Chapter 4: SOPs for Monitoring Net Carbonate Accretion ...file.iocwestpac.org/OA/SOP/Chapter 4/Chapter 4... · 3. Attach CAU to stake using a wire rope clip, tightened down with nut

Chapter 4: SOPs for Monitoring Net Carbonate

Accretion -- Using Calcification Accretion Units (CAU)

Introduction Corals and crustose coralline algae are principal components of the carbonate budget in shallow tropical reef ecosystems as they contribute to approximately 50-55 % of the carbonates present in the systems. These calcifying organisms often contribute to the buildup and maintenance of reef structures because they usually immediately recruit after disturbances. With the trends of chemical changes associated with ocean acidification such as the reduction of pH, aragonite and calcite saturation states, these marine organisms can be extremely vulnerable as the seawater carbonate parameters are approaching the levels that may not support biogenic calcification but instead drive net dissolution of marine carbonate structures.

Rates of net calcium carbonate accretion representing the amount of calcium carbonate deposited on a coral reef in a given period of time can be monitored using calcification accretion units (CAUs) which allow for recruitment and colonization of hard corals and crustose coralline algae. Total net accretion on coral reefs can be calculated by measuring the change in weight of CAUs deployed on the reef for periods of 2-3 years. By monitoring net accretion on coral reefs over a long period of time, the changes in calcification rates can be detected.

Part I: CAU Assembly Equipment

QTY (per CAU) Description 2 10.16cm x 10.16cm x 0.635cm (4” x 4” ¼”) polyvinyl chloride (PVC)

plates 3 Hex jam nut, 1/4” -20, Stainless Steel (SS), regular 2 Washers, flat, 1/4”, SS 3 Nylon spacers (½” tall, ¼” ID, ½” OD) 1 CAU Serial Number ID, leg band for pigeon 1 Lock washer, 1/4”, SS 1 Stud, full thread, 1/4” -20 x 6”, SS 1 3/8”, T316 SS All Thread (cut into 30.48cm [12”] length stainless

steel stakes and ground to a pencil tip on one side for reef penetration)

1 Wire rope clip, 3/8”, 11/16” with nuts, SS T316 *Note: see unit conversion chart on the last page for inches to millimeter conversion.

Assembly Procedures Each CAU assembly comprises of two PVC plates separated by a nylon spacer and mounted on a full thread Stud. The CAU assembly diagram in Figure 4. 1 outlines the buildout for 1 CAU, and provides multiple views of the assembled unit. Each PVC plate was sanded to provide a non-glossy surface suitable for permanent attachment and settlement of marine propagules.

Figure 4.1 CAU assembly diagram.

Part II: CAU Deployment Equipment

QTY Description 1-2 (per survey site)

Aquamend (118.29ml [4 oz.], 17.78cm [7”] stick to aid in securing stainless steel stake into reef:

1 tube - Loctite thread locker 1 tube - Aqualube saltwater grease to ease removing nuts after

long-term deployments (http://www.aqualubeky.com/grease/) 1 Heavy duty dive bag, Underwater Kinetics 2 Sledge hammer, 1.81kg (4 lbs)

2-3 Nut driver with 11/16” socket attached 5 CAUs (and 5 BMUs if applicable)

5-6 stainless steel stakes

2 sledge hammers 5 wire rope clips (10 if a l so deploying BMUs) 2 nut drivers 1 Aquamend epoxy stick (2 sticks if also deploying BMUs)

Deployment Procedures

The 5 CAUs, and tools needed for their deployment, should be carried in a heavy duty dive bag to avoid loss of gear and tools while underwater. CAUs are deployed at 5 m spacing, along the perimeter of the 15 m survey site. A team of 2-3 SCUBA divers install 5 CAUs (and 5 BMUs*) in 30-40 min.

The order of deployment for CAUs is: 1. Prepare log sheets with CAU serial numbers, location, depth, and other relevant notes prior to diving.

2. Drive stainless steel stakes into reef at appropriate intervals. Stakes must be vertical, so that when the CAU is attached that the plates of the unit are parallel to the sea surface, creating a flat surface for future marine organism recruitment.

3. Attach CAU to stake using a wire rope clip, tightened down with nut driver (effort should be made to deploy CAU plates parallel to the surface).

4. Apply 1/5 of the stick of Aquamend epoxy to aid in holding the stake to the reef (once the 2-part Aquamend is mixed, it is pliable for approx. 30 min). Figure 4.2 shows CAU installed into hard reef substrate.

*See Chapter 5 for BMU Deployment Procedures

Figure 4.2 CAU installed into hard reef substrate.

Part III: CAU Recovery/Exchange

Recovery/Exchange Procedures

CAUs are deployed on stakes secured in the reef by epoxy for long-term monitoring studies. By loosening the wire rope clips, CAUs can be removed from the stake, leaving the stake in place for use again. CAUs are individually bagged by SCUBA divers in a plastic bag (to ensure no loss of material during transportation to the laboratory from the field) and returned to the lab for processing. The new CAU can be reinstalled on the stake with a wire rope clip. Adding more epoxy may also make the stake deployment more secure.

Part IV: Laboratory Sample processing (Misa PL and Richards CR- revised July 2016) Equipment

Item Brand and Model Specs

Drying Oven Heratherm Advanced

Gravity convection; 176 L, 6.2

Protocol Oven cu ft; (OGH180, 220-240v, Interior (WxHxD) = 43.69 x 60Hz) 68.07 x 58.83cm (17.2” x

or

26.8” x 23.2”) Exterior (WxHxD) = 64.01 x

91.95 x 73.91cm (25.2” x Heratherm General 36.2” x 29.1”) Protocol Oven (OGS 180, 120v, 60Hz) Shelves: 2 supplied, 19 max

Digital SLR Camera Nikon D7000 N/A 60mm micro lens Nikon AF-S Micro

NIKKOR 60mm f/2.8 G ED

N/A

Fume hood N/A benchtop, ducted laboratory hood

Wire Mesh Shelves

Heratherm Wire Mesh Shelf (Part No.50127763)

