Electronic Supplementary Figure 4. Contour Plots Illustrating the Probability of Induction

Electronic Supplementary Table 1. Experimental conditions and results of sedative effectiveness testing. Values are reported as means ± SD; SD less than reporting conventions are reported as zero. Dashes indicate that data were not collected or available. Abbreviations are as follows: BCF: blue catfish, BFTC: Bozeman Fish Technology Center, BNT: brown trout, CCF: channel catfish, , CFAAS: Center for Fisheries, Aquaculture, and Aquatic Sciences, CMC: common carp, CTT: cutthroat trout, ET80: effective time at which 80% of fish were sedated, FHM: fathead minnow, HSB: hybrid striped bass, LKT: lake trout, NA: data not available, RBT: rainbow trout, RFCRF: Rathbun Fish Culture Research Facility, TIL: Nile tilapia, UMESC: Upper Midwest Environmental Sciences Center, WAE: walleye, YEP: yellow perch. Temperature Length (cm) / Sedative / Replicate Sets / Study Location Taxon (°C) / Dissolved Induction (min) Recovery (min) Weight (g) Dose (mg/L) Fish per Set Oxygen (mg/L) 22.5 ± 1.5 / 128 ± 13.6 ± 0.1 / 8.4 ± BFTC RBT eugenol / 25 2 / 15 1.7 ± 0.2 4.0 ± 0.9 25 0.2 MS-222 / 80 2 / 15 1.3 ± 0.2 1.7 ± 0.5

17.5 ± 2.0 / 63 ± 15.0 ± 0.1 / 8.8 ± BFTC RBT benzocaine / 40 2 / 15 1.4 ± 0.2 1.7 ± 0.5 20 0.0 MS-222 / 80 2 / 15 1.1 ± 0.1 1.5 ± 0.4

17.0 ± 2.5 / 55 ± 10.4 ± 0.1 / 9.2 ± BFTC RBT eugenol / 25 1 / 30 1.8 ± 0.4 4.7 ± 1.2 20 0.6 eugenol / 75 1 / 30 0.8 ± 0.1 5.3 ± 0.9

16.4 ± 0.0 / 8.15 ± eugenol / 25 1 / 30 1.3 ± 0.4 3.6 ± 0.8 0.0 eugenol / 75 1 / 30 0.6 ± 0.1 5.3 ± 0.8

40.0 ± 3.0 / 896 ± 10.7 ± 0.2 / 9.6 ± BFTC RBT eugenol / 25 1 / 30 1.7 ± 0.4 6.4 ± 1.9 212 0.2 eugenol / 75 1 / 30 0.8 ± 0.1 6.7 ± 1.4

16.3 ± 0.2 / 7.4 ± eugenol / 25 1 / 30 1.3 ± 0.2 4.8 ± 1.4 0.2 eugenol / 75 1 / 30 0.7 ± 0.1 6.5 ± 2.0

14.0 ± 0.2 / 8.7 ± BFTC CTT 26.5 ± 3.0 / NA eugenol / 25 2 / 15 2.1 ± 0.4 11.1 ± 3.7 0.4 benzocaine / 40 2 / 15 2.1 ± 0.5 8.8 ± 3.2 MS-222 / 80 2 / 15 1.8 ± 0.4 7.1 ± 2.2

32.5 ± 2.5 / 437 ± 24.8 ± 0.4 / 7.5 ± CFAAS HSB eugenol / 60 2 / 15 0.9 ± 0.2 7.5 ± 1.0 104 0.2 benzocaine / 150 2 / 15 1.0 ± 0.2 5.5 ± 1.4 MS-222 / 150 2 / 15 1.4 ± 0.2 5.8 ± 1.7

38.0 ± 2.0 / 716 ± 24.3 ± 0.1 / 8.0 ± CFAAS HSB eugenol / 60 2 / 15 0.9 ± 0.2 5.2 ± 1.1 114 0.2 benzocaine / 150 2 / 15 0.9 ± 0.2 4.7 ± 2.5 MS-222 / 150 2 / 15 1.5 ± 0.3 4.4 ± 1.4

