Biological Colonization on High Density Polyethylene Marine Outfall Material in Puget Sound: Year 2 Results

May 2017

Alternate Formats Available

Prepared for: King County Wastewater Treatment Division Seattle, WA

Submitted by: Kimberle Stark and Wendy Eash-Loucks King County Water and Land Resources Division Department of Natural Resources and Parks and

Jeffrey A. Lundt, P.E. King County Wastewater Treatment Division Department of Natural Resources and Parks

Biological Colonization on HDPE Outfall Material

Acknowledgements The following individuals provided species identification from photographs and their assistance was greatly appreciated: Dani Burgess (Washington State Dept. of Ecology), Megan Dethier (University of Washington), Maggie Dutch (Washington State Dept. of Ecology) , Greg Jensen (University of Washington) , and Gretchen Lambert. Global Diving retrieved the sample plates and their efforts were also appreciated, particularly given the difficulty of retrieving the plates at the deep depths.

Citation King County. 2017. Biological Colonization on High Density Polyethylene Outfall Material in Puget Sound: Year 2 Results. Prepared by Kimberle Stark, Wendy Eash-Loucks, and Jeffrey Lundt, King County Department of Natural Resources and Parks. Seattle, Washington.

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Table of Contents

Executive Summary...... v 1.0 Introduction ...... 1 1.1 Site Description ...... 1 2.0 Sampling design and methods ...... 4 2.1 Sample Retrieval and Field Methods ...... 6 2.2 Photographic Analysis ...... 8 2.3 Data Analysis ...... 10 3.0 Results ...... 12 3.1 100 foot depth ...... 12 3.2 300 foot depth ...... 13 3.3 600 foot depth ...... 14 3.4 Reference: 600 foot depth ...... 15 3.5 Results by phylum ...... 16 3.6 Statistical Analyses ...... 18 4.0 Discussion ...... 21 5.0 References ...... 23

Figures

Figure 1. Brightwater marine outfall location at Point Wells (white lines are 20 ft contours) ...... 3 Figure 2. Sample locations as indicated by blue circles (‘R’ indicates reference site) ...... 5 Figure 3. Configuration of pipe segments before (A) and after placement (B) ...... 6 Figure 4. Global Diving ROV retrieving a sample ...... 7 Figure 5. Flexible grid placed over each plate. The bare patch in the middle is where the plate was bolted to the concrete ballast...... 8 Figure 6. Average percent cover of live (total 4.2%) non-motile organisms at the 100 ft depth ...... 13 Figure 7. Average percent cover of live (total 19.6%) non-motile organisms at the 300 ft depth ...... 14 Figure 8. Average percent cover of live (total 24.6%) non-motile organisms at the 600 ft depth ...... 15

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Figure 9. Average percent cover of live (total 25.2%) non-motile organisms at the Reference 600 ft depth ...... 16 Figure 10. Average percent cover of non-motile organisms by phylum for all sites ...... 17 Figure 11. Average count of motile organisms by phylum for all sites ...... 17 Figure 12. MDS plot for percent cover of non-motile organisms (circles represent percent similarity as determined by the CLUSTER analysis) ...... 19 Figure 13. MDS plot for presence-absence data for all organisms (circles represent percent similarity as determined by the CLUSTER analysis) ...... 19 Figure 14. SIMPER results showing the contribution of the top eight taxonomic groups of organisms to the percent similarity of replicate samples ...... 20

Tables

Table 1. Organism categories...... 9 Table 2. SIMPER percent similarity results between sites and for replicates at each site...... 20

Appendices Appendix A: Replicate Data Summary Appendix B: Select Plate Photos Appendix C: Organism Photos

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EXECUTIVE SUMMARY The Brightwater Treatment Plant marine outfall includes two pipes that discharge highly treated wastewater effluent to Puget Sound. The outfall, consisting of high density polyethylene (HDPE) material, is located over a mile offshore near Point Wells and terminates at the -600 foot (ft) water depth referenced to mean lower low water (MLLW). Underwater video of the outfall pipes was recorded between 2009 and 2013 at depths between approximately -80 to -130 ft MLLW. Additional video was collected at -300 ft MLLW during this same time period. Video showed that the pipes in shallower water were first colonized by barnacles about one year after placement, followed by other organisms. In addition, motile species such as shrimps and fishes were observed utilizing the pipes as habitat.

Due to the high degree of biological colonization on the outfall pipes observed in the videos, this 10-year project was initiated in 2012 to determine if marine organisms attached to the pipe may affect the structural integrity of the HDPE material over time, and to document the diversity and abundance of marine organisms on the pipe. It is anticipated that the biological data collected as part of this project will aid state natural resource agencies in assessing the effectiveness and amount of beneficial habitat that artificial structures, such as outfall pipes, provide to various marine organisms. Biological observation results from the first sampling event in 2014 are included in this report and results from the structural integrity testing will be reported separately.