Includes 2 shelf support Max carrying capacity/shelf: 24.95kg (55 lbs)

Copy Stand Bencher Copy Mate II Halogen Copystand 900- 30SC

Overall Dimensions: 50.8cmW x 48.26cmD x 8.89cmH (20"W x 19"D x 3.5"H) Includes two (2) adjustable halogen copylights and two (2) fixed copylight arms: Light Output: 300W each, 600W total Bulb: Ushio #1000896 - JCD120V-300WC Analytical Balance Mettler-Toledo XA

Precision Balance (XA503S)

N/A

Vacuum Filtration Vacuubrand Oil-free Max pumping speed 0.4/0.5 Item Brand and Model Specs

Pump Diaphragm Pump (ME1) 50/60 Hz

cfm

Lab-grade rubber tubing

Tygon E-3603 (ACF00029)

Interior Diameter = 9.5mm (3/8”) Outer Diameter = 15.9mm

(5/8”) Wall Thickness = 3.2mm (1/8”)

Filter flask Pyrex 4000 ml Buchner funnel Buchner Funnel 110 mm,

glazed porcelain (Avogrado’s Lab Supply, Item # 33065)

Glazed porcelain Inside Diameter = 110 mm Height = 172 mm Bowl depth = 44 mm Volume = 456 ml

Rubber filter aid Rubber filter aid (Avogrado’s Lab Supply, Item # FLTR01)

Inside Diameter = 32mm (1.2”) Outside Diamter = 76mm (3”)

Squeeze bottles N/A N/A Tubing connectors Tapered Tubing

Connector 4.76mm to 7.94mm (3/16” to 5/16”) (Avogrado’s Lab Supply,Item # KT511)

Unequal 4.76mm to 7.94mm and 6.35mm to 12.7mm (3/16” to 5/16” and ¼” to ½”)

Buchner funnel mesh discs

Buchner Funnel Discs 90 mm 5/pk (Avogrado’s Lab Supply, Item # 242845-90)

Diameter = 90 mm (3.54”) Mesh size = 900 x 700 um * for use with 110 mm Buchner funnel

Filter paper Whatman Qualitative Grade 1 Filterpaper Mfr #1001-110

11 cm diameter Pore size: 11 um

Razor scraper Titan 12033 Razor blade refills Garvey (40475) 0.51mm (.02”) thickness

127 x 50.8 x 25.4mm (5” x 2” x 1”)

Glass dishes Bormioli Rocco Frigoverre (25 ½ oz) Model # 387870MB2321990

15.24 x 15.24 x 6.35cm (6” x 6” x 2.5”) Capacity = 0.74L (25.5oz)

Resealable plastic bags

Ziploc Freezer Bags - Quart size

17.78cm x 19.05cm (7” x 7.75”); Clear; 1.75 ml

Index cards

Pendaflex Oxford Index Cards, White, Blank 12.7 x 20.32cm (5” x 8”)

12.7 x 20.32 x 2.032cm (5” x 8” x 0.8”), white, acid free

Item Brand and Model Specs

Hydrochloric Acid

EMD Millipore Hydrochloric Acid (No. HX0603-75); 2.5L

Molar Mass: 36.46; Assay (HCl) approx. 36-38%

Glass bottles for 5% HCl storage

EMD Millipore Storage Jugs; 2.5 L

2.5 liter; narrow mouth with polypropylene caps

PPE N/A Lab Coat Chemical-resistant Disposable gloves Oven mitt Safety Glasses Closed-toe shoes

Pencil N/A N/A Containers for filter

N/A N/A

Tape N/A N/A 2 wrenches N/A N/A Rinse basins for plates

N/A N/A

Mortar and

N/A N/A Acid-resistant gloves

N/A N/A

Plastic mesh layer (with attached pull

N/A N/A

Spatula/teaspo

N/A N/A Drying rack for filter

N/A N/A

Preparation 1. Assign a unique ID to both plates of each Calcification Accretion Unit (CAU) using the CAU Plate ID naming convention SiteID_RecoveryYear_4-digitSerial#_PlateLocation (“U” - upper plate, “L” - lower plate)

e.g. PAL01_2012_5319_U PAL01_2012_5319_L

2. Using a pencil, pre-label the following with the CAU Plate ID (e.g. PAL01_2012_5319_U):

a) Paper tray A - blank index card 12.7 x 20.32cm (5” x 8”) folded into 12.7 x 12.7cm (5” x 5”) paper tray

b) Paper tray B - same as above

c) Filterpaper 11cm diameter, #1, >11μm particle retention (e.g. Whatman No.1001-110)

3. Insert the corresponding Filter paper into the fold of Paper tray B and store in a container. Place Paper tray A in a separate container. Store both containers away from moisture and direct sunlight. See I. Step 8 for pre- weighing instructions.

4. Print Glass Dish Labels on waterproof paper using the template in Section VII.B.1. Attach each label on one side of the glass dish with tape.

5. Print the Plate Photo ID tags on waterproof paper using the template in Section VII.B.2.

There are 4 tags, one for each surface of the CAU to be photographed: Upper Plate, Top Side Upper Plate, Bottom Side Lower Plate, Top Side Lower Plate, Bottom Side

An example of a plate photo ID: CAU_PAL01_2012_5319_U_T

6. Pre-label re-sealable plastic bags 0.95L (1 quart) with the CAU Plate ID. The filter residue will be stored in this bag for archiving.

7. Using the lab data spreadsheet, CAUTemplate_LabDataSpreadsheet.xlsx (or the CAU Data Entry tool for ESD-CREP users, see Section VII.C p. 41), enter the CAU Site IDs (e.g. PAL01) in Column A and the 4-digit CAU Serial #s (e.g. 5319) in Column B.

8. Using an analytical balance, pre-weigh the following and record the value (up to 3 decimal points) in CAUTemplate_LabDataSpreadsheet.xlsx (or the CAU Data Entry tool for ESD-CREP users, see Section VII.C p. 41):

a) Paper tray A - record the value in Column H (‘Paper Tray A Weight [g]’) b) Paper tray B + Filterpaper - record the value in Column I (‘Filter &

Paper Tray B Dry Weight [g]’)

9. To calculate the “Soak Time” of each unit, record the CAU “Deployment Date” and “Recovery Date” in Column CA and CB, respectively. Record any other additional field data in the spreadsheet (e.g. coordinates, depth, comments, etc.

