Manchester Maintenance

These are protocols to follow for weekly ocean acidification system maintenance at the NOAA Manchester Research Station from February 15, 2017 to April 7, 2017. Weekly maintenance consisted of two days at the hatchery. Tasks below are divided between the first and second day spent at the hatchery.

First Day of Week

Preliminary System Check

• Ensure seawater is flowing to all culture tanks
• Check pH on the monitors next to the tanks
  • Low pH range should be 7.18-7.22
• Confirm there is algae in algae tank
  • Ensure pumps are on and algae is flowing to the culture tanks
• Make sure there are probes in tanks
  • HOBO loggers in all culture tanks
  • One durafet and one fat mystery probe total between all three low pH tanks
  • One Durafet and one fat mystery probe total between all ambient tanks
  • Probe in the low pH header tank
• Turn space heater off in dry lab

Figure 1. pH monitors

Water Chemistry

Prime Probes

• Get salinity and temperature probes
• Obtain 1L water from any low pH tank
• Place salinity, temperature, and pH probes in 1L of low pH water
  • Check units on the probes: salinity, ºC, and mV
• Keep probes in water until ready to take discrete measurements of grab samples

Label all sampling containers

• 1L grab samples
  • Obtain eight 1L containers from the dry lab
  • Label 1L containers: H1, H2, 1, 2, 3, 4, 5, 6
• 250 mL bottle samples
  • Obtain 16 250 mL glass bottles from the dry lab
  • Label each bottle with the tank number, replicate and date
    • ex. T1 A 2/15/17 or H2 B 3/12/17

Obtain Water Samples

• Take water samples from each culture tank and header tank
• 1L samples
  • Invert container and push all the way to the front and bottom of the tank
  • Invert container again and bring the 1L container all the way to the top, filling the container
• 250 mL samples
  • Replicate A: Invert container and push all the way to the front and bottom of the tank. Invert container again and fill completely (no bubbles escaping from container). Cap container under water
  • Replicate B: Invert container at the top back of the tank. Invert container again and fill completely (no bubbles escpaing from container). Cap container under water

Add Mercuric Chloride to Bottle Samples (This step should be completed immediately after obtaining bottle samples)

• Take 16 bottle samples to the fish pathology lab
• Remove 1 mL of water from each bottle sample
• Prepare for mercury chloride poisoning
  • Put a hazardous waste label on a new ziploc bag
  • Place a few large kim wipes inside the ziploc bag
  • Place bag in fume hood
  • Turn fan and fume hood lights on
  • Wear gloves, lab coat, and goggles
• Pipet 50 µL of mercuric chlordide into each bottle
• Cap and invert bottle to ensure thorough mixing

Take Discrete Chemistry Measurements

• Select a 1L sample of water to get discrete chemistry measurements
  • Measure low pH culture tank and header water chemistry first, then for ambient culture and header tanks
• Pour some water over the probes to prime them
• Take pH (mV), temperature (ºC), and salinity measurements for selected 1L grab samples
• Repeat for remaining grab samples
• Clean 1L containers and salinity probe with freshwater and put away in dry lab

Standard Curve

• Place pH probe back into cover to prime
• Obtain two ice packs from the -80 freezer
• Pour 35 mL Tris base into a new white Falcon Tube. Label with "Tris Base" the date
• Measure temperature (ºC) and pH (mV) of Tris base
• Briefly cool Falcon Tube with Tris base. The goal is to have the Tris base about 1ºC cooler than the temperature previously measured
• Swirl Tris base
• Measure temperature (ºC) and pH (mV) once more. Repeat cooling and measuring process until there are 5 pH + temperature combinations measured
• Ideally, temperature measurements will "bound" water temperature readings from discrete measurements
  • ex. If water temperature in the culture tanks was 10ºC, there should be about two Tris base readings with temperatures above 10ºC, one reading with a temperature of approximately 10ºC, and two readings with temperatures below 10ºC
• Cap Tris base and place in Falcon Tube holder in dry lab
• Place cap back on pH probes. Leave system on, put place on "stand by"
• Clean temperature probe with freshwater and return to where it was found

