1. www.star-radioecology.org www.radioecology-exchange.org Supported by the European Commission, contract number: Fission-2010-3.5.1- 269672, and the Research Council of Norway, contract number: 209101. Reference mixture models developed for chemical toxicants work when radionuclides are in the mix C. Svendsen, N. Horemans, C. Bradshaw, H-C. Teien, R. Gilbin, A. Margerit, F. Nascimento, H. Vandenhove, S. Lofts, D. Spurgeon, STAR final dissemination Event, Aix-en-Provence, 9-11 June 2015

2. www.star-radioecology.org www.radioecology-exchange.org What does “joint effect of mixtures” really mean? Reference models developed for chemicals – IA (RA) & CA Example of U x Cd mixture analysis – general pattern found What models can be used when one stressor is not a chemical? Gamma x Cd mixture analysis – general patterns found General conclusions and other options e.g. DEB Objectives- What to expect from joint stressor effects?

3. www.star-radioecology.org www.radioecology-exchange.org What does “joint effect” really mean? How do we work out what happens if we mix these two EC50s?

4. Possible models for expected joint action of mixtures: General mistake - “Effect addition” (aka “Response Addition”) Dissimilar acting compounds → “Independent Action” (aka “Response Addition”) Similar acting compounds → “Concentration Addition” Effect Addition (aka Response Addition) LC25 + LC25 = LC50 LC40 + LC40 = LC80 LC50 + LC50 = LC100 LC40 + LC40 + LC40 = LC?? 5 x LC50 = LC?? Shame mixture: 2x Uranium LC25 = LC50?? LC 25 =2500 LC 25 =2700 2x LC 25 =5000

5. Possible models for expected joint action of mixtures: General mistake - “Effect addition” (aka “Response Addition”) Dissimilar acting compounds → “Independent Action” (aka “Response Addition”) Similar acting compounds → “Concentration Addition” So what should we expect from 2x LC50? Logically 2 x LC50 = Effects bigger than 50% Mortality, but how big? [Chemical 1] [Chemical 2] Exposure in sequence

6. Possible models for expected joint action of mixtures: General mistake - “Effect addition” (aka “Response Addition”) Dissimilar acting compounds → “Independent Action” (aka “Response Addition”) Similar acting compounds → “Concentration Addition” So what should we expect from 2x LC50? Logically 2 x LC50 = Effects bigger than 50% Mortality, but how big? [Chemical 1] [Chemical 2] Probability of surviving both chemicals: Emix = Ea+Eb-(Ea*Eb)  1-Emix = (1-Ea)*(1-Eb) = Q(unaff) mix Exposure together

7. www.star-radioecology.org www.radioecology-exchange.org Dissimilar acting compounds → “Independent Action” (IA) Developed in 1939 by Bliss Based on effects of chemicals being statistically independent I.e. It is a measure of the joint probability of effect from all the chemicals “Russian roulette with multiple guns!” you need to be lucky every time to survive – you only get killed once, but that is also enough. How do we assess the risk (h)?? “unaffected fractions”

8. www.star-radioecology.org www.radioecology-exchange.org Dissimilar acting compounds → “Independent Action” (IA) Analysis: Prob. of surviving: q mix (2xLC50) = 0.5 * 0.5 = 0.25 => Prob. Death = 1 – 0.25 = 0.75 Prediction: What Mixture gives an EC50? q1 * q2 = 0.5, If equitoxic mixture (i.e. q1=q2) => q1 = q2 = SQRT(0.5) = 0.71 So probability of no effect from each chem = 0.71 => Prob effect = 1 - 0.71 = 0.29 So for IA 2x LC29 => mix LC50 - Need only know individual effects to analyse - Need full dose response profiles to predict effect Or simply; The probability of surviving = Prob(Surv Tox(1)) * Prop(Surv Tox(2))*…*(Prob(Surv Tox(n))

9. www.star-radioecology.org www.radioecology-exchange.org Similar acting compounds → “Concentration Addition” (CA) Developed in 1926 by Loewe and Muischnek Based on chemicals having same mode of action I.e. acting on same biological pathway and strictly speaking on the same molecular target Only difference is the relative potency of chemicals – act as “dilutions of each other” How do we assess the risk?? TU = Toxic Unit - So what does that mean?