10. Oven (Heratherm OGS-180) settings:

Temperature: 60°C (140°F) Damper: level 3 (mid-high)

11. Prepare 5% HCl acid solution in 2-liter bottles One batch (20-30 units) of CAUs will require about 10-15, 2-liter bottles of 5% HCl acid solution in order to completely dissolve calcified materials. More may be needed depending on the amount of accreted CaCO3.

To dilute full-strength HCl acid (1.18 g/mL, 36.5 molar mass, 37% conc.) into 5% solution, use the recommended ratio below:

2L stock: 225 ml HCl

1775 ml deionized/distilled H2O

1L stock: 112.5 ml HCl 887.5 ml deionized/distilled H2O

Laboratory Processing Procedures

*see Section “Processing Timeline” p.?? for sample processing timeline and Appendix 1 p.?? for “Daily Task Guide”.

A. Disassembling and Photo-documentation 1. Thaw a batch of CAUs (typically 30-50 units) on the sink for at least 12 hours. Do not thaw over water. 2. Disassemble the plates of each CAU by removing the hex nuts and washers using two wrenches (one to hold the unit steady). Make sure to maintain plate identity (upper vs lower plate, top vs bottom side of plate) and the CAU Plate ID (Figure 4.4). 3. Carefully rinse each plate in a saltwater rinse basin (if saltwater is not readily available, freshwater may be used) to remove loose sediment, sand and mobile fauna. Discard any mobile fauna that may have fallen off during rinsing. 4. Fill each pre-labeled glass dish with saltwater. Again, if saltwater is not readily available, freshwater may be used. 5. Place each CAU plate with the top plate facing upwards into its corresponding glass dish (Figure 4.5). Ensure that the plate and any encrusting fauna are completely submerged in water. Add more water if necessary.

Figure 4.4 Plate Identification for Photo.

6. Attach a DSLR camera to a camera copy stand or a tripod. Arrange lighting such that the plate is fully illuminated and no glare is visible to the camera (Figure 4.6).

NOTE: If using a Nikon D7000 camera mounted on a copy stand kit with attached lights, see Section VI, p. 17 for tethering protocol and recommended settings (f/8, ISO400, AWB).

Figure 4.6 Attaching camera to a camera copy stand and light setting.

Figure 4.5 Placing CAU plates in glass dishes.

7. It is recommended to tether the camera to a computer and save the photos directly to the computer’s local drive (while using the camera’s SD card as a backup drive). Designate a folder where the CAU photoset will be stored directly. It is recommended to group the CAU photoset folders within a Site folder. E.g. PAL01 > PAL01_5319

PAL01_5320 PAL01_5321

8. Photograph the top and bottom of each plate, first with the Plate Photo ID tag and then a 2nd image without the tag (Figure 4.7).

Note: If pieces of calcified material fall off during the photographing process, retain the pieces with the plate to be used during the decalcification process

9. From the designated CAU photoset folder, confirm that both plate photos (with and without the Plate Photo ID tag) are in focus. After photographing both sides of each plate, each CAU photoset folder should have 8 photos. The photos should be later on renamed with the following naming convention:

a) With Plate Photo ID tag: CAU_SiteID_RecoveryYear_4-digitSerial#_PlateLocation (“U” - upper plate or “L” - lower plate)_PlateSurface (“T” - top plate or “B” - bottom plate)_ID.jpg

b) Without Plate Photo ID tag:

CAU_SiteID_RecoveryYear_4-digitSerial#_PlateLocation (“U” - upper plate, “L” lower plate)_PlateSurface (“T” - top plate, “B” - bottom plate).jpg

Figure 4.7 CAU plate images with ID and without ID.

Example: CAU_PAL01_2012_5319_U_T_ID.jpg – Upper Plate, Top Side with tag CAU_PAL01_2012_5319_U_T.jpg – Upper Plate, Top Side CAU_PAL01_2012_5319_U_B_ID.jpg – Upper Plate, Bottom Side with tag CAU_PAL01_2012_5319_U_B.jpg – Upper Plate, Bottom Side CAU_PAL01_2012_5319_L_T.jpg – Lower Plate, Top Side CAU_PAL01_2012_5319_L_B_ID.jpg – Lower Plate, Bottom Side with tag CAU_PAL01_2012_5319_L_B.jpg – Lower Plate, Bottom Side

10. After photographing all units, if the CAUs were submerged in seawater, carefully rinse each plate in freshwater. If the CAUs were submerged in freshwater, skip the rinsing process. 11. Be sure to remove all hex nuts and washers that may still be attached at the center of the plate. Hex nuts and washers can be covered by calcified material and difficult to see. Inspect each side of each plate to ensure that there are no washers or hex nuts present. NOTE: If a hex nut or washer is covered in calcified material, remove the hardware and chip off the calcified material from the hardware. Retain the removed material with the rest of the plate. 12. Place each plate and any dislodged pieces of calcified material on its corresponding Paper Tray A and on to a drying rack. The drying rack can be left in a fume hood to air dry at room temperature (minimum of 24 hours). The plates can continue to air dry at room temperature, indefinitely, until they are dried in the oven. NOTE: Drying at room temperature before placing in the oven can help reduce the time the plates remain in the oven. 13. Wash and rinse the glass dishes but keep the labels on. The same glass dishes will be used during the HCl acid decalcification process.

B. Determining the weight of the CAU plate

The CAU Lab Data Spreadsheet can be downloaded at: (Link to the spreadsheet) 1. After a minimum air-drying period of 24 hours, place the plate assembly (plate, dislodged pieces of calcified material and Paper Tray A) in the oven to dry at 60°C for 24 hours. NOTE: Check that the internal temperature remains consistent. If not, minor adjustments should be made to the oven temperature and damper settings.