Data Sheet Management

• Find data sheet folder in Roberts Lab cabinet in the dry lab
• Follow instructions on the inside of the folder
  • Take a photo of the data sheet (ensure the data sheet has the date on it)
  • Send photo to Laura and Yaamini through text or email
  • Laura and Yaamini will update the data sheet and keep photos in their lab notebooks

Clean Filters

• Turn off water line
• Unscrew 1 micron filter housing (farthest right)
• Spray filter and housing with freshwater
• Place 1 micron filter back in housing and screw back on
• Repeat with the other filters
• Tighten all filter housing once more
• Turn water back on
• Press red buttons and release air from filters

Bleach Algal Lines

• Turn all three algae pumps off
• Remove weights and HOBO data loggers from the ends of the water lines in the culture tanks. Keep weights and HOBOs inside the culture tank
• Remove all lines from culture tanks
• Obtain bucket labeled "Bleach"
• Add 20 mL of bleach to 5L of freshwater
  • The bucket has a line marking 10 L. Fill the bucket to half of that volume
• Move algal lines and airstone to the bleach water bucket
• Set black dial to 50 and algae pumps to 150
• Ensure bleach water is flowing through lines. Prime pumps if needed to ensure bleach water is flowling
• Obtain a bucket labeled "Algae"
• Fill bucket completely with freshwater
• After the bleach water bucket is empty, move algal lines "Algae" bucket with freshwater
• Run the entire bucket of freshwater through algal lines to ensure there is no more bleachwater in the system
• After freshwater bucket is empty, turn all three algae pumps off
• Ensure only seawater is flowing through the system
• Place lines back in culture tanks
• Put weights and HOBO loggers back on lines
• Reset dosing rates on algae pumps
  • If there is algae in the algae tank, ask PSRF what to set the pumps to
  • If there is no algae in the algae tank, keep all three pumps off. Let PSRF know you turned the pumps off

Final System Check

• Ensure seawater is flowing to all culture tanks
• Check pH on the monitors next to the tanks
  • Low pH range should be 7.18-7.22
• Confirm there is algae in algae tank
• Ensure pumps are on and algae is flowing to the culture tanks
• Make sure there are probes in tanks
  • HOBO loggers in all culture tanks
  • One durafet and one mystery probe between all three low pH tanks
  • One Durafet and one mystery probe between all ambient tanks
  • Probe in the low pH header tank
• Dry lab is clean

Figure 2. A clean dry lab

Second Day of Week

Preliminary System Check

Same as above.

Water Chemistry

Same as above.

Clean Filters

• Turn off water line
• Unscrew 1 micron filter housing (farthest right)
• Spray housing with freshwater
• Obtain a new 1 micron filter
• Place new 1 micron filter back in housing and screw back on
• Repeat with the other filters, replacing all filters
• Tighten all filter housing once more
• Turn water back on
• Press red buttons and release air from filters

Bleach Algal Lines

Same as above.

Clean Oysters

Both weeks:

Spray O. lurida with freshwater

• Remove all oyster bags from one culture tank
• Spray oysters with freshwater. Check for dead (gaping) oysters

Alternate between the following methods every other week:

Siphon Feces

• Create siphon
• For each culture tank, siphon out fecal matter
• Do not let water level get below half a tank

Fully Clean Tanks with Vortexx

• Place all C. gigas into trays
• Spray oysters down thoroughly with freshwater
  • Remove anemones, sponges, polychaete worms, etc.
  • Remove mussels and crush before washing down drain
• Spray culture tanks with freshwater
• Wipe down with Vortexx solution
• Spray culture tanks again with freshwater
• Put all oysters back in culture tanks

Final System Check

Same as above.