Methods

The University of Washington Tacoma Estuaries class conducted oceanographic surveys throughout various areas of the Puget Sound between March and May 2026.

All samples were analyzed by the Spring 2026 Estuaries team, with the exception of nutrients, which were frozen and sent to University of Washington Seattle's Marine Chemistry Lab for analysis.

R/V Welander

Captained by Dave Thoreson for sampling in Commencement Bay, Quartermaster Harbor, Colvos Passage, and East Passage

R/V Kittiwake

Captained by Eric Loss for sampling in the San Juan Channel with operation assistance by Kristi Kull

Areas Sampled

Commencement Bay: 4/10, 4/17
Quartermaster Harbor: 5/1
San Juan Islands: 5/9
Colvos & East Passages: 5/15

Equipment Used

CTD Seabird-19: Water profile
Niskin bottle: Discrete water samples
CTD rosette: Discrete water samples at multiple depths
Thoreson bottle: Discrete surface water samples
Secchi disk: Photic zone
Van Veen grab: Sediment collection
20 μm phytoplankton net
211 μm zooplankton net
335 μm manta tow net: Microplastic collection

Field Methods

In the field, we collected water samples to gather data on dissolved oxygen, nutrients, chlorophyll and phytoplankton in the lab. In order to collect discrete samples at specific depths, we used a Thoreson bottle for surface water, and a Niskin bottle for water at CTD depth. The CTD itself profiled the temperature, salinity, density, dissolved oxygen, fluorescence, transmission, and pH (San Juan only) of the water column at each sample station.

Secchi Disk

We determined the photic zone of the sample areas by lowering a Secchi disk into the water until it was just barely visible. The line attached to the Secchi disk is marked at every meter, allowing the person taking the Secchi sample to determine the depth to which light penetrates water in the sample area.

Phytoplankton

A 20 μm mesh net was lowered 10m into the water to collect phytoplankton samples. Additional phytoplankton samples were collected at the surface of each sample station using the Thoreson bottle. Each sample was appropriately labeled and placed on ice for transport back to the lab.

Dissolved Oxygen, Chlorophyll and Nutrients

The Thoreson bottle was used to collect surface samples to be tested for dissolved oxygen, chlorophyll, and nutrients concentration. Two samples were collected for dissolved oxygen and chlorophyll, while a single nutrient sample was collected. While the chlorophyll and dissolved oxygen bottles were simply filled straight from the Thoreson bottle, water for the nutrient sample was first run through a 30 mL plastic syringe with a 25 µm filter attached to the end.

Seabird 19 CTD and Niskin Bottle

The CTD and Niskin bottle were used to collect samples at depth. The Niskin bottle was opened and attached to a 200 m line, with the CTD attached about a meter below it. The CTD was then powered on and lowered under the surface of the water to soak for three minutes. Once soaked, the CTD was lowered into the water until it was a few meters from the bottom of the sample site. A messenger weight was then attached to the line and dropped into the water. Once it reached the end of the line, it triggered the release of the cords holding the Niskin bottle open, causing the top and bottom stoppers to shut and trap a depth water sample inside. The Niskin and CTD were then raised back to the surface and hoisted back onto the boat. The CTD was then turned off, and samples of dissolved oxygen, chlorophyll, and nutrients were collected from the Niskin bottle in the same manner as the surface samples.

CTD rosette

Aboard the R/V Kittiwake, the team used a CTD rosette. In addition to the CTD profile, this equipment configuration allows the capture of discrete samples in the surrounding Niskin bottles at multiple depths.

Sediment

Sediment samples were collected by Van Veen grab at certain stations by allowing the Van Veen to drop to the bottom of the water column. Once it hit sediment, the line was yanked sharply, triggering the Van Veen to close and scoop up a sample in the process. A small amount of sediment was scooped from the Van Veen into a plastic ziptop bag and placed on ice for transport back to the lab.

Microplastics

A microplastic sample was collected using a 335 µm manta tow net. The net was lowered over the side of the boat and towed behind it off to one side at a speed of 2 knots for 15 minutes. After being removed from the water, the net was rinsed down from the outside into a sieve to remove larger debris

Thoreson Bottle

The Thoreson bottle was used to collect discrete samples to assess for dissolved oxygen, chlorophyll, nutrients, and phytoplankton concentration at the surface.

Niskin Bottle

The Niskin bottle was mounted above the CTD for casts from the R/V Weelander. Samples from the Niskin bottle were used to assess dissolved oxygen, chlorophyll, and nutrients at the bottom.

Dissolved oxygen

Researchers collected and fixed dissolved oxygen samples in the field in accordance with the Winkler method.