2. After 24 hours, remove the plate assembly and allow it to come to room temperature for 1 hour. 3. Weigh the entire plate assembly and record the weight in the CAU Lab Data Spreadsheet. 4. On the lab data spreadsheet (or CAU Data Entry tool for ESD-CREP users), record the weight to at least 3 decimal places under Column O (‘Drying 1 [24 h] [paper tray + CAU plate] [g]’). 5. Return the plate assembly in the oven. 6. Dry the CAU plates for another 24 hours. 7. Remove the plate assembly and allow it to come to room temperature for 1 hour. 8. Weigh the entire plate assembly and record the weight in the CAU Lab Data Spreadsheet. 9. On the lab data spreadsheet (or CAU Data Entry tool for ESD-CREP users), record the 2nd weight to at least 3 decimal places under Column Q (‘Drying 2 [48 h] [paper tray + CAU plate] [g]’). 10. Compare the current weight from the previous weight:

a) If the difference in weight is less than 0.1g, the plates are considered dry and no further drying needs to occur.

Note: On the lab data spreadsheet (or CAU Data Entry tool for ESD-CREP users), conditional formatting has been applied to the columns labeled Mass Lost (g). The cells turn green if the difference in weight is less than 0.1g.

b) If the difference in weight is greater than 0.1 g, return the plates to the oven for another 24 hours.

11. Repeat IV.B Steps 5-10 until the difference in weight is less than 0.1 g.

C. Decalcification of CaCO3 in Hydrochloric Acid 1. Ensure that the plate is fully dry (mass lost between last two weighing will have been less than 0.1g) and the weight of the plate assembly and Paper Tray A has been recorded. 2. Place the individual plates in the pre-labeled glass dishes. Keep Paper Tray A and place under the glass dish.

3. Chip off large pieces of calcified materials (e.g. CCA, coral, shells, etc) and grind using a mortar and pestle. Return the pulverized forms in the glass dish. 4. Fill the glass dish midway with 5% HCl acid solution and/ until the entire CAU plate is fully submerged in acid. Add HCl acid slowly as the solution may overflow when bubble formation/decalcification begins. 5. When decalcification has slowed (12-24 hours): 5.1 Scrape leftover material from the CAU plates with a razor blade into the glass dish (Figure 4.8 a). 5.2 Grind pieces of calcified materials that may still be present and return in the glass dish (Figure 4.8 b). 5.3 Using acid-resistant gloves, sort through the scraped fleshy material in the glass dish and feel for any calcified materials (Figure 4.8 c). 5.4 Some calcified material can be completely covered by a boundary layer of fleshy material. If present, manually tease the fleshy material and grind the calcified material to allow decalcification to continue. 5.5 If there are more calcified materials in the dish, but no obvious decalcification occurring (no bubble formation), decant the used acid solution and drain the fleshy material into another pre-labeled container (beaker, glass dish, etc). Add new 5% HCl acid solution in the first glass dish to continue dissolving the remaining calcified materials.

Figure 4.8 Processing Sample on the CAU plates after the decalcification has slowed.

a b c

6. Repeat IV.C, Steps 5b-e until all CaCO3 is fully dissolved. Additional 5% HCl acid solution may need to be added 2-4x, depending on the amount of CaCO3 present. 7. Using 5% HCl acid in a squeeze bottle, thoroughly rinse the tools (gloves, mortar and pestle, razor/scraper) into the glass dish. 8. When the CAU PVC plates have been scraped clean (fleshy/calcified material completely removed from either side and edges of the plate), rinse all sides of the plates with 5% acid into the glass dish. 9. Place the CAU PVC plate on its corresponding Paper Tray A. When dry, label the plate with the CAU Plate ID (e.g. PAL01_2012_5319_U) using a permanent marker (Figure 4.9). NOTE: Paper Tray A can be discarded after the CAU PVC plates have been labeled.

10. Scrub and rinse the CAU PVC plates in freshwater. Pat to dry. 11. Place the rinsed CAU PVC plates in the oven at 60°C for 24 hours. 12. Remove the plate from the oven and allow to come to room temperature for 1 hour. 13. Weigh the CAU PVC plate. 14. On the lab data spreadsheet, record the weight to at least 3 decimal places under Column M (‘CAU PVC Dry Weight [g]’). 15. Bind the CAU PVCs together using painter’s/masking tape and put away for archiving.

D. Vacuum filtration of fleshy material 1. Using a 4-liter Buchner funnel, a plastic mesh layer (with attached pull string) and the corresponding pre-weighed filter paper, vacuum filter the acid bath and remaining fleshy material for each CAU plate. Use a freshwater squeeze bottle to rinse contents of the glass dish into the funnel.

Figure 4.9 Placing CAU plates on Paper Tray A to dry.

2. If the volume of fleshy material is sufficient to clog the filter paper, a 2nd pre-labeled and pre-weighed filter paper (see IV.D, Step 2, a-c) can be used to filter the remaining volume of acid bath and fleshy material: 2.1 Label an additional filterpaper and paper tray with its corresponding CAU Plate ID (e.g. PAL01_2012_5319_U)

2.2 In the lab data spreadsheet, record the weight of the 2nd filterpaper + paper tray to at least 3 decimal places in Column K(‘Addt’l Filter & Paper Tray B Dry Weight [g]’).

3. If filtering has slowed down, use a spatula/teaspoon to gently scrape the surface of the filterpaper to allow more solution to filter through. Filtering can take >5 mins depending the amount of fleshy material in the acid bath. 4. When filtration is completed, place the filterpaper on its corresponding Paper Tray B and on to a drying rack (Figure 4.10). The drying rack can be left in a fume hood to air dry at room temperature (minimum of 24 hours). The filterpaper can continue to air dry at room temperature, indefinitely, until they are dried in the oven.

E. Determining the weight of the fleshy material 1. After a minimum air-drying period of 24 hours, place the filterpaper assembly (filterpaper and Papery Tray B) in the oven to dry at 60ºC for 24 hours. NOTE: Check that the internal temperature remains consistent. If not, minor adjustments should be made to the oven temperature and damper settings. 2. After 24 hours, remove the filterpaper assembly and allow it to come to room temperature for 1 hour.