36.5 ± 1.5 / 715 ± 17.2 ± 0.3 / 9.6 ± CFAAS HSB eugenol / 48 1 / 10 1.5 ± 0.2 5.7 ± 1.0 113 0.3 eugenol / 60 1 / 10 1.7 ± 0.2 6.5 ± 0.9 eugenol / 72 1 / 10 1.48 ± 0.10 11.68 ± 4.85 Electronic Supplementary Table 1 (continued). Temperature Length (cm) / Sedative / Replicate Sets / Study Location Taxon (°C) / Dissolved Induction (min) Recovery (min) Weight (g) Dose (mg/L) Fish per Set Oxygen (mg/L) 36.5 ± 1.5 / 715 ± 22.1 ± 0.2 / 9.2 ± CFAAS HSB eugenol / 48 1 / 10 0.9 ± 0.0 7.0 ± 1.1 113 0.1 eugenol / 72 1 / 10 0.9 ± 0.2 6.0 ± 1.5

23.5 ± 1.0 / 161 ± 16.1 ± 0.1 / 9.8 ± CFAAS HSB eugenol / 48 1 / 10 1.8 ± 0.4 13.4 ± 3.4 25 0.0 eugenol / 60 1 / 10 1.7 ± 0.2 12.9 ± 2.0 eugenol / 72 1 / 10 1.8 ± 0.2 14.4 ± 1.8

22.1 ± 0.1 / 8.5 ± eugenol / 48 1 / 10 0.8 ± 0.1 4.4 ± 1.2 0.1 eugenol / 60 1 / 10 1.0 ± 0.2 5.2 ± 0.9 eugenol / 72 1 / 10 1.0 ± 0.1 4.8 ± 1.0

36.5 ± 1.5 / 712 ± 16.6 ± 0.3 / 9.7 ± CFAAS HSB benzocaine / 120 1 / 10 1.6 ± 0.1 9.6 ± 1.9 87 0.2 benzocaine / 150 1 / 10 1.3 ± 0.1 8.0 ± 1.4 benzocaine / 180 1 / 10 1.3 ± 0.1 8.7 ± 0.6

24.7 ± 0.2 / 7.6 ± benzocaine / 120 1 / 10 1.2 ± 0.3 6.3 ± 1.4 0.2 benzocaine / 180 1 / 10 1.0 ± 0.1 4.9 ± 0.8

28.0 ± 3.5 / 134 ± 22.8 ± 0.9 / 8.7 ± CFAAS CCF eugenol / 60 4 / 10 1.4 ± 0.2 NA 63 0.3 benzocaine / 150 4 / 10 1.4 ± 0.2 NA

20.5 ± 2.5 / 60 ± 24.4 ± 0.2 / 7.5 ± CFAAS BCF eugenol / 60 2 / 15 1.6 ± 0.3 7.7 ± 1.2 24 0.2 benzocaine / 150 2 / 15 1.6 ± 0.4 6.9 ± 1.2 MS-222 / 150 2 / 15 1.6 ± 0.3 5.3 ± 0.9

22.0 ± 2.5 / 77 ± 24.0 ± 0.2 / 7.6 ± CFAAS BCF eugenol / 60 2 / 15 1.4 ± 0.4 5.4 ± 1.8 34 0.2 benzocaine / 150 2 / 15 1.6 ± 0.9 6.2 ± 1.2 MS-222 / 150 2 / 15 1.8 ± 0.6 3.8 ± 1.0

25.7 ± 0.6 / 7.7 ± CFAAS TIL 11.0 ± 1.0 / 25 ± 6 eugenol / 60 2 / 15 1.8 ± 0.6 3.8 ± 1.0 0.1 benzocaine / 150 2 / 15 1.1 ± 0.4 3.0 ± 1.5 MS-222 / 150 2 / 15 2.3 ± 0.6 3.1 ± 0.7

Electronic Supplementary Table 1 (continued). Study Length (cm) / Temperature (°C) / Sedative / Replicate Sets / Induction Recovery Taxon Location Weight (g) Dissolved Oxygen (mg/L) Dose (mg/L) Fish per Set (min) (min) UMESC BNT 31.0 ± 2.5 / 320 ± 73 12.9 ± 0.7 / 9.4 ± 0.8 eugenol / 25 2 / 15 1.8 ± 0.3 7.1 ± 1.8 benzocaine / 40 2 / 15 2.1 ± 0.3 6.0 ± 1.7 MS-222 / 80 2 / 15 1.5 ± 0.3 4.6 ± 1.8

UMESC WAE 13.5 ± 1.0 / 20 ± 6 18.2 ± 0.2 / 9.4 ± 0.1 eugenol / 40 2 / 15 2.0 ± 0.4 10.2 ± 2.1 benzocaine / 80 2 / 15 2.0 ± 0.4 10.1 ± 2.8 MS-222 / 80 2 / 15 2.6 ± 0.6 6.7 ± 1.3

