histology and bulk tissue anlysis of freshwater mussels show that Ra-226 accumulates within calcium granules in mussel soft tissues to values greater than the external Ra-226 [Jefferee and Simpson, 1984, ######], [Brenner, Smoak et al 2007, Limnol Oceanogr 52, 1614-1623]. Alpha track imaging of Ra-226 decay overlaying histological images of the mussel localized alpha particle tracks to the calcium granules (Ra-226) and nearby soft tissues possibly from additional alpha tracks emitted from decay of the Ra-226 daughters: the mobile Rn-222 gas and Po-218, Po-214 and Po-210 or other alpha emitting radionuclides.
A shift from oxy/deoxy hemocyanin to methemocyanin would be a sign that the exposed snail's physiology has changed and thus may be a measureable early event in Giant pond snails exposed to radiolysis products generated from decay of internalized/accumulated Ra-226.
Hemocyanin also displays a Bohr effect akin to that of hemoglobin that may be important to how the snail brain detects and responds to low oxygen environments and motivates individulas to move to the water/air interface and initiate pulminate breathing.
For non-human biota adverse outcomes would be those outcomes that impact individual and population level risk criteria such as growth, survival and reproductive success. Since L. stagnalis is pulmonated (i.e., contains lung-like organs), when dissolved oxygen is low, it can add to its oxygen uptake via a respiratory pneumostome “hole” acting as a flow path at the air/water surface to obtain oxygen from the air . The stress of low oxygen conditions, therefore, is displayed as a behavioural adaptation that enables continued growth and survival.
Hemocyanin and Radiation Effects on Oxygen Binding Capacity
The copper-containing protein hemocyanin is a major component of the snail's hemolymph involving oxygen binding and delivery to cells akin to that of mammalian hemoglobin. In the presence of calcium, the hemocyanin from L. stagnalis hemolymph cooperatively binds oxygen in the pH range 6.8-8.5 with a Hill coefficient of 3.5-4.0 and a small Bohr effect at lower pH  . Other blue-blooded species with structurally different hemocyanin proteins complexes display different Hill coefficients for oxygen binding [Gonzalez, Nova, Del Campo et al. 2017, BBA Proteins and Proteomics].
In general, ionizing radiation lowers the oxygen binding of hemocyanins in the crab species Limulus and in welk species Busycon   that typically have limiting G-values in irradiated oxygen-free media of 0.026 and 0.044, respectively , however, oxygen binding capacity can be preserved if irradiation occurs in the presense of hydroxyl radical scavengers.
Thre is some quesiton on whether oxygen binding capacity is lost when hemocyanin is in its oxy-state or in its deoxy-state at the time of irradiation . In one explanation, the effect of ionizing radiation on hemocyanin oxygen binding is thought to be due to the un-scavenged hydrated electrons produced , however, the levels of the radiolysis product, hydrogen peroxide, may be important . Unlike what is measured for hemocyanin from Limulus, ionizing radiation has a dual effect on the hemocyanin from the welk Busycon via the protein's copper moiety: at low doses, and thus low hydrogen peroxide yield, the copper within the hemocyanin molecule becomes oxidized, thus lowering the hemocyanin oxygen binding capacity. At higher doses, the corresponding higher yields of hydrogen peroxide act to reduce the copper moiety, thus restoring the oxygen binding capacity of any hemocyanin molecules in the met‑form.
Measuring Oxygen Binding by Hemocyanin
In transitioning from its deoxygenated form to its oxygenated form, the hemocyannin protein changes from colorless to blue with an UV-visible absorption peak at ~348 nm  29 . For this reason, species with hemocyanin are referred to as “blue blooded”. In its met‑hemocyanin form, the oxidized copper moiety cannot bind oxygen and its UV-visible absorption peak at 348 nm disappears, while a peak around 380-410 nm appears .
A possible early sign that internalized Ra-226 may be impacting the snail physiology is through an oxidizing effect of ionizing radiation on hemocyanin and the oxygen binding capacity of this protein. A change in oxygen binding capacity by this protein may be interpreted as a low oxygen environment, where levels of dissolved oxygen in the water medium fall below what is sufficient for cutaneous respiration. If this is the case, further biological events may manifest in affected snails by employing comparatively more pulminate breathing with increased Ra-226. Possible longer-term impacts of such events, with associated behavioural cues, would be reduced overall metabolism, slower growth and a longer time needed to reach reproductive maturity.
Summary of the AOP
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
|Type||Event ID||Title||Short name|
|MIE||1686||Deposition of Energy||Energy Deposition|
|KE||1632||Increase in reactive oxygen and nitrogen species (RONS)||Increase in RONS|
|KE||2073||increase oxidation of the di-copper moiety of the hemocyanin active site||methemocyanin formation (decrease overall oxygen binding capacity)|
|KE||2074||Decreased, oxygen binding capacity by hemocyanin||Decrease overall oxygen binding capacity (methemocyanin formation)|
|KE||2075||Cognitive, sensed as hypoxic/low oxygen environment||Hemocyanin Bohr effect decrease|
|KE||2076||Increase, hemocyanin mRNA||Increase, hemocyanin mRNA|
|KE||2077||Increase, pulmonate breathing||behavioral change leading to possible reduced feeding opportunity|
Relationships Between Two Key Events (Including MIEs and AOs)
|Energy Deposition leads to Increase in RONS||adjacent||High||Low|
|Increase in RONS leads to methemocyanin formation (decrease overall oxygen binding capacity)||adjacent||Moderate||Low|
|methemocyanin formation (decrease overall oxygen binding capacity) leads to Decrease overall oxygen binding capacity (methemocyanin formation)||adjacent||Moderate||Low|
|methemocyanin formation (decrease overall oxygen binding capacity) leads to Hemocyanin Bohr effect decrease||adjacent||Not Specified||Not Specified|
|Hemocyanin Bohr effect decrease leads to behavioral change leading to possible reduced feeding opportunity||adjacent||Not Specified||Not Specified|
|behavioral change leading to possible reduced feeding opportunity leads to Decrease, Growth||adjacent||Not Specified||Not Specified|
|Decrease overall oxygen binding capacity (methemocyanin formation) leads to Increase, hemocyanin mRNA||adjacent||Not Specified||Not Specified|
|Decrease, Growth leads to Decreased, Reproductive Success||adjacent||Not Specified||Not Specified|
Life Stage Applicability
|All life stages||Not Specified|
Overall Assessment of the AOP
Domain of Applicability
All blue blooded species
Essentiality of the Key Events
Oxidation of copper moieties in copper bearing proteins
Connecttion levels of biological organization -- omics
Changes in behaviour affecting growth/survival/reproductive success
Known Modulating Factors
|Modulating Factor (MF)||Influence or Outcome||KER(s) involved|
Considerations for Potential Applications of the AOP (optional)
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