Figure 4.10 Placing filterpaper on Paper Tray B and frying rack.

3. Weigh the entire filterpaper assembly (Figure 4.11). 4. On the lab data spreadsheet, record the weight to at least 3 decimal places under Column AS (‘Drying 1 [24 h] [paper tray + filterpaper] [g]’). 5. Return the filterpaper assembly in the oven. 6. Dry the filterpaper for another 24 hours. 7. Remove the filterpaper assembly and allow it to come to room temperature for 1 hour.

8. Weigh the entire filterpaper assembly. 9. On the lab data spreadsheet, record the 2nd weight to at least 3 decimal places under Column AU(‘Drying 2 [24 h] [paper tray + filterpaper] [g]’). 10. Compare the current weight from the previous weight:

a) If the difference in weight is less than 0.1g, the plates are considered dry and no further drying/weighing needs to occur.

NOTE: On the lab data spreadsheet, conditional formatting has been applied to the columns labeled Weight of Fleshy Materials [g]. The cells turn green if the difference in weight is less than 0.1g.

Figure 4.11 weighing the entire filterpaper assembly.

b) If the difference in weight is greater than 0.1 g, return the plates to the oven for another 24 hours.

11. Repeat IV.E Steps 5-10 until the difference in weight is less than 0.1 g. 12. After recording the final filterpaper assembly dry weight, place the filterpaper and Paper Tray B in its corresponding re-sealable bag for archiving (Figure 4.12).

Figure 4.12 Filterpaper assembly and Paper Tray B in re-sealable bags for achieving.

Processing Timeline Approximately 50 units can be processed as a batch (at ESD-CREP, this is equivalent to CAUs collected from 2 islands). The processing of each batch (<50 units) is typically completed in 4 weeks. Below is an example of a typical processing timeline

Sun M T W Th F Sat Prep Prep Def Proc AD AD Week 1

PREP, DISASSEMBLE

AD

OD

W/O W/ O

W/ Deca

l

W/ Deca

l

Deca l

Week 2 WEIGH PLATES, DECALCIFY

Deca l

Sc/ Deca

l

Sc/ Deca

l

Filt

Filt

Filt

AD

Week 3 DECALCIFY, FILTER

AD W/O W/O W/ O W/O W/O Week 4

WEIGH FILTERPAPERS

Prep

Prep labels, dilute acid, etc

Man Hours 8 x 2

Def Defrost; prep lab 2

Proc AD OD

Process: Dismantle and photograph Air-dry Oven-dry

7

Decal Sc/Decal

Filt

Decalcify (sit in 5%HCl bath) Scrape and decalcify Vacuum filtration process

4 7 x 2 7 x 3

W/O Weigh (and oven-dry) 1 x 7

W/Decal Weigh and decalcify (pour HCl; pulverize large pieces) 7 x 2

85

Part V: Camera tethering and CAU plate photo-documentation (based on NOAA equipment) Camera Tethering and CAU Plat Photo-documentation Procedures NOTE: Refer to the brand/model user documentation/manual if using a different camera and setup

A. Installing the Software For ESD-CREP users, the installation instructions and software product key can be accessed in the network folder T:\general\ProjectGroup\Ocean Acidification\CAUs\Analysis\CAU Processing SOP, Data sheet, Labels/CAU SOPs 2015/ CAUSOP_NikonD7000Settings.pdf

B. Connecting the Camera to the Computer 1. Connect Nikon DSLR camera (Nikon D7000 with AF-S Micro NIKKOR 60mm f\2.8G ED lens) to the computer via USB mini cable (Figure 4.13).

2. Start the Nikon Camera Control Pro 2 application. 3. Turn the camera ON. 4. Once connected, the connection status of the camera in use will be displayed on the Camera Control Pro window (Figure 4.14).

Figure 4.13 Connecting camera to the computer.

5. If using the software for the FIRST TIME: 5.1 Click on “Exposure 1” under the Camera Control tabs and change the settings of the following (Figure 4.15):

Shutter speed: 1/50 sec Aperture: f/8

5.2 Click on “Exposure 2” under the Camera Control tabs and change the settings of the following (Figure 4.16):

ISO Sensitivity: ISO 400 White Balance: Auto Option: Normal

Figure 4.14 pop up window showing connection status.

Figure 4.15 Pup-up window shows setting up value for “Exposure 1”.

5.3 Click on “Storage” under the Camera Control tabs and change the settings of the following (Figure 4.17):

Image Quality: JPEG fine JPEG Compression: Optimal quality Image size: Large (4928x3264) Record to: PC Card Record Mode: Backup

Figure 4.16 Pup-up window shows setting up value for “Exposure 2”.

Figure 4.17 Pup-up window shows setting up value for “Storage”.

5.4 Click on “Image Processing” under the Camera Control tabs and change the settings of the following (Figure 4.18):

Picture Control: Standard High ISO NR: On (High)

NOTE: the above settings need to be set each time the software is reinstalled and the above settings will need to be modified if a new camera model/lens are used.

C. CAU Plate Photo-documentation 1. Ensure that the camera’s Mode dial is set to “M” (Manual) (Figure 4.19).

Figure 4.18 Pup-up window shows setting up value for “Image Processing”.

Figure 4.19 Setting camera’s Mode dial to M (Manual).

2. Ensure that the camera lens’ A-M switch is set to “M/A” (Autofocus w/

manual override) (Figure 4.20).

3. Connect the Nikon DSLR camera to the copier stand, then connect the camera to the computer via mini USB cable (Figure 4.21).

4. Start the Nikon Camera Control Pro 2 application 5. Turn the camera ON 6. Once connected, the connection status of the camera in use will be displayed on the Camera Control Pro main window (Figure 4.22).

Figure 4.20 Setting camera lens’ A-M to M/A (Autofocus w/ manual override).

