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.

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", 
                "0.1 mg/L R1", 
                "0.1 mg/L R2", 
                "0.1 mg/L R3", 
                "1 mg/L R1", 
                "1 mg/L R2", 
                "1 mg/L R3",
                "10 mg/L R1", 
                "10 mg/L R2", 
                "10 mg/L R3", 
                "100 mg/L R1", 
                "100 mg/L R2", 
                "100 mg/L R3", 
                "1000 mg/L R1", 
                "1000 mg/L R2", 
                "1000 mg/L R3")
animal_id <- c("DM012024_C01", 
               "DM012024_C02", 
               "DM012024_C04", 
               "DM012024_C02*", 
               "DM012024_C07", 
               "DM012024_C15", 
               "DM012024_C16", 
               "DM012024_C06",
               "DM012024_C13",
               "DM012024_C10", 
               "DM012024_C13*", 
               "DM012024_C09", 
               "DM012024_C19*", 
               "DM012024_C20", 
               "DM012024_C08", 
               "DM012024_C21", 
               "DM012024_C22",
               "DM012024_C23")

treatment_assignments <- data.frame(
  Conditions = sample(conditions),
  animal_ID = sample(animal_id)
)
print(treatment_assignments)

     Conditions     animal_ID
1   0.1 mg/L R2 DM012024_C19*
2     1 mg/L R3  DM012024_C22
3    Control R3  DM012024_C20
4    10 mg/L R3  DM012024_C08
5   100 mg/L R3  DM012024_C23
6    10 mg/L R1  DM012024_C04
7    10 mg/L R2  DM012024_C07
8    Control R1  DM012024_C13
9  1000 mg/L R3  DM012024_C10
10  100 mg/L R2  DM012024_C06
11  0.1 mg/L R3  DM012024_C21
12    1 mg/L R2  DM012024_C15
13    1 mg/L R1  DM012024_C02
14   Control R2 DM012024_C02*
15 1000 mg/L R1  DM012024_C16
16  100 mg/L R1  DM012024_C09
17  0.1 mg/L R1  DM012024_C01
18 1000 mg/L R2 DM012024_C13*

#save assignments in csv for downstream use as metadata
write.csv(treatment_assignments, file = "treatment_assignments_LC50_E1.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:

3 replicates
225 mL per jar
6 conditions
Account for 10% loss of water during transferring
Additional 15 mL required for analytical chemistry

\[(3 * 225 * 6) + (15*6) + (0.1*(3*225*6)) + (0.1*(15*6)) = 4,554\]

Need 4,419 mL of artificial seawater total for exposure, particularly, 759 mL of artificial seawater per condition.

Calculations

Stock solution of NiCl₂ (10 g/L) made 04/08/24.

NiCl₂ (5g)
Suspended in MilliQ water (500 mL)

0.1 mg/L

\[ C_1*V_1 = C_2*V_2\] \[ 10 g/L * V_1 = 0.0001g/L*0.759L\] \[V_1 = 0.00000759 L = 7.59 uL\] Spike 759 mL of aerated, artificial seawater (16 C) with 7.59 uL of stock NiCl₂ solution. Change in salinity negligible (+/- 1 PPT).

1 mg/L

\[ C_1*V_1 = C_2*V_2\] \[ 10 g/L * V_1 = 0.001g/L*0.759L\] \[V_1 = 0.0000759 L = 75.9 uL\]

Spike 759 mL of aerated, artificial seawater (16 C) with 75.9 uL of stock NiCl₂ solution. Change in salinity negligible (+/- 1 PPT).

10 mg/L

\[ C_1*V_1 = C_2*V_2\] \[ 10 g/L * V_1 = 0.01g/L*0.759L\] \[V_1 = 0.000759 L = 759 uL\]

Spike 759 mL of aerated, artificial seawater (16 C) with 759 uL of stock NiCl₂ solution. Change in salinity negligible (+/- 1 PPT).

100 mg/L

\[ C_1*V_1 = C_2*V_2\] \[ 10 g/L * V_1 = 0.1g/L*0.759L\] \[V_1 = 0.00759 L = 7.59 mL\]

Spike 759 mL of aerated, artificial seawater (16 C) with 7.59 mL of stock NiCl₂ solution. Change in salinity negligible (+/- 1 PPT).

1000 mg/L

\[ C_1*V_1 = C_2*V_2\] \[ 10 g/L * V_1 = 1g/L*0.759L\] \[V_1 = 0.0759 L = 75.9 mL\]

Spike 759 mL of aerated, artificial seawater (16 C) with 75.9 mL of stock NiCl₂ solution. Change in salinity negligible (+/- 1 PPT).