Experimental Planning

Animal Handling Prior to Exposure

Divided animals into separate system, 3 days prior to start of exposure, 8 days acclimated to the recirculating system.

Only 9 individuals had properly adhered systems. Will reduce the number of replicates.

Randomize Treatment

Here we will have R randomly assign each animal to a treatment.

set.seed(3349)

conditions <- c("Control R1", "Control R2", "Control R3", "5 mg/L R1", "5 mg/L R2", "5 mg/L R3", "45 mg/L R1", "45 mg/L R2", "45 mg/L R3")
animal_id <- c("S295C001", "S290C001", "S291C001", "S287C001", "S275C001", "S277C001", "S283C001", "S301C001", "S286C001")
treatment_assignments_exp3 <- data.frame(
  Conditions = sample(conditions),
  animal_ID = sample(animal_id)
)
print(treatment_assignments_exp3)

  Conditions animal_ID
1  5 mg/L R2  S275C001
2  5 mg/L R3  S295C001
3 Control R1  S277C001
4 Control R3  S283C001
5  5 mg/L R1  S290C001
6 45 mg/L R3  S291C001
7 Control R2  S286C001
8 45 mg/L R1  S287C001
9 45 mg/L R2  S301C001

#save assignments in csv for downstream use as metadata
write.csv(treatment_assignments_exp3, file = "treatment_assignments.csv")

Nickel Chloride Dilutions

Review resources prior to handling nickel chloride:

Nickel chloride SDS.
Chemical resistance of gloves.

All handling of stock solution and treatments to be done in a chemical fume hood. Once spiked water has been distributed, add animals, parafilm jars, place in secondary container, and move to environmental chamber.

Required Volume of Artificial Seawater

Calculations for spiking artificial seawater. 225 mL of seawater required to mostly submerge glass slide in wide mouth 16 oz Kerr mason jar.

Considerations:

4 replicates
225 mL per jar
3 conditions
Account for 10% loss of water during transferring

\[(4*225 * 3) + (0.1*(4*225*3)) = 2,970\]

Need 2,970 mL of artificial seawater total for exposure, particularly, 990 mL of artificial seawater per condition.

Calculations

Stock solution of NiCl₂ made by undergraduate Gina Jones on 02/08/23.

NiCl₂
Suspended in MilliQ water (0 PPT)

Low Dose: 5 mg/L

\[ C_1*V_1 = C_2*V_2\] \[ 1 g/L * V_1 = 0.005g/L*0.99L\] \[V_1 = 0.00495 L = 4.95 mL\] Spike 990 mL of aerated, artificial seawater (16 C) with 4.95 mL of stock NiCl₂ solution. Change in salinity negligible (+/- 1 PPT).

High Dose: 45 mg/L

\[ C_1*V_1 = C_2*V_2\] \[ 1 g/L * V_1 = 0.045g/L*0.99L\] \[V_1 = 0.04455 L = 44.55 mL\] Spike 990 mL of aerated, artificial seawater (16 C) with 44.55 mL of stock NiCl₂ solution. Change in salinity negligible (+/- 1 PPT).