MODELLING THE DYNAMIC TRITIUM TRANSFER TO FARM ANIMALS. EXTENSION TO WILD MAMMALS AND BIRDS Anca Melintescu PhD “Horia Hulubei” National Institute for Physics and Nuclear Engineering, Bucharest- Magurele, ROMANIA firstname.lastname@example.org, email@example.com 2nd Meeting of the EMRAS II Working Group 7, “Tritium”, Chatou, France, 28–29, September 2009 MODELLING OF TRITIUM TRANSFER IN ANIMALS Simplified models and experimental data base • Models in use are schematic and non-validated or are empirically derived and cannot be used out of initial data set; • Use one compartment for OBT with halftime given by total organic carbon one; • Animal products contribute significantly to the diet - reliable dynamical models are needed; • Sparse experimental data - old experiments insufficiently reported; • BUT very good experimental data and model for rat (experiments done by H. Takeda, NIRS, Japan); • Need a different approach based on comparative metabolism and OBT-C links; Animals bioenergetics • Review of past results of 3H and 14C transfer modelling 1 dE 1 dM in mammals → necessity to have a common approach E=mc2 → based on energy needs and on the relation between E dt M dt energy and matter (well established in Atomic and Quantum Physics) • Knowledge on animal metabolism and nutrition GE in food • Metabolism = countless chemical processes going on continuously inside the body that allow life and normal GEf functioning • These processes require energy from food DE • Energy is derived from the digestion of several compounds, including carbohydrates and fats. Excess GEug dietary protein can also be used as an energy source, ME but it is a costly practice. • Gross energy, Digestible energy, Metabolisable Basal Met. Energy, Net energy Maint. Met. • Maintenance metabolism (basal+heat of digestion), Heat of Dig. lost as heat Cold Therm. • Heat needed for cold thermogenesis, activity and losses in processes of growth, production and Used for work, reproduction Growth, re-prod • Energy stored (deposited, retained) in the products of growth, lactation (egg) and reproduction NE • Daily Energy Expenditure (Field Metabolic Rate) • Food must include maintenance protein • Field Metabolic Rate (FMR, MJ kg-1 d-1) = the net daily energy expenditure of animals - depends on the level of nutrition, taxon, diet, environment FMR= a*BWb ? b ~0.75 or 0.67 or ? BW – body weight (kg) a, b – scaling coefficients • Specific Metabolic Rate (SMR, MJ kg-1 d-1) = the daily energy expenditure per unit fresh body mass • Relative Metabolic Rate or the energy turnover rate (ReMR, d-1) = ratio of SMR and the energy content of the body, determined by the body composition (protein, lipids, and carbohydrates): FMR SMR Re MR EBW * BED BED ReMR - used also for loss rate of organic matter (as in ontogenetic growth) EBW - the empty body mass (kg) defined as the live-weight less the mass of the gastrointestinal contents; BED - the body energy density (MJ kg-1 fw) - depends on body composition BED=flipid*39.6+fprotein*23.7+fcarbohydrate*17.7 Body Size : Surface Area Ratio and Energy Demand Comparison of Endotherms 8 (B/M)=aM1/4 Allometric relation 7 Mass-specific metabolic rate (ml O2/gram/hour) Shrew 6 5 4 3 Harvest mouse 2 Mouse Flying squirrel 1 Bat Cat Dog Human Horse Elephant 0 0.01 0.1 1 10 100 1000 10,000 Mass (kg) Derivation of a generic model based on energy metabolism tested with experiments MAGENTC - MAmmal GENeral Tritium and Carbon transfer • Complex dynamic model developed by us in the last four years in an international collaboration for H-3 and C-14 in mammals • full description given in: D. Galeriu, A. Melintescu, N. A. Beresford, H. Takeda, N.M.J. Crout, “The Dynamic transfer of 3H and 14C in mammals – a proposed generic model”, Radiation and Environmental Biophysics, (2009) 48:29–45 • A key element in any model of