Figure 4.21 Connecting the camera to the copier stand.

a) b)

7. Go to the Tools menu to display the Transfer Options window (Figure 4.23).

8. Click the Browse button next to the “Destination folder” (Figure 4.24a). For ESD-CREP users, navigate to the appropriate CAU site and unit folder location (e.g. Documents\CAU Analysis\ASRAMP 2015\PHOTO ANALYSIS\SAMOA\SWAINS\SWA03\SWA03_368) where the images will be downloaded as they are taken and click OK (Figure 4.24b).

9. On the Camera Control Pro main window, click on the “LV” button at the

Figure 4.22 Pop-up window shows status of camera once connected.

Figure 4.23 Opening “Transfer Options” window via “Tools” menu.

Figure 4.24 a) “Transfer Option” window and b) “Browse For Folder” window.

bottom-right corner to display the Live View window (Figure 4.25). Note: the camera controls and folder location options cannot be modified while the Live View window is displayed.

10. Center the glass dish with the plate under the camera and using the Live View display, ensure that all corners of the plate are visible in the frame (Figure 4.26). Note: both copier stand lights should be switched on at this time and that any external light sources are minimized (i.e., window shades are drawn, overhead/ceiling lights are switched off).

Figure 4.25 Setting up “Camera Control Pro” window.

11. Using the “AF and Shoot” button on the Live View window, photograph the top of the plate, first with the Plate Photo ID tag and then a second image without the tag (Figure 4.27).

12. Flip the plate and photograph the bottom of the plate, again with Plate Photo ID tag and then a second image without the tag. 13. From the designated CAU unit folder, confirm that both plate photos (with and without the Plate Photo ID tag) are in focus.

14. Repeat for the next set of plates/surfaces for each unit. 15. After all 8 images have been taken and saved to the unit folder (e.g. SWA03_368), click on the “LV” button at the bottom-right corner to turn off Live View.

Figure 4.26 “Live View Display” of the camera of a CAU plate.

Figure 4.27 Photos of the top plate with and without ID.

16. On the Camera Control Pro main window, go to the Tools menu to display the Transfer Options window. Click on the Browse button next to the “Destination folder” and navigate to the next CAU site and unit folder (e.g. SWA03_394) (Figure 4.28). Repeat steps C. 7-13.

17. At the end of the plate photo documentation session, ensure that all CAU plate photos have been taken and saved to their appropriate local drive folders. 18. Backup the images by copying the Island parent folder (e.g. Documents\CAU Analysis\ASRAMP 2015\PHOTO ANALYSIS\SAMOA\SWAINS) to the network drive (e.g. T:/general/ProjectGroup/OceanAcidification/CAUs/Analysis/2015_CAUAnalysis/ASRAMP 2015/PHOTO ANALYSIS/SAMOA/SWAINS) and to the external hard drive. If prompted, select “Overwrite”. 19. Turn off the camera and disconnect it from the mini USB cable. On the Camera Control Pro main window, select Exit from the File menu. 20. Rename all plate photos with the following naming convention (Figure 4.29):

a) With Plate Photo ID tag: CAU_SiteID_RecoveryYear_4-digitSerial#_PlateLocation (“U”-upper plate or “L”-lower plate)_PlateSurface (“T”-top plate or “B”-bottom plate)_ID.jpg

b) Without Plate Photo ID tag: CAU_SiteID_RecoveryYear_4-digitSerial#_PlateLocation (“U” - upper plate, “L” - lower plate)_PlateSurface (“T” - top plate, “B” - bottom plate).jpg

Figure 4.28 Opening “Transfer Options” (a) window via “Camera Control Pro” (b).

(a) (b)

Example:

CAU_SWA03_2015_368_U_T_ID.jpg – Upper Plate, Top Side with tag CAU_SWA03_2015_368_U_T.jpg – Upper Plate, Top Side CAU_SWA03_2015_368_U_B_ID.jpg – Upper Plate, Bottom Side with tag CAU_SWA03_2015_368_U_B.jpg – Upper Plate, Bottom Side CAU_SWA03_2015_368_L_T_ID.jpg – Lower Plate, Top Side with tag CAU_SWA03_2015_368_L_T.jpg – Lower Plate, Top Side CAU_SWA03_2015_368_L_B_ID.jpg – Lower Plate, Bottom Side with tag CAU_SWA03_2015_368_L_B.jpg – Lower Plate, Bottom Side

21. Copy the newly-renamed images to the network folder by copying the Island parent folder (e.g. Documents/CAU Analysis/ASRAMP 2015/PHOTO ANALYSIS/SAMOA/SWAINS) to the network drive (e.g. T:/general/ProjectGroup/Ocean Acidification/CAUs/Analysis/2015_CAU Analysis/ASRAMP 2015/PHOTO ANALYSIS/SAMOA/SWAINS) and to the external hard drive. If prompted, select “Overwrite”

Figure 4.28 Plate photo’s name format.

Appendix 1: Daily Task Guide Sample processing schedule

6-8 hour work day 4-5 hour work day 3-4 hour work day 1-2 hour work day

Sun M T W Th F Sat ISLAND

1

25 26 27 28 29 30 31 PREP, DISASSEMBLE, 10/26 - 10/30 1 2 3 4 5 6 7 WEIGH PLATES, DECALCIFY, 11/2 - 11/6 8 9 10 11 12 13 14 DECALCIFY, FILTER, 11/9 - 11/13

15 16 17 18 19 20 21 WEIGH FILTERPAPERS, 11/16 - 11/20

ISLAND 2

22 23 24 25 26 27 28 PREP, DISASSEMBLE, 11/23 - 11/27 29 30 1 2 3 4 5 WEIGH PLATES, DECALCIFY, 11/30 - 12/4 6 7 8 9 10 11 12 DECALCIFY 12/7 - 12/11

13 14 15 16 17 18 19 FILTER, 12/14 - 12/18 20 21 22 23 24 25 26 WEIGH FILTERPAPERS, 12/21 - 12/25

27 28 29 30 31 1 2 HOLIDAY BREAK ISLAND

3

3 4 5 6 7 8 9 PREP, DISASSEMBLE, 1/4 - 1/8 10 11 12 13 14 15 16 WEIGH PLATES, DECALCIFY, 1/11 - 1/15 17 18 19 20 21 22 23 DECALCIFY, FILTER, 1/18 - 1/22 24 25 26 27 28 29 30 WEIGH FILTERPAPERS, 1/25 - 1/29

