Sublethal effects salmonid olfaction and pesticides Keith Tierney

Sublethal effects: salmonid olfaction and pesticides Keith Tierney & Amber Taylor

Water quality criteria n What data do we use to establish acceptable pesticide levels? n NOAEL n Are n the tests used adequate? E. g. salmon – what if they survive tests but have other “sublethal effects”?

n Streamliving stage: fry & parr n Can a salmon survive if it cannot smell?

Salmonid olfaction n Olfactory rosette n Olfactory nerve n Olfactory bulb

Salmonid olfaction n The rosette: n Up to 2 million cells on 1 o and 2 o lamellae n G-protein coupled receptors; Adenylyl cyclase; c. AMP activated channels Moran et al. , 1992

Salmonid olfaction n 2 classes of receptor cells: n n n Microvillar Ciliatated At least 2 classes of receptors: n n n Amino acid Bile salts Nucleotides? Moran et al. , 1992

Ag Cl electrode Salmonid olfaction n Electro- olfactogram 2 sec odorant pulse Moran et al. , 1992

Salmonid olfaction n Behaviorally-relevant odors… Predator as source amino acid L-serine Prey as source Alarm pheromone

Salmonid olfaction Predator as source amino acid L-serine dose-response 6 5 EOG (fraction of 10 -5) * * 4 3 2 1 * 10 min recovery 0 1 e-6 1 e-5 1 e-4 1 e-3 1 e-2 L-serine [M] 1 e-1 1 e+0 1 e+1

Predator as source amino acid L-serine Pesticides and olfaction n Known effects (I): IPBC - carbamate fungicide ~25, 000 kg used per annum in BC n 40% drop in EOG w/ 0. 1μg/L exposure n n n (coho parr; Jarrard et al. 2004) Water quality criteria: n 1. 9 µg/L or ~20 x NOAEL

Predator as source amino acid L-serine Pesticides and olfaction n Known effects (II): n n Simazine + Atrazine – ~18, 000 kg used in BC per annum 10% EOG drop w/ 1 μg/L Simazine, 12% Atrazine, 17% for combination n (Moore & Lower 2001) 0. 0005% LC 50 Water quality criteria: n 10 µg/L or ~10 x NOAEL

Predator as source amino acid L-serine Pesticides and olfaction n Known n Copper… a success story? n n CCA – copper is extremely toxic to olfactory epithelium 20% EOG drop w/ 1 μg/L Copper n n effects (III): (Baldwin et al. 2003) Water quality guidelines? n 2 μg/L – almost at LOAEL

Reduction in juvenile coho salmon electro-olfactogram during sublethal exposure to Glyphosate K. B. Tierney, P. S. Ross, H. E. Jarrard, K. R. Delaney, C. J. Kennedy

Glyphosate n Used Predator as source amino acid L-serine primarily by forestry sector n Active ingredient of Roundup© n >125, 000 kg used per annum in BC n Glyphosate is highly water soluble (12 g/L at 25ºC) but binds soil tightly n Not actively avoided by salmonids n (Folmar, 1976)

Predator as source amino acid L-serine Glyphosate n *Suspected* ACh. E inhibitor n n IC 50= 714 m. M (El-Demerdash et al. 2001) 96 hr LC 50 [95% CI] n n 620 [607 -638] mg/L for 50 g carp (Neškovic et al. 1996) 130 mg/L [108 -156] for 1 g rainbow trout (Johnson & Finley 1980) n 20 mg/L [17 -27] for 5 g chinook salmon (Mitchell et al. 1987) n 22 mg/L [12 -38] for 12 g coho salmon (Mitchell et al. 1987) n Dose chosen ~ ½ LC 50 10 mg/L

Glyphosate and EOG n Predator as source amino acid L-serine Dose-response 10 mg/L Glyphosate, 30 min exposure 10 s 6 5 * * 4 3 2 1 01 e-61 e-51 e-41 e-31 e-21 e-1 1 e+0 1 e+1 fractional EOG 5 1 m. V 2 min Gly. Exp. 6 EOG Pre Glyphosate EOG Post Glyphosate 4 3 2 1 0 1 e-6 1 e-5 1 e-4 [Serine] M 1 e-3 1 e-2