UMESC YEP 10.5 ± 1.0 / 11 ± 3 18.0 ± 0.0 / 9.8 ± 0.1 eugenol / 40 2 / 15 3.0 ± 0.9 9.7 ± 2.5 benzocaine / 80 2 / 15 2.1 ± 0.5 7.9 ± 2.1 MS-222 / 80 2 / 15 3.5 ± 0.6 6.9 ± 2.1

UMESC YEP 7.5 ± 1.0 / 4 ± 2 18.1 ± 0.2 / 9.7 ± 0.1 eugenol / 40 1 / 10 2.0 ± 0.4 8.1 ± 2.3 eugenol / 60 1 / 10 1.7 ± 0.4 8.6 ± 0.9 eugenol / 80 1 / 10 1.6 ± 0.2 10.1 ± 0.8

UMESC YEP 7.5 ± 1.0 / 4 ± 2 18.4 ± 0.1 / 9.6 ± 0.1 benzocaine / 80 1 / 10 1.9 ± 0.4 7.3 ± 1.3 benzocaine / 100 1 / 10 1.4 ± 0.3 6.0 ± 1.2 benzocaine / 120 1 / 10 1.1 ± 0.2 6.7 ± 1.6

UMESC CMC 7.5 ± 1.0 / 7 ± 4 18.5 ± 0.1 / 8.6 ± 0.1 eugenol / 40 1 / 10 1.8 ± 0.3 5.9 ± 0.8 benzocaine / 80 1 / 10 1.7 ± 0.4 5.9 ± 1.6 MS-222 / 80 1 / 10 1.9 ± 0.3 4.7 ± 1.1

UMESC FHM 7.0 ± 0.5 / 4 ± 1 18.5 ± 0.1 / 8.7 ± 0.1 eugenol / 40 1 / 10 1.4 ± 0.4 6.0 ± 1.1 benzocaine / 80 1 / 10 1.2 ± 0.2 5.1 ± 1.6 MS-222 / 80 1 / 10 1.0 ± 0.2 5.3 ± 1.0

UMESC LKT 12.0 ± 2.0 / 15 ± 7 13.6 ± 0.2 / 10.0 ± 0.2 eugenol / 25 4 / 10 3.2 ± 0.4 NA benzocaine / 40 4 / 10 3.0 ± 0.5 NA

RFCRF WAE 22.5 ± 1.5 / NA 20.9 ± 0.1 / 10.2 ± 0.1 eugenol / 60 4 / 10 0.9 ± 0.2 1.9 ± 0.6 benzocaine / 150 4 / 10 0.7 ± 0.1 1.5 ± 0.2 MS-222 / 150 4 / 10 0.9 ± 0.1 1.2 ± 0.3 1 Mean of samples collected from baths prior to use and after exposures were complete; each sample was analyzed in duplicate. Electronic Supplementary Table 2. Source water quality by taxon and study location. Abbreviations are as follows: BCF: blue catfish, BFTC: Bozeman Fish Technology Center, BNT: brown trout, CCF: channel catfish, , CFAAS: Center for Fisheries,

Aquaculture, and Aquatic Sciences, CMC: common carp, CTT: cutthroat trout, ET80: effective time at which 80% of fish were sedated, FHM: fathead minnow, HSB: hybrid striped bass, LKT: lake trout, NA: data not available, RBT: rainbow trout, RFH: Rathbun Fish Hatchery, TIL: Nile tilapia, UMESC: Upper Midwest Environmental Sciences Center, WAE: walleye, YEP: yellow perch. Study Alkalinity Hardness Ammonia Nitrite Nitrate Taxon pH Location (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) BFTC RBT 8.1 169 244 NA NA NA CTT 8.0 168 212 NA NA NA

CFAAS HSB 7.9-8.8 54-124 54-138 0.2-0.9 0.0-0.2 9.5-13.3 BCF 7.9-8.6 124-141 138-142 <0.1-0.2 <0.1-0.1 5.6-13.4 TIL 8.9 599 58 0.1 <0.1 12.2 CCF 8.6 112 55.2 0.0 0.1 5.6

UMESC BNT 8.0 125 178 NA NA NA WAE 7.8 132 176 NA NA NA YEP 8.0-8.1 127-132 170-176 NA NA NA CMC 8.1 127 170 NA NA NA FHM 8.1 127 170 NA NA NA LKT 7.8 124 180 NA NA NA