ISLAND

4

31 1 2 3 4 5 6 PREP, DISASSEMBLE, 2/1 - 2/5 7 8 9 10 11 12 13 WEIGH PLATES, DECALCIFY, 2/8 - 2/12

14 15 16 17 18 19 20 DECALCIFY, FILTER, 2/15 - 2/19 21 22 23 24 25 26 27 WEIGH FILTERPAPERS, 2/22 - 2/26

Sample Processing Daily Tasks

WEEK 1 Monday - Tuesday, Week 1 1. Fold, label and weigh paper trays and filter papers. 2. Print glass dish labels and plate photo labels. 3. Label quart-sized resealable plastic bags. 4. Dilute acid to 5% and restock as needed. Wednesday, Week 1 1. Prepare glass dishes for each CAU plate. 2. Attach glass dish labels to each glass dish using regular clear tape (Figure A1).

3. Arrange glass dishes between two carts in alphabetical order (island, site, serial # and plate) and fill the dishes with freshwater/seawater (Figure A2).

Figure A1 Glass dishes with label.

4. Arrange oven wire mesh shelves in the fume hood elevated by a drying rack; arrange paper trays on the wire mesh shelves in alphabetical order (Figure A3).

5. Place CAUs in the lab sink and thaw overnight (Figure A4).

Thursday, Week 1 1. Disassemble CAUs (orient each plate on the dish with the top surface facing up) (Figure A5).

Figure A2 Arranging glass dishes on carts in alphabetical order.

Figure A3 Paper trays and wire mesh shelves arranged in the fume hood.

Figure A4 Thawing CAU plates overnight.

2. Set up plate, camera and light position according to Figure A6. Photograph each plate surface on the copy stand (with and without the plate photo ID tags; see SOPs).

3. If using seawater, rinse plate with freshwater and place each plate on their corresponding paper trays in the fume hood. 4. Air dry overnight. 5. Rinse glass dishes (do not remove dish labels) and dry on the cart. Friday PM, Week 1

Figure A5 Disassembled CAU plates in the glass dishes.

Figure A6 Camera and CAU plate positions for photography.

1. Place wire mesh shelves with plates in the rolling rack. 2. Arrange the shelves between the two drying ovens (pre-set to 75C with damper settings at level 3 to achieve a uniform 60C internal temp). * For the initial drying, CAU plates are best dried continuously over the weekend WEEK 2 Monday, Week 2 1. Pull plates from the oven at 8:00 AM and cool to room temperature in the fume hood for 15-30 minutes. 2. Weigh each plate (with the paper tray) and enter into data spreadsheet or the CAU Data Entry tool. 3. Return CAUs to the oven. Tuesday, Week 2 1. Repeat Monday’s tasks. 2. Take note of the average difference in weight of all plates. 3. For ESD-CREP users, if a majority of the upper plates are almost dry (weight difference of 0.2 -0.5), coordinate with NIST representatives to schedule CCA subsampling for the following day when applicable 4. Line up upper plate glass dishes on the bench top in alphabetical order; arrange lower plate glass dishes in the right fume hood (Figure A7).

Wednesday, Week 2

Figure A7 Lining up upper plate glass dishes on the bench top.

1. Pull plates from the oven and weigh. 2. If majority of the upper plates are NOT dry, return all upper plates to oven. 3. If all upper plates ARE dry, place each plate (with paper tray) inside its corresponding glass dish on the bench top. 4. For ESD-CREP users, if applicable, call/email NIST representatives to confirm that subsampling can occur today 5. If majority of the lower plates ARE dry, place each plate in their respective glass dish in the fume hood, place paper tray under each dish. Return the rest of the non-dry lower plates to the oven. 6. Start decalcifiying the dry plates: slowly pour 5% HCl acid in each dish and allow the plate to sit in acid for 12-24 hours. 7. Optional: if the plates have a good amount of calcified material, pry the calcified material off and pulverize with a mortar and pestle prior to pouring HCl acid in the dish Thursday, Week 2 1. Pull the remaining plates from the oven and weigh. 2. Place the remaining CAUs into their respective dishes and pour 5% HCl acid on each and allow to sit in acid for 12-24 hours. 3. For plates that have already been soaked in acid for 12-24 hours, scrape the accreted materials from the PVC plates using a scraper (Figure A8).

4. Place the clean PVC plates on their corresponding paper trays and air dry on a baking tray (Figure A9).

Figure A8 Scraping the accreted materials from the PVC plates.

5. For dishes that have large pieces of calcified materials (CCA/coral), pulverize these pieces using a mortar and pestle or a blender. Friday, Week 2 1. Find dishes that still have PVC plates and scrape off accreted materials. 2. Find dishes that still have large pieces of calcified materials and pulverize pieces via mortar and pestle or blender. 3. If some dishes still contain calcified materials that have settled, decant acid solution into a pre-labeled (serial number and plate identity, e.g. “650_U” is sufficient) plastic container and pour more 5% HCl acid into the glass dish (Figure A10).

Figure A9 PVC plates on paper trays to air dry.

Figure A10 Acid solution in plastic containers and settled calcified materials in glass dishes

4. Make sure that all dishes have “new” acid in them before the end of the day. WEEK 3 Monday - Friday, Week 3 1. Check for solid calcified materials in each dish. 2. Repeat tasks from Week 2, Friday. 3. Arrange wire mesh shelves in one of the fume hoods/bench top. 4. Set aside the dishes that have completely decalcified materials next to the Filtration station (Figure A11).

Figure A11 Setting dishes that have completely decalcified materials next to the filtration station.

5. Find the corresponding paper tray+filterpaper and place next to dishes that are ready for filtration. 6. Filter the decalcified materials (Figure A12).