Twenty-seven approximately 2-ft2 (1-ft by 2-ft) portions of HDPE pipe material were placed in 2012 by a remotely operated vehicle (ROV) at the -100 ft, -300 ft, and -600 ft MLLW depths adjacent to the south outfall pipe and at a -600 ft reference site further away from the outfall. The pipe material will remain in place for predefined time intervals: 2, 5, and 10 years. Three replicate pipe material segments and one backup segment were placed at each depth and for each time interval.

The 2-year time interval samples were retrieved in September 2014 and percent cover (for non-motile species) and counts (motile species) determined for each plate. Although most of the surface on the plates was covered at each site, large differences were seen in the number and type of organisms present between the four sites. Overall, the shallowest 100 ft depth had the fewest organisms, both in percent cover and diversity, while the two 600 ft depth sites (including the reference site) had the most diversity and highest abundance. The two 600 ft sites were similar to each other and depth appeared to be a determining factor for the distribution of many organisms.

Of note is that all four of the tunicates identified to species for this project were documented beyond their published depth range. This is likely a consequence of the difficulty in sampling at these depths and lack of studies focused on organisms at the depths of this project, rather than an expansion of depth range for these species.

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Two years after placement, non-motile organisms have colonized the majority of the available surface area on the plates with motile invertebrates also present. This confirms the observations seen in the underwater video footage of the outfall pipes at the 100 and 300 ft depths where both the pipes and concrete anchors were rapidly colonized by barnacles, then by other species. Although the plates at the shallowest 100 ft depth were almost completely covered with barnacles and barnacle scars, very few barnacles were alive, likely due to predation by fishes and other invertebrates. It is expected that recruitment of new barnacles will occur, along with other organisms.

Additional video footage of the outfall pipes and settling plates will be obtained when the next set of plates are retrieved in 2017, five years after placement. This video, along with video recorded on an annual basis since 2009, will allow for an assessment of habitat use of the pipes by a variety of marine organisms, including fishes. Video results will be reported in a separate document and together with the results from this study, will allow for a more comprehensive assessment of marine organisms utilizing the pipe structures for habitat.

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1.0 INTRODUCTION King County’s Brightwater Treatment Plant, completed in 2012, includes a marine outfall that discharges highly treated effluent to Puget Sound. The outfall starts near Point Wells and extends approximately 5,000 ft west from the shoreline, terminating at the -600 foot (ft) water depth referenced to mean lower low water (MLLW). The outfall consists of twin, 63-inch diameter pipes each with a 250 ft long diffuser at the end of the pipe (Figure 1). The diffusers are staggered for an overall length of 500 ft. The outfall pipes are composed of high-density polyethylene (HDPE). To counteract the buoyancy of the HDPE, the outfall pipes are anchored to the seafloor by concrete collars. Outfall construction was completed in October 2008, however, effluent was not discharged until the plant was completed in November 2012.

From 2009 to 2013, underwater video of the outfall pipes and concrete collars was collected annually during eelgrass monitoring surveys at depths between approximately - 80 to -130 ft MLLW. Additional outfall video during these surveys was also collected at - 300 ft MLLW. Video showed that the pipes in shallower water (i.e., the -80 to -130 ft MLLW depths) were first colonized by barnacles about one year after placement, followed by other organisms. Several other sessile species, such as anemones, calcareous tube worms, and bivalves, also attached to the pipes and concrete collars. In addition, motile species such as shrimps, squat lobsters, sea stars, and fishes, were observed utilizing the pipes and anchors as habitat. By 2013, video showed that the pipes and concrete collars were almost entirely covered with marine organisms at the shallower depths and mostly covered at the deeper depth. Colonization was slower at the deeper depths; however, epibenthic crustaceans such as shrimp (spot prawns in particular) and squat lobsters were more abundant at the -300 ft MLLW depth.

In response to the amount of biota attached to the oufall, this 10-year project was initiated in 2012 to determine if marine organisms attached to the pipe affect the structural integrity of the HDPE material over time, and to document the diversity and abundance of marine organisms on the pipe. It is anticipated that the biological data collected as part of this project will aid state natural resource agencies in assessing the effectiveness and amount of habitat that artificial structures provide to various marine organisms.

Biological observation results from the first sampling event in 2014 are reported here and results from the structural integrity testing will be reported separately. Details of the structural integrity analyses are provided in the project Sampling and Analysis Plan (King County, 2012).

