1 SUPPLEMENTAL DATA Table S1 Chronology of uranium chronic toxicity studies for freshwater species, which report concentration-response relationships based on ecologically relevant endpoints (e.g. growth, development, reproduction, survival). Species Reference1 Test type, toxicity measure reported Peer-reviewed studies Pre 2000 Daphnia magna 21-d survival / reproduction, NOEC Poston et al. (1984) Hydra viridissima 96-h population growth rate, NOEC Hyne et al. (1992) Melanotaenia splendida inornata 7-d survival, NOEC Holdway (1992) Mogurnda mogurnda 7-d (+ 7-d post-exposure) survival, NOEC Holdway (1992) Moinodaphnia macleayi 5-d survival, NOEC H. viridissima 96-h population growth rate, BEC10 Markich and Camilleri (1997) H. viridissima 96-h population growth rate, BEC10 Riethmuller et al. (2000) Chlorella sp. 1 72-h cell division rate, BEC10 Franklin et al. (2000) Chlorella sp. 2 72-h cell division rate, BEC10 Charles et al. (2002) Lemna gibba 21-d growth rate, NOEC M. macleayi 3 brood reproduction, NOEC Hyne et al. (1993) 2000 – present Mkandawire and Dudel (2002) Semaan et al. (2001) Catostomus commersoni 30-d growth, NOEC Liber et al. (2004a) Chlorella sp. 1 72-h cell division rate, NOEC Hogan et al. (2005) Lemna aequinoctialis 96-h population growth rate, MDEC Charles et al. (2006) D. magna 21-d growth / reproduction, NOEC / EC10 Zeman et al. (2008) Danio rerio 15-d development / growth, NOEC L. aequinoctialis 96-h population growth rate, NOEC / IC10 Hogan et al. (2010) Amerianna cumingi 96-h egg production, NOEC / IC10 Hogan et al. (2010) D. magna Multigeneration, growth/reproduction, NOEC M. mogurnda 28-d growth / survival, IC10 Cheng et al. (2010) Chlorella sp. 1 72-h cell division rate, IC50 Trenfield et al. (2011) H. viridissima 96-h population growth rate, IC50 Trenfield et al. (2011) Euglena gracilis 96-h cell division rate, IC50 Trenfield et al. (2012) Bourrachot et al. (2008) Massarin et al. (2010) Non peer-reviewed studies2 Pre 2000 Ceriodaphnia dubia 2000 – present 7-d (3 brood) reproduction, NOEC Pickett et al. (1993) 2 1 Salvelinus namaycush 141-d development / survival, NOEC Liber et al. (2004b) Selenastrum capricornutum / Pseudokirchneriella subcapitata 72-h cell division rate, IC25 / IC50 / NOEC Vizon SciTec Inc. (2004) Lemna minor 7-d growth, IC25 / IC50 Vizon SciTec Inc. (2004) C. dubia 7-d (3 brood) reproduction, IC25 / IC50 / NOEC Vizon SciTec Inc. (2004) Hyalella azteca 14-d survival / growth Vizon SciTec Inc. (2004) Oncorhynchus mykiss 30-d hatching / viability Vizon SciTec Inc. (2004) Esox lucius 65-d development / growth / survival, NOEC Liber et al. (2005) S. capricornutum / P. subcapitata 72-h cell division rate, IC25 / IC50 / NOEC Liber et al. (2007) Chlamydomonsa reinhardtii 72-h cell division rate, IC25 / IC50 / NOEC Liber et al. (2007) Cryptomonas erosa 6-d cell division rate, IC25 / IC50 / NOEC Liber et al. (2007) D. magna 21-d reproduction, IC25 / IC50 / NOEC Liber et al. (2007) C. dubia 7-d (3 brood) reproduction, IC25 / IC50 / NOEC Liber et al. (2007) Simocephalus serrulatus 21-d reproduction, IC25 / IC50 / NOEC Liber et al. (2007) Chironomus tentans 28-d survival / growth / emergence, IC25 / IC50 / NOEC Liber et al. (2007) Corynoneura spp. 28-d survival / emergence, IC25 / IC50 / NOEC Liber et al. (2007) H. azteca 28-d survival / growth, IC25 / IC50 / NOEC Liber et al. (2007) See reference list below for details. 