radionuclide transfer in animals is the loss rate (half-time) from the body or organs; • There are too few experimental data for 14C and 3H from which one could derive these values, and we therefore advance the working hypothesis that the loss rate of organic compounds (organic carbon, OBH or OBT) from the body or organs can be linked with the energy turnover rate. • The model has 6 organic compartments and distinguishes between organs with high transfer and metabolic rate (viscera), storage and very low metabolic rate (adipose tissue), and „muscle‟ with intermediate metabolic and transfer rates. • Some organs have high metabolic activities and will therefore have high 3H and 14C transfer rates. • Liver, kidney, heart, and the gastrointestinal tract use about 50 % of the basal metabolic requirements whilst typically contributing less than 10 % of the body mass; these organs are included as a combined “viscera” compartment. • Blood is separated into red blood cells (RBC) and plasma as plasma is the vector of metabolites in the body (and also as a convenient bioassay media). • The remaining tissues are bulked into one model compartment („remainder‟) in order to achieve mass balance. • The organic compounds of 3H and 14C enter the body via the stomach and they are mostly absorbed from the small intestine and a simplified transfer through gastrointestinal tract is used to reproduce the delay between intake and absorption. • The stomach and small intestine compartment refers to the content, as an input pathway, whilst the stomach and small intestine walls are included in the viscera, having high metabolic rate. Modelling approaches • The metabolisable fraction of dietary intakes of organic tritium and carbon are transferred to systemic body compartments; the remainder is excreted. In the case of dietary tritium, the exchangeable fraction is transferred directly to body water and only the non- exchangeable fraction enters blood plasma; • Ingested HTO is assumed to be immediately mixed in the body water compartment • The transfer rates between compartment and blood plasma are given by RMR. The transfer rates from blood plasma to model compartments are assessed using the mass balance of the stable analogues (include net growth); • Transfers include the net flux after the digestion and transformation of dietary compounds in protein, lipids or carbohydrates; • Transfer rate to urine (organic) given by mass balance (urine dry matter production, plasma organic content); • Transfer rate between body HTO and plasma OBT given by hydrogen metabolism (equilibrium value of OBH derived from free H); • Transfer rate for respiration (or body HTO) by mass balance of stable nuclide: intake assumed correlated with energy needs; • Organ composition assumed similar to humans (cf. Geigy tables and other models) • Plasma composition (OBC,OBT) same for all mammals (cf. Baldwin 1995); • All model compartments have a single component (no fast-slow distinction) Model tests with experimental data on rats • Complete database for 3H and 14C transfer, obtained from experiments with Wistar strain rats thanks to H. Takeda (NIRS, Japan) • Studies included: – continuous 98 days intakes of 14C and OBT contaminated food or HTO; - acute intakes of HTO or 14C and 3H labelled glucose, leucine, glycine, lysine, and oleic and palmitic acids. • Available data include 14C, OBT and HTO measurements in visceral organs, muscle, adipose tissue, brain, blood and urine. • For the acute studies data on labelled organic compounds in proportions typical of normal rat food were combined. • Model parameters not obtained from the study were estimated from the literature: - organ mass, - whole body and - organ energy expenditure. • The intakes of OBT (metabolisable and non-exchangeable fractions) and organic 14C were estimated from the known food