1.1 Site Description The Brightwater Treatment System parallel marine outfall pipes leave the shoreline in the southwest corner of unincorporated Snohomish County at Point Wells. Running just south of due west, the 5,000 ft long pipes discharge south of the County line in King County. Point Wells is a natural point of land that was reinforced with a rubble seawall by the railroad

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more than 100 years ago. The site borders are Puget Sound to the west, the terminal dock for the Paramount Asphalt and Petroleum Storage Facility to the north, and the Burlington Northern Santa Fe railroad to the east. Other land uses within a 0.25-mile of radius of the outer perimeter of the site include single-family residences to the southeast, and a forested terrace and ravine to the east and northeast.

The marine outfall pipes are buried beneath the seafloor landward to a water depth of -80 ft MLLW. From -80 ft MLLW to where the pipes terminate at -600 ft MLLW, the pipes lie on the seafloor. For the portion of the outfall lying on the seafloor, the pipe lengths are approximately 4,255 ft for the north pipe and 4,505 ft for the south pipe.

Sediment at the outfall terminus consists primarily of fine-grained (silt and clay) material characteristic of a silty, depositional environment, with total fines ranging from 50 to 60% (King County, 2008). Sediment at the shallower outfall depths consists primarily of sand with only a small amount of fines (<2%) (King County, 2009). There is little to no hard substrate (cobble size or larger) available in the immediate vicinity of the pipes.

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Figure 1. Brightwater marine outfall location at Point Wells (white lines are 20 ft contours).

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2.0 SAMPLING DESIGN AND METHODS This project entails placement of HDPE pipe material at multiple depths and sampling the material at set time intervals for biological colonization and structural integrity. HDPE pipe material was placed near the seafloor in September 2012 at the -100 ft, -300 ft, and -600 ft depths proximal to the south outfall pipe. As the -600 ft depth is located near the diffuser portion of the outfall, a reference site at -600 ft (herein referred to as 600 ft reference) but away from potential effluent impacts was also included (Figure 2). The pipe material will remain in place for pre-defined time intervals: 2, 5, and 10 years.

Forty approximately 2-ft2 (1-ft by 2-ft) portions of HDPE pipe material were placed by a remotely-operated vehicle (ROV) at all four sites. Three replicates were placed at each depth and for each time interval plus one additional portion as a backup, for a total of 10 pipe segments at each depth. Replicates are included due to inherent biological variability and to mitigate this variability to the extent feasible.

As the pipe material is buoyant, each portion was bolted to a solid concrete block in order for the pipe material to be contained within the water column and not resting directly on the seafloor, thereby replicating to the extent practical the pipe/weight configuration. Each block was placed within an aluminum tube which was welded to an aluminum frame to remain upright in the correct orientation and placed no further than 15 ft south of the outfall pipe. The pipe material was oriented in a vertical direction to represent the shape of the outfall pipe and to minimize sedimentation. Figure 3 shows the configuration of the pipe segments placed at each depth.

Although it is not expected that effluent will have an effect on biota colonization of the pipe material at the -600 ft MLLW depth or the other two depths further away from the diffusers, a reference site at the same depth as the diffusers was included in this study. Effluent dilutions within 80 ft of the discharge are expected to be between 377 to 950:1 dependent on various conditions and not expected to have a measurable effect on ambient salinity. Effluent dilutions beyond 800 ft from the discharge ports are expected to be between 400 to 6,000:1. The -300 ft MLLW and -100 ft MLLW sites are over 3,400 ft and 4,400 ft east of the end of the outfall, respectively. As only the -600 ft MLLW depth is located near a diffuser, the 2-ft2 portions of the pipe material were placed along the same -600 ft MLLW bathymetric contour 300 ft south of the outfall pipe. As with the other three sites, three replicates were placed at the reference site for each time interval and one backup segment. No reference sites were used for the shallow depths as they are located far from the diffusers.

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Figure 2. Sample locations as indicated by blue circles (‘R’ indicates reference site).

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Figure 3. Configuration of pipe segments before (A) and after placement (B).

2.1 Sample Retrieval and Field Methods The first set of samples were retrieved September 15–16, 2014 for the 2-year time interval. An ROV deployed by Global Diving & Salvage, Inc. was used to retrieve three replicates at each depth. Figure 4 shows retrieval of one of the samples. Immediately following retrieval, a plate was placed in a large tub in order to recover motile organisms that detached from the plate. Each plate was assessed for total percent cover by placing a flexible mesh grid

King County Science and Technical Support Section 6 May 2017 Biological Colonization on HDPE Outfall Material over the top side of the plate and estimating the biota coverage in each grid cell. Only the top (convex) side of the plate was assessed as this side more closely replicates the configuration of the outfall pipes and settlement conditions. It was anticipated that settlement on the plates would differ between the convex and concave sides; however, organisms would have little opportunity to settle on the inside (concave side) of the outfall pipes due to the closed pipe ends and freshwater flow within the pipes. A flexible grid was necessary as the plates were cut from pieces of curved pipe, thus, a flat mesh grid would not remain flush on the plate. The mesh grid contained 21 cells, each 3.94 inches × 3.54 inches (10 cm × 9 cm) (Figure 5). To the extent possible, macroscopic biota were identified and enumerated in each grid cell and recorded on field sheets.