Glyphosate and EOG Predator as source amino acid L-serine 1. 0 Drop occurs within 5 -min EOG fractional PRE 0. 8 0. 6 0. 4 1 mg/l Glyphosate 100 mg/l Glyphosate 0. 2 0. 0 0 20 40 Time (min) 60 80

Behavior study: Predator as source amino acid L-serine making the EOG meaningful Prey as source Alarm pheromone n Does a decrease in EOG mean anything? Smell thresholds – How much of a drop in EOG is meaningful to behavior ? n Use behaviors to make EOGs more ecologically relevant n Commonly used assay: Alarm response n

Behavior study: making the EOG meaningful Prey as source Alarm pheromone n Alarm pheromone: 1. 0 EOG (V) 0. 8 0. 6 L-Serine -3 0. 4 0. 2 0. 0 0 100 200 300 Skin extract (mg/L) 400 500

Behavior study: making the EOG meaningful Prey as source Alarm pheromone n Alarm pheromone: n n ‘Schreckstoff’ – means ? Released from club cells of damaged conspecifics Causes sheltering and/or flight responses Effects heightened with predator presence and loss of vision… n How do we isolate olfaction from the other senses?

Behavior study: making the EOG meaningful 1. Number of fish swimming 4. Swimming to the bottom n Methods 5. Dashing Prey as source Alarm pheromone 2. Swim speeds of each fish 3. Net-to-gross displacement (NGDR) of quantifying alarm response: 7. Aggregation (shoal cohesion) 8. Swimming against the wall 6. Freezing 9. Line crossings 10. Sheltering (hiding) 13. Time spent motionless 12. Frequency of food strikes 16. Surfacing activity 15. Attraction to skin solution 11. Moving closer to the substrate and away from the area where the alarm substance was injected 14. Time spent in the lower, middle, and upper third of the water column 18. # Food items eaten 17. Time to capture 1 st food 19. # Individuals eating

Behavior study: preliminary results # active* Prey as source Alarm pheromone 5 (a) Control 4 3 2 1 0 (c) Alarm [44. 5 ppm] 5 4 3 2 1 0 (d) Alarm [48. 2 ppm] 1 d repeat (b) Alarm [5. 7 ppm] 0 100 200 300 400 0 Seconds (s) *moving >1 cm/s 100 200 300 400

Behavior study: making the EOG meaningful 1. Number of fish swimming n Methods 5. Dashing Prey as source Alarm pheromone we will use: 7. Aggregation (shoal cohesion) 8. Swimming against the wall 6. Freezing 13. Time spent motionless

Behavior study: in the dark… almost Prey as source Alarm pheromone n 800 nm IR light and IR-sensitive surveillance cameras on an array of 12 tanks n Experimental groups: Alarm pheromone alone Pesticide alone Alarm pheromone & pesticide Control 1. 2. 3. 4. Hypothesis: group 3 is no different from 2 and 4

Behavior study: Prey as source Alarm pheromone n In addition to ‘behavioral stress’… we measure ‘physiological stress’ response n Cortisol n n Lactate, Glucose, Na+ K+ n n osmotic balance and energy metabolism E. g. increase Lac, decrease gluc. Hematocrit, Leucocrit

Predator as source amino acid L-serine Pesticides, a reminder: Prey as source Alarm pheromone n Known effects (I): IPBC - carbamate fungicide ~25, 000 kg used per annum in BC n 40% drop in EOG w/ 0. 1μg/L exposure n n (coho parr; Jarrard et al. 2004) Water quality criteria: n 1. 9 µg/L or ~20 x NOAEL n 96 hr LC 50 = 95 μg/L coho smolts n

Behavior study: Prey as source Alarm pheromone n Results… we don’t know yet! n What we expect to learn: n IPBC concentration required to eliminate alarm response… n What levels are safe for the environment?

end n Questions? n Future 498/499 students? Email ktierney@sfu. ca n Drop by SSB 6125 n Phone (604) 291 -5634 n