composition. Results of model test with rat data (no calibration) Average and standard deviation of predicted to observed ratios in rat viscera, muscle, blood, adipose tissue and whole body (except bone and skin) for the six forms of intake Organ 14C chronic 14C acute OBT OBT HTO HTO acute chronic acute chronic Viscera 1.12 ± 0.15 0.51 ± 0.4 1.06 ± 0.15 0.67 ± 0.43 ± 0.07 0.87 ± 0.34 0.56 Muscle 1.25 ± 0.14 0.81 ± 1.23 ± 0.21 0.90 ± 0.40 ± 0.09 1.02 ± 0.38 0.29 0.37 Adipose 1.11 ± 0.15 0.61 ± 0.97 ± 0.2 0.75 ± 0.3 ± 0.1 1.33 ± 0.3 0.12 0.13 Whole 1.12 ± 0.27 0.4 ± 0.1 0.88 ± 0.12 0.38 ± 0.37 ± 0.09 0.62 ± 0.18 blood 0.03 Whole-body 1.18 ± 0.08 0.7 ± 0.1 1.08 ± 0.11 0.8 ± 0.1 0.36 ± 0.08 1.09 ± 0.18 Data error ?! Representative results, no calibration Model predictions and experimental observations for rat muscle following acute intakes of food labelled with 14C or OBT Model tests with cow data (no calibration) • Several exposures – Single HTO intake – Continuous HTO intake – Continuous OBT intake • Cow mass, feed and water intake, milk and urine production taken from experiments • All other model parameters taken from literature – no calibration with tritium data Results of model test with cow data (no calibration) Model performance for dairy cow; NA: not calculated/available Experiment R2 Milk total 3H Milk OBT Urine HTO Mean standard deviation P/O (range presented in parenthesis) Cow_P 0.97 2.60 ± 1.7 1.68 ± 0.8 2.90 ± 2.34 (0.8 -1.9) (0.5 - 2) Cow_C 0.89 0.97 ± 0.08 0.73 ± 0.17 0.97 ± 0.06 (0.9 -1.4) (0.65 - 1.7) Cow_H3 0.67 1.02 ± 0.15 0.49 ± 0.12 1.36 ± 0.42 (0.9 - 1.5) (0.4 - 0.9) Cow_H 0.88 1.45 ± 0.59 1.86 ± 0.38 NA (0.6 - 2.3) (0.55 - 2.12) Representative results, no calibration Experimental data and model predictions for OBT in milk after OBT fed for 26 days. Experimental data were reported only after stop dosing 100000 milk_OBT_present milk_OBT_TRIF milk OBT concentration BQ/L milk_OBT_UFOTRI 10000 milk_OBT_exp 1000 100 0 50 100 150 tim e (d) Model tests on sheep data (no calibration) • Scottish Blackface female sheep – acute intake of 14C- and 3H- labelled glucose and acetate • The experiment provides approximate information on the transfer from feed to organs. • We added a sub-model for growth (from 27 kg at the beginning of exposure to 47 kg after one year) - Organs‟ masses growth were taken from experiment and literature • The model considered normal marked food intake: protein + fat + carbohydrates (not only carbohydrates as in experiment); • The study did not include labelled protein, although production of protein by rumen bacteria may have led to some labelled protein being present • Model results are sensitive to the growth rate in the day of intake Representative results, no calibration Dynamics of organic 14C (left) and OBT (right) in sheep muscle after an intake of labelled glucose and acetate. 0.006 1.00E-02 predicted predicted normalised concentration normalised concentration 0.005 observed observed 0.004 1.00E-03 0.003 0.002 1.00E-04 0.001 0 1.00E-05 140 240 340 440 540 140 240 340 440 540 time (d) time (d) Model tests with pig data (no calibration) • Data on organ OBT concentrations are available for a gestating sow fed OBT for 84 days and who died before delivery. Results of model test with pig data (no calibration) Organ P/O blood 1.17 muscle 1.7 viscera 1.4 • Initial body composition was adjusted to be close to lean or fat genotype considering the lipid content of muscle according with experimental information on inter-muscular fat for the contrasting genotype PP, SL and MS. • The results show a clear distinction between meat concentrations of genotypes at the time of killing, the fat MS genotype having the highest value and PP the lowest. OBT concentration 0.6 1 0.9 0.5 SLmuscle_conc 0.8 concentration 0.4 0.7 SLwhole_conc adipSL 0.6 0.3 musSL MSmuscle_con Bq/kgfw 