Figure 4. Global Diving ROV retrieving a sample.

Digital photographs were taken in .jpg format of each sample at a minimum of three megapixel (MP) resolution. At least one photograph was taken of the entire plate and multiple photos were taken zoomed in on each grid cell. Site, depth, replicate, and grid cell identifiers were placed in each cell prior to each photograph.

Upon completion of photographs and organism identification and enumeration, any large biota (i.e., anemones) and motile organisms (i.e., crabs) were detached and returned to the water. The pipe samples remained outside to dry for several weeks. As much organic material as possible, including barnacle shells, was removed to prepare the plates for the structural integrity tests.

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Figure 5. Flexible grid placed over each plate. The bare patch in the middle is where the plate was bolted to the concrete ballast.

2.2 Photographic Analysis All organisms were identified to the class level at a minimum and whenever possible, identified to the species level. References used for identification included Kozloff (1996), Lamb and Hanby (2005), and Jensen (2014). Photographs of some species were sent to the following taxonomic experts for identification down to the family or species level; Gretchen Lambert-tunicates, Greg Jensen-crustaceans; Dani Burgess/Maggie Dutch-polychaetes, and Megan Dethier-molluscs.

Image J®, an image processing software created by the National Institutes of Health, was used to process the photographs. The software was used to determine percent cover of non-motile species and/or taxonomic groups by calculating area and pixel value statistics of each grid cell. The pixel area of each grid cell was determined and then compared to the pixel area for each target species/taxonomic group within that grid cell to derive the percent cover value for that species/group. To estimate total percent cover of the entire grid cell, a plugin grid feature set at a resolution of 80,000 pixels2 per point was used as an overlay.

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Only photos taken with the camera positioned directly above the grid cell (90 degree angle to the plate) were used to calculate pixel area in order to minimize perspective error. Motile organisms, such as crabs and errant polychaetes, were counted but not used in total percent cover estimates.

Organisms attached to the plate (non-motile) were placed into one of 20 taxonomic categories in Table 1 below, including an ‘unidentified’ category. Motile organisms were placed into one of eight categories. The snail Trichotropis cancellata was not placed into the general ‘gastropod’ category as it was readily identifiable and abundant at one depth. For the tube-dwelling amphipod Ericthonius rubricornis, percent cover was estimated based on the area of the tubes not the actual organism. Many of these amphipods were out of the tubes on the plate, in the tub, or had jumped outside the tub area entirely; therefore, to obtain a more accurate comparison between plates, the area of the tubes was calculated.

Percent cover results were averaged and counts summed for all 21 grid cells to derive values for each plate. For motile species, observations from the field sheets were used to adjust results from the photographic analysis as needed. Results from the three replicates at each site were averaged and data analyses are described below.

Table 1. Organism categories.

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2.3 Data Analysis Following photographic identification, summary and multivariate statistical analyses of species composition were calculated for all sites. Organisms were summarized by the 28 taxonomic categories listed in Table 1 as well as at a higher taxonomic level (Phylum). Percent cover was determined for each replicate and all three replicates averaged to derive a total percent cover by site.

Two multivariate statistical analyses were conducted using Primer v6: non-metric multi- dimensional scaling (MDS) and hierarchical clustering (CLUSTER). MDS is a statistical tool that enables the comparison of complex biological community data. This ordination technique allows for the visualization of data in a two-dimensional space. Replicates and sites that most closely resemble each other in organism presence and abundance are grouped closer together and those more dissimilar are oriented further apart (i.e., the farther the points are from each other, the less similar they are in organism assemblages). The MDS values were used to produce a Bray-Curtis similarity matrix, which was then used to produce a plot (Shepard diagram) based on the rank-ordered similarity score of each sample. As is common for proportional data, the percent cover data were arcsine square- root transformed prior to data analysis to stabilize the variance and minimize weighting of the highly abundant and less common taxa. The percent cover data for the 12 replicates (4 sites, 3 replicates each) were compared to one another to create the MDS plots as well as conduct the CLUSTER analysis. The MDS plots were created using the best 2-D configuration with the stress set at 0.05.