2 Non peer-reviewed studies were included where they have been cited in the peer-reviewed literature and they did not present research subsequently published in peer-reviewed publications. References for Table S1 Bourrachot S, Simon O, Gilbin R. 2008. The effects of waterborne uranium on the hatching success, development, and survival of early life stages of zebrafish (Danio rerio). Aquat Toxicol 90:29-36. Charles A, Markich S, Stauber J, de Filippis L. 2002. The effect of water hardness on the toxicity of uranium to a tropical freshwater alga (Chlorella sp.). Aquat Toxicol 60:61-73. Charles AL, Markich, Scott J and Ralph P. 2006. Toxicity of uranium and copper individually, and in combination, to a tropical freshwater macrophyte Lemna aequinoctialis. Chemosphere 62:12241233. 3 Cheng KL, Hogan AC, Parry DL, Markich SJ, Harford AJ, van Dam RA. 2010. Uranium toxicity and speciation during chronic exposure to the tropical freshwater fish, Mogurnda mogurnda. Chemosphere 79:547-554. Franklin NM, Stauber JL, Markich SJ, Lim RP. 2000. pH-dependent toxicity of copper and uranium to a tropical freshwater alga (Chlorella sp.). Aquat Toxicol 48:275-289. Hogan AC, van Dam RA, Markich SJ, Camilleri C. 2005. Chronic toxicity of uranium to a tropical green alga (Chlorella sp.) in natural waters and the influence of dissolved organic carbon. Aquat Toxicol 75:343-353. Hogan A, van Dam RA, Houston MA, Harford AJ, Nou S. 2010. Uranium exposure to the tropical duckweed Lemna aequinoctialis and pulmonate snail Amerianna cumingi: fate and toxicity. Arch Environ Contam Toxicol 59:204-215. Holdway DA. 1992. Uranium toxicity to two species of Australian tropical fish. Sci Total Environ 125:137-158. Hyne RV, Rippon GD, Ellender G. 1992. pH-dependent uranium toxicity to freshwater hydra. Sci Total Environ 125:159-173. Hyne RV, Padovan A, Parry DL, Renaud SM. 1993. Increased fecundity of the cladoceran Moinodaphnia macleayi on a diet supplemented with a green alga, and its use in uranium toxicity tests. Aust J Mar Fresh Res 44:389-399. Liber K, Stoughton S, Rosaasen A. 2004a. Chronic uranium toxicity to White Sucker fry (Catostomus commersoni) Bull Environ Contam Toxicol 73: 1065-1071. Liber K, Stoughton S, Janz D. 2004b. Uranium toxicity to early life-stage lake trout (Salvelinus namaycush). Report for Saskatchewan Environment, Shield EcoRegion-Uranium Mining Unit, Prepared by the Toxicology Centre, University of Saskatchewan, Saskatoon, Canada. 4 Liber K, Stoughton S, Janz D. 2005. Uranium toxicity to early life-stage northern pike (Esox lucius). Report for Saskatchewan Environment, Shield EcoRegion-Uranium Mining Unit, Saskatoon, Canada. Prepared by the Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada. Liber K, deRosemond S, Budnick K. 2007. Uranium toxicity to regionally-representative algae and invertebrate species. Saskatoon, SK, submitted to AREVA Resources Inc. and Cameco Corporation, final report. Markich SJ, Camilleri C. 1997. Investigation of metal toxicity to tropical biota: Recommendations for revision of the Australian water quality guidelines. Supervising Scientist Report 127. Supervising Scientist, Department of Sustainability, Environment, Water, Population and Communities, Darwin, Australia. Pickett