0.5 viscSL c adipMSC 0.2 0.4 musMSC MSwhole_conc 0.3 viscMSC 0.1 adipPPM 0.2 musPPM 0 0.1 viscPPM 0 50 100 150 0 body mass 0 50 100 150 empty body mass kg Conclusions for MAGENTC • Despite simplifications, the model tests are encouraging for tissues and milk for a range of animals. • Without parameter optimization, the model predictions are within a factor of 3 of the reported values in all cases. • Some improvements could be made to the model in the future, in order to increase the predictive power: 1. Incorporation of an understanding of ruminant digestion to clarify the exchangeable fraction of net organic intake; 2. Incorporation of fast and slow compartments for each organ/organs group, if a general rule can be obtained from animal science and physiology research; 3. Inclusion of up-to-date knowledge on organ specific metabolic rate (in vivo) for animals; there has been considerable progress in the use of modern noninvasive techniques such as Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI) for metabolic studies. Extension of the current model to wild mammals and birds • Full description is given in: A. Melintescu, D. Galeriu, “Using energy metabolism as a tool for modelling the transfer of 14C and 3H in animals”, submitted to Radiation and Environmental Biophysics Extension to wild mammals • Clear need to explicitly consider non-human biota within radiological assessments (ICRP 2007); • ICRP proposes the use of Reference Animals and Plants; • We have past experience to assess the concentration ratio for specific animals for tritium and 14C in the frame of European projects (EPIC, FASSET) for routine emissions; full details are given in: D. Galeriu, N.A. Beresford, A. Melintescu, R. Avila, N.M.J. Crout, “Predicting tritium and radiocarbon in wild animals”, International Conference on the Protection of the Environment on the Effects of Ionising Radiation, Stockholm, Sweden, 6 –10 Oct. 2003, P. 186-189, IAEA-CN-109/85 • Data for radionuclides in wild animals are sparse and a number of approaches including allometry have been proposed to address this issue • Unlike to laboratory or housed farm animals, wild mammals and birds are subjected to large environmental and dietary variability for which they must adapt. • Our definition of biological halftime has been used in order to explore the range for wild mammals; full details given in: D. Galeriu, A. Melintescu, N.A. Beresford, N.M.J. Crout, H. Takeda, “14C and tritium dynamics in wild mammals: a metabolic model”, Radioprotection, Suppl. 1, Vol. 40 (2005), S351-S357, May 2005 • There are many studies demonstrating allometric (mass dependent) relations for basal metabolic rate, daily energy expenditure and organs‟ masses. • For DEE there is considerable evidence of taxon specific allometric relationships, but dietary habits can still have a large influence for rodents with herbivorous, omnivorous or granivorous diets. • DEE depends on environmental temperature (small mammals in the Arctic have a 2 fold higher DEE for the same body mass compared with animals in Mediterranean climates). 10000 Visceral percentage of body mass granivore omnivore 35 herbivore Rodentia 30 1000 DEE (kJ d-1) 25 20 15 100 10 5 0 10 0.01 1 100 10000 10 100 1000 10000 Body mass kg Body mass (g) Variation with body mass in the mass of DEE (kJ d-1) for granivorous, carnivorous, visceral organs expressed as a percentage of and herbivorous diets, compared with an allometric whole body mass. relationship for rodents. • The biological halftime does not only depend on animal mass but also on taxon either. • For the same body mass, taxon and diet may affect the biological half time with a factor 2. Biological half times for Carbon (and OBT) units days Mass (kg) 0.03 0.1 1 5 10 30 300 Animal Biological half-times Terrestrial 3.1 4.8 11.1 19.8 25.4 37.8 - mammals Mesic