In addition to MDS, a hierarchical cluster analysis (CLUSTER) was conducted on the Bray- Curtis similarity matrix to provide additional information on the degree of similarity of replicates and sites in relation to one another by linking samples based on their similarity. The CLUSTER analysis was done in conjunction with a SIMPROF (similarity profile) test. The SIMPROF test determines which sites are statistically similar to one another, creating clusters. The SIMPROF test calculates a series of similarity profile permutations that looks for statistically significant verification of genuine clusters, in this case clusters or replicate samples. This analysis included 1000 permutations and a pre-defined significance level of five percent. These additional analyses were used in conjunction with the MDS to create a more informative visual for interpretation in which circles are drawn around clusters based on their degree of similarity.

Arcsine log-transformed percent cover replicate data were then used to conduct an ANOSIM (analysis of similarities) test to determine if there was a significant difference between community compositions between sites. This test generates an R value that is scaled to lie between -1 and +1, with a value of 0 representing no difference between sites.

A SIMPER (similarity percentages) analysis was also conducted on the arcsine square root transformed percent cover data for each replicate. This test evaluates which taxonomic groups were principally responsible for the site grouping. The test deconstructs the Bray- Curtis similarities between sites and among replicates into percentage contributions from each taxomic group towards the overall similarity.

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Finally, the percent cover and count data (motile and non-motile organisms) were combined and converted to presence/absence data (given either a 0 or 1). Replicates were then compared to one another using a Bray-Curtis similarity matrix and the CLUSTER, MDS, and SIMPROF tests described above were conducted.

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3.0 RESULTS Summary results from all sampling locations are provided below. A more detailed summary table for each replicate is provided in Appendix A and representative plate photos from each location are provided in Appendix B. Select organism photos are provided in Appendix C.

3.1 100 foot depth Plates at the shallowest 100 ft depth had the least diversity of all the depths, with eight non-motile and two motile taxonomic categories identified. All the plates from this depth were covered with a high percentage of barnacle scars or dead barnacles and contained very few live barnacles (ranged between 0 and 0.49% cover, average of 0.21%). Although average total percent cover was 56.3% due to the abundance of dead barnacles (52.1%), average percent cover for live organisms was only 4.2%. Barnacle settlement was substantial at this depth, however, predation by other invertebrates and fish, surfperch in particular, likely limited the presence of live barnacles. Video from previous outfall monitoring surveys showed large schools of surfperch present at this depth.

The coral bryozoan, Lichenopora sp. (phylum Bryozoa) was the most abundant live organism at this depth and was more abundant at 100 ft than the deeper depths. The jingle Pododesmus macrochisma (phylum Mollusca) and branched bryozoan Crisia sp. were the next most abundant organisms at this depth. Figure 6 shows the abundance of non-motile organisms at the 100 ft depth in the 10 most abundant categories across all depths. A small amount of the seaweed Ulva spp. (1.1%) was found attached to one of the three replicates at this depth but not at the deeper depths due to light limitations. The other notable organism at this depth was the presence of eight Glebocarcinus oregonensis (pygmy rock crab, family Cancridae) crabs on the plates (average 2.7). The only other motile organisms seen at this depth were two errant polychaetes, one identified in the genus Phyllodoce.

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Figure 6. Average percent cover of live (total 4.2%) non-motile organisms at the 100 ft depth.

3.2 300 foot depth Thirteen non-motile and five motile taxonomic groups of organisms, were identified at the 300 ft depth. Figure 7 shows the abundance of non-motile organisms in the 10 most overall abundant categories for all the depths. All three types of bryozoans (branched, coral [Lichenopora sp.], and encrusting [including Schizoporella unicornis]) were found on the plates at this depth with encrusting bryozoans the most abundant. Overall, a tube worm in the family Sabellariidae (Neosabellaria cementarium) was the most abundant organism at this depth and calcareous tube worms (family Serpulidae) were also abundant. The percent cover for calcareous tube worms was determined from the area of the outside tube and it is likely that not all the tubes contained live worms. Four solitary tunicates, Chelysoma productum, were seen on one of the replicates. There were very few live barnacles at this depth (0.03%). Total percent cover for this depth averaged 47.9%, mainly due to the presence of dead barnacles.

Motile organisms with the highest numbers at this depth were errant polychaetes (mean =3) and the hairy checkered snail Trichotropis cancellata (mean=13). Several of the errant ploychaetes that were able to be identified down to species were the scaleworm Harmothoe imbricata. For Trichotropis cancellata, two of the replicates each had 18 snails and the third replicate had 4 snails. Trichotropis cancellata is known to be a kleptoparasite and steal food from calcareous tube worms, which were also abundant at this depth (Pernet and Kohn 1998).