7. Place the solid residue+filterpaper on their corresponding paper trays and on to the wire mesh shelves (Figure A13).

8. Ensure that the wire mesh shelves are left in the fume hood/s over the weekend to prevent the filterpapers from absorbing too much moisture. WEEK 4 Monday, Week 4 1. Place wire mesh shelves with filterpapers+paper tray in the rolling rack 2. Arrange the shelves between the two drying ovens (pre-set to 75C with damper settings at level 3 to achieve a uniform 60C internal temp). 3. Label and rinse each PVC plate; discard the paper trays. 4. Pat the PVC plates dry, arrange on wire mesh shelves and place in the oven to dry for 24 hours. Tuesday, Week 4 1. Pull filterpapers from the oven at 8:00AM and cool to room temperature in the fume hood for 15-30mins. 2. Weigh each filterpaper (with the paper tray) and enter into data spreadsheet or the CAU Data Entry tool.

Figure A13 Placing the residue and filterpaper on paper trays.

Figure A12 Filtering the decalcified materials.

3. Return filterpapers to the oven. 4. Pull PVC plates from the oven, cool to room temperature for 15-30 mins, weigh and enter reading to data spreadsheet. 5. Bind PVC plates with painter’s tape and store in action packers to be reused. Wednesday-Friday, Week 4 1. Pull filterpapers from the oven at 8:00AM and cool to room temperature for 1 hour. 2. Weigh each filterpaper (with the paper tray). 3. Return CAUs to the oven. 4. When a filterpaper is considered dry, place the filterpaper+paper tray in its corresponding pre-labeled quart-sized resealable plastic bag (Figure A14).

5. Place all plastic bags in a larger pre-labeled (“ISLAND YYYY”) plastic bag and store in the Dry Archive room.

Figure A14 Dried filterpapers+paper trays in resealable plastic bags.

Appendix 2: Labels Glass dish label template

BATCH/SITE 1 BATCH/SITE 2

SITE_00_1_0000_ U

SITE_00_1_0000 _L

SITE_00_1_0000_ U

SITE_00_1_0000 _L

SITE_00_2_0000_ U

SITE_00_2_0000 _L

SITE_00_2_0000_ U

SITE_00_2_0000 _L

SITE_00_3_0000_ U

SITE_00_3_0000 _L

SITE_00_3_0000_ U

SITE_00_3_0000 _L

SITE_00_4_0000_ U

SITE_00_4_0000 _L

SITE_00_4_0000_ U

SITE_00_4_0000 _L

SITE_00_5_0000_ U

SITE_00_5_0000 _L

SITE_00_5_0000_ U

SITE_00_5_0000 _L


SITE_00_1_0000_ U

SITE_00_1_0000 _L

SITE_00_1_0000_ U

SITE_00_1_0000 _L

SITE_00_2_0000_ U

SITE_00_2_0000 _L

SITE_00_2_0000_ U

SITE_00_2_0000 _L

SITE_00_3_0000_ U

SITE_00_3_0000 _L

SITE_00_3_0000_ U

SITE_00_3_0000 _L

SITE_00_4_0000_ U

SITE_00_4_0000 _L

SITE_00_4_0000_ U

SITE_00_4_0000 _L

SITE_00_5_0000_ U

SITE_00_5_0000 _L

SITE_00_5_0000_ U

SITE_00_5_0000 _L


SITE_00_1_0000_ U

SITE_00_1_0000 _L

SITE_00_1_0000_ U

SITE_00_1_0000 _L

SITE_00_2_0000_ U

SITE_00_2_0000 _L

SITE_00_2_0000_ U

SITE_00_2_0000 _L

SITE_00_3_0000_ U

SITE_00_3_0000 _L

SITE_00_3_0000_ U

SITE_00_3_0000 _L

SITE_00_4_0000_ U

SITE_00_4_0000 _L

SITE_00_4_0000_ U

SITE_00_4_0000 _L

SITE_00_5_0000_ U

SITE_00_5_0000_L

SITE_00_5_0000_U

SITE_00_5_0000_L

Plate photo ID tag template

SITE / BATCH 1 SITE / BATCH 2 Site REA_00_1 CAU Serial # 0000 Recovered: YYYY Upper Plate, Top Side

Site REA_00_1 CAU Serial # 0000 Recovered: YYYY Upper Plate, Bottom Side

Site REA_00_1 CAU Serial # 0000 Recovered: YYYY Upper Plate, Top Side


Site REA_00_1 CAU Serial # 0000 Recovered: YYYY Lower Plate, Top Side

Site REA_00_1 CAU Serial # 0000 Recovered: YYYY Lower Plate, Bottom Side



































Appendix 3: Standard-metric Wrench Conversion Chart

Bolt Diameter Standard Metric 1/8" 5/16" 8 mm 3/16" 3/8" 10 mm 1/4" 7/16" 11 mm 5/16" 1/2" 13 mm 3/8" 9/16" 14 mm 7/16" 5/8" 16 mm 1/2" 3/4" 19 mm 9/16" 13/16" 21 mm 7/8" 22 mm 5/8" 15/16" 24 mm 1" 25 mm 3/4" 1-1/8" 29 mm 1-1/4" 32 mm 7/8" 1-5/16" 34 mm 1-3/8" 35 mm 1" 1-1/2" 38 mm 1-5/8" 41 mm 1-1/8" 1-11/16" 43 mm 1-3/4" 45 mm 1-1/4" 1-7/8" 48 mm 1-3/8" 2" 51 mm 1-1/2" 2-1/4" 57 mm 1-5/8" 2-1/2" 64 mm 1-3/4" 2-5/8" 67 mm 2-3/4" 70 mm 1-7/8" 2-15/16" 75 mm 3" 76 mm 2" 3-1/8" 80 mm 3-1/4" 83 mm 2-1/4" 3-1/2" 89 mm

3-3/4" 95 mm 2-1/2" 3-7/8" 99 mm 4" 102 mm 2-3/4" 4-1/4" 108 mm 4-1/2" 114 mm 3" 4-5/8" 118 mm 3-1/4" 5" 127 mm 3-1/2" 5-3/8" 137 mm 5-1/2" 140 mm 3-3/4" 5-3/4" 146 mm 6" 152 mm 6-1/2" 165 mm 7" 178 mm