rodents 2.8 4.1 8.4 13.7 - - - Carnivores 5.5 6.7 9.4 12.0 13.3 15.7 Granivores 4.9 10.0 - - - - - Herbivores 3.1 4.8 10.8 19.2 24.5 36.2 81.8 Insectivores 3.8 6.1 14.6 26.8 - - - Omnivores 3.7 5.4 11.4 19.2 - - - • Our model needs as input the Basal Metabolic Rate (BMR), the field energy expenditure (FMR), organ mass and organ Specific Metabolic Rate (SMR). • Body mass is not the only predictor of BMR, but body temperature, taxon, diet and climate are also important; • A gap in the database for wild animals is the assessment of maintenance energy needed per kg tissue and time unit, the so called specific metabolic rate (SMR) for organs in basal and active states. • Due to adaptation to various environmental constraints it is possible that the organ metabolism of wild mammals to differ from domesticated ones. • The organ mass for wild mammals also is less documented than for farm animals and the intraspecific variability can be higher. This explains why our predicted BMR values are sometimes close to observed values, but there are cases of 50 % discrepancies. • In practice we have considered the relative contribution of organs to whole body BMR and use the experimental BMR in the model input values. Species Mass (kg fw) Measured BMR (MJ d-1) Estimated BMR (MJ d-1) Hare (Lepus carpensis) 2.9 0.78 0.79 Jackal (Canis mesomelas) 2.8 0.7 1.05 Racoon (Procyon lotor) 2.2 0.5 0.76 Puma (Felis capensis) 9.6 1.9 1.5 Wild cat (Felis ocreata) 2.7 0.5 0.52 Chipmunk (Tamias 0.0075 0.045 0.07 striatus) We reassessed all input data and also select red deer as a large herbivore. We include two rodents (lemming and chipmunk), a small herbivore - rabbit and a carnivore – red fox. The lemming from Arctic regions is modelled with enhanced energy needs. As much as possible input data correspond to same habitat, diet, temperature and subspecies for each considered mammal. The effect of a coherent selection of model parameters is exemplified for chipmunk, for which we considered mixed literature data but also measured BMR and FMR of the same population (Quebec - personal data from Careau). Model inputs Animal Latin name Mass (kg) BMR Mass fractions BMR FMR (MJ day-1) (MJ day-1) (MJ day-1) adipose muscle viscera lemming Lemmus 0.06 0.045 0.35 0.28 0.15 0.042 0.19 trimucronatus chipmunk Tamias 0.096 0.052 0.15 0.4 0.22 0.081 0.12 striatus chipmunk Tamias 0.0915 0.0675 0.15 0.4 0.22 0.078 0.17 C striatus rabbit Lepus 1.8 0.57 0.1 0.43 0.13 0.573 1.3 californicus red fox Vulpes vulpes 6 1.1 0.15 0.45 0.13 1.43 4.5 red deer Cervus 107 11.7 0.1 0.43 0.12 12.4 24.5 (elk) elaphus Model results Animal Mass Fast half- Slow half- Fast Effective half- Transfer factor (kg) time time (day) contribution in time (day) (day kg-1) (day) retention lemming 0.06 4.2 52 0.8 5.2 36.88 chipmunk 0.096 4.4 69.3 0.91 4.76 47.75 chipmunk 0.0915 3.1 55.4 0.926 3.32 34.6 C rabbit 1.8 7.4 79.8 0.87 8.44 3.35 red fox 6 8.1 147.6 0.91 8.76 1.51 red deer 107 25.2 227.2 0.83 29.6 0.21 Concentration ratios in different model compartments Animal whole body adipose muscle viscera remainder lemming 0.70 1.38 0.32 0.28 0.79 chipmunk 0.48 1.26 0.32 0.28 0.76 chipmunkC 0.49 1.34 0.32 0.28 0.76 rabbit 0.44 1.19 0.32 0.28 0.57 red fox 0.38 1.03 0.24 0.21 0.50 red deer 0.45 1.28 0.32 0.28 0.55 Short term dynamics of 14C in whole body (generalised coordinates) lemming chipmunk chipmunkC rabbit redfox reddeer 0.1 norm whole conc 0.01 1 2 3 4 5 6 7 8 9 10 T*RMR Generalised coordinates: Normalised concentration=Whole body conc *Mature mass T*RMR – non-dimensional time = time * mature RMR Despite these shortcomings, the results presented above are less uncertain than for many other radionuclides and can provide useful results for biota radioprotection. Extension of the current model to birds • The model developed for mammals is based on energy metabolism and body