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Figure 7. Average percent cover of live (total 19.6%) non-motile organisms at the 300 ft depth.

3.3 600 foot depth Fourteen non-motile and three motile taxonomic groups were identified at the 600 ft depth. Figure 8 shows the abundance of non-motile organisms in the 10 most overall abundant categories. Both branched and encrusting bryozoans were found at this depth, with encrusting byrozoans being the most abundant organism with an average of 12.3% cover. The coral bryozoan, Lichenopora sp. was seen at the two shallower depths; however, it was not seen at this depth. Bivalves, primarily the clam Hiatella arctica, were the next most abundant organism at this depth with an average of 4.0% cover. The tube-dwelling amphipod Erichthonius rubricornis was relatively abundant at this depth but was not seen at the two shallower depths. Unlike the shallower depths, spiny pink scallops, Chlamys hastata, were seen at this depth in small numbers. The scallops were recently settled as most were less than one centimeter in diameter. A total of nine solitary tunicates, including Chelysoma productum, Styela gibbsii, and Boltenia villosa, were seen on two of the replicates at this depth. Total percent cover at this depth averaged 60%.

Few motile organisms were seen on the plates at this depth. One Majoidae crab, Chorilia longipes, was seen on each of the three replicates.

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Figure 8. Average percent cover of live (total 24.6%) non-motile organisms at the 600 ft depth.

3.4 Reference: 600 foot depth Fifteen non-motile and seven motile taxonomic groups of organisms were identified at the reference site. Figure 9 shows the abundance of non-motile organisms in the 10 most abundant categories. Encrusting bryozoans were the most abundant organisms at this site (8.2%) followed by clams (4.6%) comprised primarily of Hiatella arctica. As with the 600 ft depth, tube-dwelling amphipods were also fairly abundant at the reference site as well as the jingle bivalve clam. Both branched and encrusting bryozoans were seen at this site but not coral bryozoans, which were observed at the shallower depths. Three scallop species, Chlamys hastata, C. rubida, and Delectopecten vancouverensis, were seen on two of the three replicates with an average 0.8% cover at this site. As with the 600 ft depth, the scallops at the reference site were likely recently settled as most were less than one centimeter in diameter. A total of 16 solitary tunicates were seen at this site including the two species that were able to be identified, Chelysoma productum and Corella willmeriana. Total percent cover at this site averaged 67.8%, the highest of all four sites.

The 600 ft reference site had the most motile organisms of all the four sites, which is notable considering these organisms, as well as those at the 600 ft depth site, remained on the plates during the retrieval process from the sea floor to the surface. Organisms in five different phylums were recovered from this site. Two replicates had four and five errant polychaetes respectively, with an average of three for all replicates. Most of the errant polychaetes were scale worms in the order Phyllodocida. A flatworm (phylum Platyhelminthes) and two ribbon worms (Tetrastemma nigrifrons) in the phylum Nemertea were also seen at this site. Similar to the 600-ft depth, one Majoidae crab, Chorilia longipes, was seen on two of the three replicates at the reference site.

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Figure 9. Average percent cover of live (total 25.2%) non-motile organisms at the Reference 600 ft depth.

3.5 Results by phylum Figures 10 and 11 show the percent cover of non-motile and motile count of organisms by phylum for all four sites, respectively. Non-motile organisms were present in a total of eight phyla. Bryozoa was the most represented phylum, primarily due to the amount of encrusting bryozoans at both the 600 ft and 600 ft reference sites. Organisms in the second highest percent cover phylum, Mollusca (organisms in six categories), were present at all four sites. Annelida was third most represented phylum by percent cover, but only due to the amount of worms present at the 300 ft depth.

Motile organisms were present in a total of five phyla. Mollusca was the most predominant phylum due to the number of Trichotropis cancellata snails at the 300 ft depth. Arthropoda was the next most abundant phylum due mainly to the presence of crabs and amphipods at the 100 and 600 ft reference sites. Only one organism in each of the Platyhelminthes and Echinodermata phyla were observed at the 600 ft reference site and 300 ft depth, respectively.

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Figure 10. Average percent cover of non-motile organisms by phylum for all sites

Figure 11. Average count of motile organisms by phylum for all sites.

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3.6 Statistical Analyses Figure 12 shows the results of the MDS using the percent cover data. The MDS plot is a two- dimensional interpretation of the complex biological community data for which samples that are more similar to one another are grouped closer together and those that are further from one another are less similar in their organism assemblage. The depths exhibited distinct groupings with the two 600 ft depths grouping together.

