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DocuSign Envelope ID: D07924E6-3D94-4398-AO66-B683COBAB33E <br /> 1.2 Test Results <br /> The 25% effect concentration (IC25) and No Observed Effect Concentration (NOEC) were <br /> calculated for lethal and sub-lethal effects for the first two rounds of C. dubia chronic tests. In <br /> some cases, the IC25 for reproduction calculated by the statistical software was determined to <br /> be anomalous due to smoothing error. As appropriate, the IC25 was recalculated manually per <br /> EPA methodology (EPA 2002) and the smoothed IC25 is reported. No significant effects on <br /> survival were detected in the round one 1 g ethanol/g SO4 C. dubia test, however, significant <br /> effects were detected in the 0.5 g and 0.25 g ethanol/ g SO4 round 1 and 2 C. dubia tests, <br /> respectively (Table 1). Statistically significant effects were detected for reproduction in all <br /> treatments. In addition, the 0.25 g ethanol/g SO4 test had a percent minimum significant <br /> difference (PMSD) of 62.4%, which exceeds the upper boundary set by the EPA of 47%, <br /> indicating high variability in reproduction, however, high PMSD does not invalidate a test when <br /> toxicity is present. Control performance criteria were met in rounds one and two for all C. dubia <br /> tests. Control performance in the Round two test was slightly poorer than usual for GEI which <br /> may have led to slightly poorer performance throughout the test. <br /> The third round of C. dubia testing did not meet control performance criteria for survival (<80% <br /> survival) or reproduction (<60% of the control organisms had 3 broods in 8 days) indicating <br /> invalid test results. However, based on professional judgment, it appeared that toxicity was <br /> likely present even if control performance criteria were met based on the results of the round <br /> three P. promelas test. <br /> Table 1: Summary of chronic WET results. IC25 and NOEC below 100% indicates statistically <br /> significant effects (i.e. toxicity). Percent survival and percent effects columns <br /> summarize data from the 100% treated concentration. Higher survival equates to <br /> less toxicity; higher effects equates to more toxicity. <br /> Treatment Survival Reproduction/Growth <br /> g SO4 Ethanol/ Species o 0/a�g ) ICzs NOEC /a Survival ICzs NOEC Effects <br /> 0.5 65.7 80 0 36.4 20 99.3 <br /> 1.0 65.0 100 60 42.3 40 99.5 <br /> 0.25 43.3 40 0 48.6 40 100 <br /> 0.5 with lime pre- C. dubia <br /> N/A <100* 0 N/A <100* 100 <br /> softening <br /> 0.5 without lime N/A <100* 0 N/A <100* 100 <br /> pre-softening <br /> 0.5 N/A 100 72.5 N/A <100 49.3 <br /> 1.0 N/A 100* 75.0 N/A 100* 34.7 <br /> 0.25 N/A 100 95 N/A <100 24.7 <br /> 0.5 with lime pre- P.promelas <br /> N/A <100 45 N/A <100 75.4 <br /> softening <br /> 0.5 without lime N/A <100 0 N/A <100 100 <br /> pre-softening <br /> *Control performance criteria not met. <br /> The pass/fail requirement is based only on the hypothesis test, therefore, only hypothesis <br /> testing was used for statistical analysis of the chronic P. promelas tests (Table 1). For the first <br /> two rounds of testing, no significant effects on survival were detected for any of the treatments, <br /> GEI Consultants,Inc. 2 February 2021 <br /> Ecological Division Whole Effluent Toxicity Test Report <br /> Stantec-Elk Ridge Mining and Reclamation,LLC <br />