composition with the assumption that the turnover rate of organics is linked to energy turnover rate. • There are not reasons to restrict the model to mammals, if the assumptions are qualitatively correct. • The allometry of basal metabolic rate of birds has close mass exponent to mammals. • After a selection of good data and correction for phylogenetic bias, we found: BMR = 303*M-0.33 (mass in kg and metabolic rate in kJ day-1). • There is no difference between passerine and non passerine and the higher values for birds comparing to mammals are explained by higher body temperatures. • The scaling exponent of BMR in captive birds (0.670) is significantly lower than in wild-caught birds (0.744) due to phenotypic plasticity. • The scaling exponents of FMR for birds and mammals were not significantly different: birds: FMR = 1.02 M0.68, mammals: FMR = 0.68 M0.72 Disregarding the effect of increased body temperature we compare our model BMR to experimental data Comparison between BMR model and experimental data for birds 1.2 1 BMR calc/BMR exp 0.8 0.6 0.4 0.2 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Body mass (kg) For small birds we under predict with 20-40 %. With one exception (Arenaria interpres) all are passerine with higher body temperature than other birds. We conclude that our mammals SMR, corrected for body temperature, can help as a first attempt to expand the model to birds. For food chain modelling, laying hens and broilers are of special concern and there are not experimental data for eggs or meat contamination with 3H and 14C. We considered a tritium intake (1 Bq day-1) for 60 days in both forms (HTO or OBT). Dynamics of tritium in eggs after HTO or OBT intake 10 Concentration (Bq/kg) 1 OBT(OBT) OBT(HTO) 0.1 Total(OBT) 0.01 Total(HTO) 0.001 0 50 100 150 200 250 Time (d) OBT concentration in eggs is predicted to increase rapidly in the first 7 days corresponding to the duration of egg formation, and slowly thereafter, due to contribution of recycled body OBT. We observe that the OBT concentration in egg, after stop dosing decreases in the first days with a half-time of about 5 days and slower later (halftime of about 40 days), due to contribution of body reserves. Total tritium in eggs is 2 times higher when the intake is OBT, but share of OBT is about 75 % for OBT intake and only 9 % for HTO intake. In order to obtain directly the transfer factor, intake has been fixed at 1 Bq day-1, while for concentration ratio, intake was 1 Bq kg-1 dry matter or 1 Bq L-1 of water. Transfer factor for tritium in broiler Concentration ratio for tritium in broiler 100 1 OBT (HTO) T (HTO) OBT (OBT) 10 T (OBT) Transfer factor (1/kg) Concentration ratio OBT (HTO) 1 0.1 T (HTO) OBT (OBT) T (OBT) 0.1 0.01 0.01 0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160 Time (d) Time (d) In the case of fast growing broiler, at the market weight of about 2 kg (42 days old) the model predicts lower transfer factors (TF) than for the equilibrium case The predicted concentration ratios (CR) for our fast growing broiler are close to those obtained for “equilibrium” . In absence of any experimental data or previous modelling assessments, our results give a first view on the transfer of 3H and 14C in birds. CONCLUSIONS • We developed research grade model for plants and animals based on process level, pointing out that model inputs can be obtained using Life Science research in connection with National Research on plant physiology and growth, soil physics, and plant atmosphere interaction, as well as animal physiology, nutrition and metabolism; • We re-use these knowledge with a very low cost, but spending time to learn basics from these fields → Interdisciplinary Research; • Classical compartmental models can be derived and appropriate parameters for each case can be obtained in this way. Thank you!