JB, Specht WL, Keyes JL. 1993. Acute and chronic toxicity of uranium compounds to Ceriodaphnia dubia, Rep. No. WSRC-RP-92-995. Prepared by the Department of Energy under contract DE-AC09-89SR 18035. Poston TM, Hanf RW, Simmons MA. 1984. Toxicity of uranium to Daphnia magna. Water Air Soil Poll 22:289-298. Massarin S, Alonzo F, Garcia-Sanchez L, Gilbin R, Garnier-Laplace J, Poggiale J. 2010. Effects of chronic uranium exposure on life history and physiology of Daphnia magna over three successive generations. Aquat Toxicol 99:309-319. Mkandawire M, Dudel GE. 2002. Uranium attenuation from tailing waters by floating macrophyte Lemna gibba L. In Merkel JB, Planer-Friedrich B, Wolkersdorfer C, eds, Uranium in the aquatic environment Berlin, Springer: 623-630. Riethmuller N, Markich S, Parry D, van Dam R. 2000. The effect of true water hardness and alkalinity on the toxicity of Cu and U to two tropical Australian freshwater organisms. Supervising Scientist Report 155. Supervising Scientist, Department of Sustainability, Environment, Water, Population and Communities, Darwin, Australia. 5 Semaan M, Holdway DA, Van Dam R. 2001. Comparative sensitivity of three populations of the cladoceran Moinodaphnia macleayi to acute and chronic uranium exposure. Environ Toxicol 16:365-376. Trenfield MA, Ng JC, Noller BN, Markich SJ, Van Dam RA. 2011. Dissolved organic carbon reduces uranium bioavailability and toxicity. 2. Uranium[VI] speciation and toxicity to three tropical freshwater organisms. Environ Sci Technol 45:3082-3089. Trenfield MA, Ng JC, Noller BN, Markich SJ, van Dam RA. 2012. Dissolved organic carbon reduces uranium toxicity to the unicellular eukaryote Euglena gracilis. Ecotoxicology 21:1013-1023. Vizon SciTec Inc. 2004. Final report on the toxicity investigation of uranium to aquatic organisms. CNSC Project No.: R205.1. Zeman FA, Gilbin R, Alonzo F, Lecomte-Pradines C, Garnier-Laplace J, Aliaume C. 2008. Effects of waterborne uranium on survival, growth, reproduction and physiological processes of the freshwater cladoceran Daphnia magna. Aquat Toxicol 86:370-378. 6 Table S2 Stepwise regression relationships between toxicity measures for uranium and key physico-chemical variables, for Chlorella spp., Hydra viridissima, and both species combined. Species / toxicity Predictor measure Model equation Model details (r2, P) Chlorella spp. (n = 20) IC10 DOC IC10 = 36.24 + 10.4[DOC] 0.68, <0.001 IC50 DOC IC50 = 74.10 + 21.0[DOC] 0.74, <0.001 DOC, hardness IC50 = 53.58 + 22.6[DOC] + 0.44[hardness] 0.82, <0.001 (DOC), 0.008 (hardness) Hydra viridissima (n = 14) IC10 DOC IC10 = 50.31 + 7.4[DOC] 0.48, 0.004 IC50 DOC IC50 = 103.74 + 16.4[DOC] 0.59, 0.001 IC10 DOC IC10 = 42.58 + 9.0[DOC] 0.62, <0.001 IC50 DOC IC50 = 87.35 + 18.9[DOC] 0.68, <0.001 DOC, hardness IC50 = 60.77 + (21.0[DOC]) + (0.47[hardness]) 0.79, <0.001 (DOC), <0.001 (hardness) Combined (n = 34) 7 A. B. 120 pH 5.7 pH 6.5 100 Percent of control growth rate Percent of control growth rate 120 80 60 40 20 0 0 mg/L DOC 2.6 mg/L DOC 3.4 mg/L DOC 4.1 mg/L DOC 8.1 mg/L DOC 100 80 60 40 20 0 0 50 100 150 200 250 300 0 100 Uranium ( g/L) 200 300 400 Uranium ( g/L) C. Percent of control growth rate 120 0 mg/L DOC 1.0 mg/L DOC 5.1 mg/L DOC 10.2 mg/L DOC 20.4 mg/L DOC 100 80 60 40 20 0 0 200 400 600 800 1000 1200 1400 1600 Uranium (µg/L) D. E. 120 0 mg/L DOC 1.0 mg/L DOC 4.7 mg/L DOC 9.5 mg/L DOC 100 Percent of control growth rate Percent of control growth rate 120 80 60 40 20 0 8 mg/L hardness 40 mg/L hardness 100 mg/L hardness 400 mg/L hardness 100 80 60 40 20 0 0 100 200 300 400 500 600 0 100 200 Uranium ( g/L) 300 400 500 600 700 Uranium ( g/L) Figure S1 Concentration-response plots for uranium toxicity to Chlorella sp. 1 based on data from (A) Franklin et al. [18], individual replicates plotted; (B) Hogan et al. [19], individual replicates plotted; (C) Trenfield et al. [20], Suwannee River Fulvic Acid as the DOC source; means standard error; and (D) Trenfield et al. [20], Sandy Billabong DOC as the DOC source; means standard error; and Chlorella sp. 2 based on data from (E) Charles et al. [21], means standard error. Model details are presented in Table 1. 800 8 A. B. 120 Percent of control growth rate Percent of control growth rate 120 100 80 60 40 20 Dry season Wet season 0 0 100 80 60 40 20 0 100 200 300 400 0 200 Uranium ( g/L) 400 C. 1000 1200 120 6.6 mg/L hardness 165 mg/L hardness 330 mg/L hardness 100 Percent of control growth rate Percent of control growth rate 800 D. 120 80 60 40 20 0 100 80 60 40 20 0 0 200 400 600 800 1000 1200 0 100 Uranium ( g/L) 200 300 400 500 600 Uranium ( g/L) E. F. 120 0 mg/L DOC 0.9 mg/L DOC 4.9 mg/L DOC 9.7 mg/L DOC 19.5 mg/L DOC 100 80 60 40 20 0 Percent of control growth rate 120 Percent of control growth rate 600 Uranium ( g/L) 0 mg/L DOC 0.9 mg/L DOC 4.8 mg/L DOC 9.7 mg/L DOC 100 80 60 40 20 0 0 200 400 600 Uranium (µg/L) 800 1000 0 100 200 300 400 500 600 Uranium (µg/L) Figure S2 Concentration-response plots for uranium toxicity to Hydra viridissima based on data from (A) Hyne et al. [22], means standard error for dry season, means only for wet season; (B) Markich & Camilleri [23], means standard error; (C) Riethmuller et al. [24], means standard error; (D) Riethmuller et al. [24], means standard error; data for water hardness and alkalinity of 165 mg/L and 102 mg/L (as CaCO 3), respectively; (E) Trenfield et al. [20], Suwannee River Fulvic Acid as the DOC source; means standard error; and (F) Trenfield et al. [20], Sandy Billabong DOC as the DOC source; means standard error. Model details are presented in 9 Table 2. 10 A. B. 120 Test 1 Test 2 Test 3 Test 4 100 80 Percent of control survival Percent of control reproduction 120 60 40 20 100 80 60 40 20 0 0 0 20 40 60 80 100 0 120 50 100 C. 200 D. 120 Percent of control reproduction 120 Percent of control reproduction 150 Uranium ( g/L) Uranium ( g/L) 100 80 60 40 Laboratory population (Test 1) Laboratory population (Test 2) Laboratory population (Test 3) 20 0 100 80 60 40 20 Wild population (Test 1) Wild population (Test 2) 0 0 10 20 30 Uranium ( g/L) 40 50 0 10 20 30 40 50 Uranium ( g/L) Figure S3 Concentration-response plots for uranium toxicity to Moinodaphnia macleayi based on data from (A) unpublished data from the Environmental Research Institute of the Supervising Scientist from tests undertaken in 1992, means standard error; (B) Hyne et al. [25], means standard error; survival data; (C) Semaan et al. [26], means standard error; tests using an established laboratory population; and (D) Semaan et al. [26], means standard error; tests using a wild population. Model details are presented in Table 3. 60