RFCRF WAE 9.9 64 97.2 NA NA NA Electronic Supplementary Table 3. Experimental conditions and results of factorial experiment to assess the effects of temperature and sedative dose. Values are reported as means ± SD. Nominal Temperature (°C) / Dose Length (cm) / Induction Recovery Temperature (°C) Dissolved Oxygen (mg/L)1 (mg/L) Weight (g) (min) (min) 10 9.8 ± 0.2 / 11.0 ± 0.1 20 14.7 ± 1.0 / 32.8 ± 6.8 2.5 ± 0.5 5.8 ± 1.1 40 1.7 ± 0.3 7.8 ± 1.8 60 1.7 ± 0.1 10.1 ± 2.6 80 1.7 ± 0.2 11.0 ± 2.2 100 1.5 ± 0.2 13.3 ± 3.2 120 1.4 ± 0.2 15.4 ± 3.0

18 17.8 ± 0.3 / 9.0 ± 0.2 20 13.7 ± 1.0 / 30.9 ± 7.2 3.9 ± 0.8 5.4 ± 2.2 40 2.0 ± 0.5 6.7 ± 2.3 60 1.6 ± 0.3 8.0 ± 4.0 80 1.5 ± 0.2 8.6 ± 2.1 100 1.5 ± 0.8 9.2 ± 4.0 120 1.3 ± 0.2 9.9 ± 2.8

24 24.2 ± 0.5 / 7.0 ± 0.7 20 13.7 ± 0.9 / 31.3 ± 7.2 2.4 ± 0.6 2.6 ± 1.0 40 1.0 ± 0.3 2.8 ± 1.6 60 1.0 ± 0.3 3.5 ± 1.7 80 1.0 ± 0.1 3.3 ± 0.8 100 0.8 ± 0.1 4.7 ± 1.9 120 0.8 ± 0.0 3.5 ± 0.8

28 27.1 ± 0.3 / 7.3 ± 0.4 20 14.7 ± 1.1 / 35.9 ± 9.1 5.2 ± 0.8 4.4 ± 0.8 40 1.5 ± 0.3 3.7 ± 1.2 60 1.1 ± 0.1 3.8 ± 1.6 80 1.0 ± 0.1 5.2 ± 1.7 100 0.8 ± 0.1 5.1 ± 2.0 120 0.6 ± 0.1 4.4 ± 1.1 1 Values reflect exposure baths only. Electronic Supplementary Figure 4. Contour plots illustrating the probability of induction occurring within 2 min (A), recovery occurring within 5 min (B), and both events occurring (C) among hybrid Striped Bass (HSB) sedated with different concentrations of eugenol at different water temperatures. P-values for factors exerting significant effects on event probabilities are provided. 28 Probability of 0.2 induction Probability within 2 min 0.4 Legend 26 0.6 0.8 0.0 0.2 24 0.4 0.6 0.8 22 1.0 0.4 0.6 0.8 0.2 20

T 0.4 0.6 0.8 e m 16 0.2 p e r a t u 14 r e

( ° C

12 0.6 0.4 Dose P-value < 0.001 0.2 Temperature P-value < 0.001 A 10 20 40 60 80 100 120 Dose (mg eugenol/L)

Electronic Supplementary Figure 4A. 28 0.6 Probability of recovery 0.8 within 2 min 26 0.8 0.0 0.8 0.2 24 0.4 0.8 0.8 0.6 0.8 0.8 0.8 0.6 1.0 22 0.6 0.6 0.6 0.4 0.4 20 0.4

0.4 0.2 0.2 18 0.2 T e Probability m 16

p Legend e r a 0.2 t u 14 r e Electronic Supplementary Figure 4B.

( ° C

) 12 Dose P-value = 0.006 Temperature P-value < 0.001 B Dissolved Oxygen P-value < 0.001 10 20 40 60 80 100 120 Dose (mg eugenol/L) 28 Probability of 0.6 induction 0.2 0.8 within 2 min 26 0.4 and recovery 0.8 0.8 within 5 min 0.8 0.0 24 0.2 0.8 0.8 0.8 0.4 0.6 0.6 22 0.6 0.6 0.8 0.4 0.6 1.0 0.4 0.4 20 0.2 0.4 0.2 0.2 0.2 18 T e

m 16 Probability p e Legend r a t u 14 r Electronic Supplementary Figure 4C. e

( ° C )

12 Dose P-value < 0.001 C Temperature P-value < 0.001 10 20 40 60 80 100 120 Dose (mg eugenol/L)