Nutrients

Nutrients samples were filtered to remove suspended solids before being frozen and transported to the marine chemistry lab at UW Seattle for analysis.

Chlorophyll

Chlorophyll samples were collected as a proxy measurement of phytoplankton concentration.

Van Veen Grab Sampler

The Van Veen grab sampler was used to gather sediment samples in Commencement Bay and Quartermaster Harbor.

Secchi Disk

The Secchi disk was used to gauge water clarity at the surface.

Phytoplankton net

Researchers gathered a surface layer phytoplankton sample at each station using a 20 μm plankton net.

Zooplankton Net

On the R/V Kittiwake, researchers collected zooplankton samples using a 211 μm tow net. Because zooplankton typically descend during daylight hours, the net was lowered to a depth of 75 m then raised to 25 m, where it was closed to prevent zooplankton from escaping. The net contents were rinsed down the net and into into the cod end. The contents were qualitatively assessed at the Friday Harbor lab.

Microplastics Sampling

Researchers conducted microplastics sampling in Friday Harbor from the R/V Kittiwake using a 335 μm tow net. As large pieces of organic matter (seaweed, sticks, etc.) were removed, they were rinsed into the sample to ensure that any microplastics stuck to their surfaces were retained for analysis in the lab. Remaining organic material was removed chemically in the UW Tacoma oceanography lab.

Lab Methods

Phytoplankton Net Samples

Once phytoplankton net samples had been returned to the lab, a 1 mL pipette was used to extract a sample of phytoplankton from the sample jar, which was then placed on a Sedgewick-Rafter Counting Chamber. The counting chamber was then placed under the compound microscope and examined under 100x magnification. For these samples, a single transect was counted. Each phytoplankton present within that transect was identified using provided handbooks and identification sheets and recorded on a provided checklist. Depending on the species, a plankton would be counted as an entire individual or by individual cells (Huber 2012).

Phytoplankton Surface Samples

Once surface samples had been returned to the lab, each was fixed with 1 mL of 37% formalin and allowed to sit under the fume hood at least overnight. After the fixed samples had settled, each was decanted down to 10 mL. One mL of the remaining sample was loaded into a Sedgewick-Rafter Counting Chamber in the same fashion as the phytoplankton collected via net. The phytoplankton were placed on the compound microscope at 100x magnification and counted one transect at a time until at least 100 individuals or cells had been counted. If the threshold of 100 phytoplankton had been reached mid-transect, counting continued until the end of the transect was reached.

Chlorophyll

Chlorophyll samples had to be run as soon as possible after returning to the lab, as they could not be kept for more than six hours. Samples were analyzed according to the method outlined in A Practical Handbook of Seawater Analysis (Strickland 1972).

Dissolved Oxygen

Dissolved oxygen samples were assessed through titration using the methods outlined in The accuracy of the Winkler method for dissolved oxygen (Carpenter 1965). The amount of thiosulfate required to complete each titration was recorded on paper, then input into an Excel spreadsheet along with the volume of each flask used to collect the discrete samples in order to calculate the amount of dissolved oxygen present in each sample.

Sediments and Total Organic Compounds

Sediment samples were run according to the methods outlined in the Beckman Coulter Particle Size Analyzer LS13 320 user manual and the standard operating procedure set forth by Roca and Greengrove (2020). A small amount of the sediment sample was placed in a blast oven and weighed to determine the amount of total organic compounds present.

Microplastics (San Juan only)

A beaker containing the manta net sample was placed in the oven at 90℃ for 24 hours to allow water to evaporate from the sample. A wet peroxide oxidation method was then run on the sample at 75℃ until the majority of organic material had been dissolved. Salt was added according to the volume of solution; the solution was allowed to settle in a density separator for 24 hours before being sieved. Once the sieved sample had dried for 24 hours, the remaining organic material was removed and plastics were categorized according to type, size, and color. All plastics were placed into a vial and weighed to determine the amount of microplastics present.

Zooplankton (San Juan only)

In order to get a clear picture of the kinds of zooplankton present around San Juan Island, 1 mL of water collected using the zooplankton net was decanted into a petri dish using a pipette. The sample was viewed at 40x magnification under a dissecting microscope. Zooplankton were categorized by species and discrete counts of each were recorded on a spreadsheet. This process was repeated with another 1 mL of sample for each station, for a total of 2 mL of water sampled from the two stations where zooplankton were collected.

UW Tacoma Laboratory

Oceanography Lab at University of Washington - Tacoma

Friday Harbor Labs

Laboratory equipment was brought from UW Tacoma to the laboratory space at Friday Harbor in order to assess samples gathered aboard the R/V Kittiwake.

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