Percent cover data were also converted to presence-absence and combined with the motile organism presence-absence data. The MDS plot created from these data is shown in Figure 13. The resulting MDS plots of presence-absence data and percent cover data (arcsine transformed) showed similar groupings of replicates by depth. The percent similarity between the replicates using presence-absence data was consistent with transformed percent cover data results. A common measure used to evaluate the closeness of fit between the MDS ordination distances and the actual dissimilarities is the stress level. The stress level for both MDS analyses was low at 0.05 which is considered excellent to good (Clarke, 1993). A large stress level (>0.2) result indicates the ordination distances never reached a reasonable representation of the dissimilarity matrix.

The CLUSTER analyses conducted using both the percent cover and the presence-absence data were similar to each other and indicated little difference between the 600 ft depth site and reference site, with replicates for each of these sites grouping together. The CLUSTER results are represented by the circles around the samples in Figures 12 and 13. The results of the SIMPROF test showed that the 100 ft replicates were not significantly different from each other but were significantly different than the other sites. The ANOSIM results comparing all four sites and then the three depths (100, 300, and 600 ft) showed that site was significant (p=0.002) and depth was also significant (p=0.001). An ANOSIM ran just between the 600 ft and 600 ft reference sites showed no significant difference (p=0.3).

A SIMPER analysis was conducted using the Arcsine transformed percent cover data to show which of the 19 taxonomic categories (the unidentified category was excluded) explained the similarity or dissimilarity between replicates and sites and to what extent. Results of the SIMPER analysis showed 90 % of the similarity of all sites can be explained by 8 of the 19 taxonomic categories. The 100 ft site had the least diversity overall, and only three categories contributed to 90% of the similarity between replicates. By contrast, six taxonomic categories explain 90% of the similarity between replicates at the reference site. Figure 14 shows the SIMPER results for the eight categories at each site and the percent contribution of those categories to the similarity between the replicates. SIMPER analysis was also conducted between sites to determine how similar the sites were relative to each other. Results of this analysis were similar to the other tests in that the 100 ft site was not similar to the 600 ft or 600 ft reference sites and both 600 ft sites were most similar to each other. Table 2 provides the SIMPER results for the replicates at each site as well as the similarity percentage between sites.

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Figure 12. MDS plot for percent cover of non-motile organisms (circles represent percent similarity as determined by the CLUSTER analysis).

Figure 13. MDS plot for presence-absence data for all organisms (circles represent percent similarity as determined by the CLUSTER analysis).

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100 ft 300 ft

600 ft Ref: 600 ft

Figure 14. SIMPER results showing the contribution of the top eight taxonomic groups of organisms to the percent similarity of replicate samples.

Table 2. SIMPER percent similarity results between sites and for replicates at each site. Site 100 ft 300 ft 600 ft Ref:600 ft 100 ft 57.7 30.6 17.6 14.5 300 ft -- 73.1 47.2 45.1 600 ft -- -- 77.2 68.1 Ref:600 ft ------62.7

indicates the average similarity between replicates

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4.0 DISCUSSION This report documents the diversity and abundance of marine organisms on HDPE pipe material in Puget Sound at three depths after two years. Portions of the pipe material were placed at four sites ranging from 100 to 600 ft MLLW near the Brightwater outfall in 2012. Plates were retrieved in 2014 and after two years, much of the surface on all the plates was covered by non-motile organisms.

Although much of the surface on the plates was covered at each site, large differences were seen in the number and type of organisms present between the four sites. Overall, the shallowest 100 ft depth had the fewest organisms, both in percent cover and diversity, while the two 600 ft depth sites (including the reference site) had the most diversity and highest abundance. The plates at the 100 ft depth were primarily covered with barnacles (both dead and barnacle scars) and little else, whereas both the 600 ft and 600 ft reference plates contained at least one organism in most of the major taxonomic groups seen during this sampling event.

Depth appeared to be a determining factor for the distribution of many organisms, including the coral bryozoan Lichenopora sp. This species was most prevalent at the 100 ft depth, less abundant at 300 ft, and not present at either the 600 ft or 600 ft reference sites. A small cluster of the green alga Ulva sp. was observed attached to a plate at the 100 ft depth, which is likely near the depth limit for this alga due to an insufficient amount of light to allow photosynthesis. Cancridae crabs (the pygmy rock crab Glebocarcinus oregonensis) were more abundant at the 100 ft depth and were not seen at the 600 ft depths, even though 600 ft is well within the depth range for this species. It is possible that pygmy rock crabs (and other motile species) were present on the 600 ft plates but fell off during the relatively lengthy transit to the surface.