10. www.star-radioecology.org www.radioecology-exchange.org Similar acting compounds → “Concentration Addition” (CA) Analysis: If 50% effect is caused by a mixture with “M” other than 1 => non concentration additivity: 1)M = 1, toxicants are concentration additive 2)M < 1, toxicants more than concentration additive ( Synergism ) 3)M > 1, toxicants less than concentration additive ( Antagonism ) Prediction: 50% Effect from: 1/2EC50 + 1/2EC50, 1/4EC50 + 3/4EC50, 1/6EC50 + 5/6EC50 25% Effect from: 1/2EC25 + 1/2EC25, 1/4EC25 + 3/4EC25, 1/6EC25 + 5/6EC25 BUT, EC50 + EC50 = ?? (> EC50 that is all), EC25 + EC50 = ?? (>EC50), => (ECx values are needed) X = 50 A binary mixture follows CONCENTRATION ADDITIVITY IF: When mixture causes 50% effect, M = C1/EC50 1 + C2/EC50 2 = 1

11. www.star-radioecology.org www.radioecology-exchange.org Concentration Addition (CA) for a mixture response surface We need ISOBOLs = “line of same value” (e.g. weather charts, soil conc., etc.) Bristol Metal smelter Soil [Cd] mg/kg (dw)

12. www.star-radioecology.org www.radioecology-exchange.org Concentration Addition (CA) for a mixture response surface We need ISOBOLs = “line of same value” (e.g. weather charts, soil conc., etc.)

13. www.star-radioecology.org www.radioecology-exchange.org Concentration Addition (CA) for a mixture response surface [Chem 1] [Chem 2] EC 50 For a binary mixture that follows ADDITIVITY: M = C1/EC50 1 + C2/EC50 2 = 1 (The EC50 Isobol) ¼ + ¾ = 1 ¾ + ¼ = 1 0 + 1 = 1 1 + 0 = 1 ½ + ½ = 1 EC50 2 EC50 1

14. www.star-radioecology.org www.radioecology-exchange.org Comparing CA and IA isobols CA generally conservative over IA Unless slopes < 1.3

15. To calculate the predicted effects to compare data against: CA:We need full DRCs to allow calculation of all single chem ECx values IA: We need the effects caused by the individual chemicals in each mixture (DRC ideal, but not needed if design is factorial) Determining if data follows the simple principles of CA or IA, lies between them, or there is overt Synergy or antagonism, is not too hard..BUT, a good guide motto for all natural philosophers is, " Seek simplicity and distrust it. " Alfred North Whitehead (1861 –1947) British mathematician who became an American philosopher Comparing data against CA and IA predictions

16. 1) CA & IA 2) S/A3A) DL or 3B) DR Add parameter “a”Add parameter “b” More detailed testing of data against CA and IA predictions Jonker, et. al. 2005, ET&C, 24:2701-2713: “MIXTOX model”

17. www.star-radioecology.org www.radioecology-exchange.org Conclusions STAR U x Cd mixture experiments across Species L. minor:antagonism identified compared to CA or IA S. salar: antagonism identified compared to IA C. elegans: strong antagonism identified compared to CA and IA additivity In most cases there is a dose ratio deviation with Antagonism generally strongest where Cd dominates toxicity.

18. www.star-radioecology.org www.radioecology-exchange.org What model if one stressor is not a chemical (and have little effect)? Example of STAR Gamma x Cd mixture with Daphnia “ Concentration Addition ” Assumptions: The chemicals have same mode of action (same biological pathway / molecular target) Only difference is the relative potency of chemicals → “dilutions of each other” Needs: Dose Response Curves (DRC) for both compounds to allow full analysis “Independent Action” Assumptions: Based on effects of chemicals being statistically independent - joint probability of effect Needs: Effects know for the individual stressors being mixed (including no effect) Gamma - not a chemical, no effects (or DRC) obtainable for some endpoints ) => IA works best (no real violation of assumptions) => CA math can work, but assumptions violated (same molecular mech. / dilution).

19. www.star-radioecology.org www.radioecology-exchange.org Conclusions STAR Gamma x Cd mixtures across Species L. minor:antagonism identified compared to IA (and CA) D. magna: antagonism identified compared to IA P. Subcapitata: antagonism identified compared to IA Conclusions STAR Gamma x FA mixtures across Species D. magna: Effects were relatively well described by IA

20. www.star-radioecology.org www.radioecology-exchange.org The mixture reference models of CA and IA applies as well to radio- ecotoxicology as any other field of ecotoxicology Both CA and IA reference models often were close to observed effects Ignoring the “joint effects” would seriously underestimate effects The deviations seen generally had the same patterns across species: U x Cd – mainly antagonism (especially where Cd dominates toxicity) Gamma x Cd – mainly antagonism at high dose levels, BUT with some general evidence of synergy at lower doses. Gamma x FA – Close to IA The deviations from the mixture models were of a level that has significance in biological terms – Possible to integrate these using DEB. Even where full effects curves could not be obtained for e.g. Gamma the IA model could still be used as a mixture reference model for joint effect Needs factorial designs Main conclusions and highlights