Conversely, some species were only present at the deeper depths or found in larger numbers than at shallower depths. Annelids, both calcareous tube worms (family Serpulidae) and tube-dwelling polychaetes (family Sabellariidae) were abundant only at the 300 ft depth. The tube-dwelling amphipod Erichthonius rubricornis was relatively abundant on all replicates for both the reference and 600 ft sites but was not seen at the shallower depths. Tunicates were more abundant at the 600 ft sites than the 300 ft depth and were not present at all at 100 ft. It should be noted that all four of the tunicates identified to species (Chelysoma productum, Corella willmeriana, Styela gibbsii, and Boltenia villosa) were observed beyond their expected depth range (Lamb and Hanby 2005, Gotshall 2005, Lambert and Sanamyan 2001). This is likely a consequence of the difficulty in sampling at these depths and lack of studies focused on epifauna at the depths of this project, rather than an expansion of depth range for these species.

The clam Hiatella arctica was present in small numbers at the 300 ft depth but abundant at both 600 ft depth sites. Three scallop species (Chlamys hastata, C. rubida, and Delectopecten vancouverensis) and the Majoidae crab Chorilia longipes were also present at the 600 ft depth sites but were not seen at shallower depths. Based on their small size, the scallops

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were juveniles and had likely settled within a few months prior to sampling (NPFMC 2005). Organism assemblages were similar between the 600 ft and the 600 ft reference sites, although the reference site had slightly higher percent cover and abundances for most of the organism categories. Based on these results, it does not appear that the effluent has had a substantial impact on the biological colonization and composition of the plates.

As stated in the previous section, the snail Trichotropis cancellata was abundant at the 300- ft depth, which was likely due to the abundance of calcareous tube worms at this depth. Although this snail is a suspension feeder (Harbo 1997), it is also known to have a second mode of feeding style called kleptoparasitism, where it diverts and eats particles captured by calcareous tube worms (Pernet and Kohn 1998).

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5.0 REFERENCES

Clarke, K.R. 1993. Non-parametric multivariate analyses of changes in community structure. Australian Journal of Ecology. Vol 18:117-143.

Gotshall, D.W. 2005. Guide to Marine Invertebrates: Alaska to Baja, California, 2nd Ed. (revised). Sea Challengers. 117 p.

Harbo, R.M. 1997. Shells and Shellfish of the Pacific Northwest, a Field Guide. Harbour Publishing, Canada. 270 p.

Jensen, G.C. 2014. Crabs and Shrimps of the Pacific Coast: A Guide to Shallow-Water Decapods from Southeastern Alaska to the Mexican Border. MolaMarine Publishing, Bremerton, WA. 240 p.

King County. 2008. Brightwater marine outfall diffuser baseline sediment characterization final report. Prepared by Scott Mickelson, King County Dept. of Natural Resources and Parks, Seattle, WA.

King County. 2009. Brightwater Treatment System marine outfall baseline benthic community characterization: construction trench results of the 2007 sampling event. Technical memorandum prepared by Scott Mickelson, King County Dept. of Natural Resources and Parks, Seattle, WA.

King County. 2012. Sampling and analysis plan for HDPE biological and structural integrity assessment of the Brightwater marine outfall, April 2012. Revised 2014. Prepared by Kimberle Stark and Jeff Lundt, King County Dept. of Natural Resources and Parks, Seattle, WA.

Kozloff, E.N. 1996. Marine Invertebrates of the Pacific Northwest. University of Washington Press, Seattle, WA. 539 p.

Lamb, A. and B.P. Hanby. 2005. Marine Life of the Pacific Northwest. Harbour Publishing, Canada. 398 p.

Lambert, G. and K. Sanamyan. 2001. Distapla alaskensis sp. (Ascidiacea, Aplousobranchia) and other new ascidian records from south-central Alaska, with a redescription of Ascidia columbiana (Huntsman, 1912). Can. J. Zool. 79: 1766-1781.

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North Pacific Fishery Management Council (NPFMC). 2005. Fishery management plan for the scallop fishery off Alaska. North pacific Fishery Management Council, Anchorage, AK.

Pernet, B. and A.J. Kohn. 1998. Size-related obligate and facultative parasitism in the marine gastropod Trichotropis cancellata. Biol. Bull. 195: 349-356.

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Appendix A: Replicate Data Summary

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Table A-1 Individual replicate data for all locations.

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Appendix B: Select Plate Photos

Figure B-1 100 ft depth plate and grid photos.

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Figure B-2 300 ft depth plate and grid photos.

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Figure B-3 600 ft depth plate and grid photos.

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Figure B-4 Reference: 600 ft depth plate and grid photos.

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Appendix C: Organism Photos

Serpulid worm Harmothoe sp. Eulalia quadrioculata Phylum: Annelida Phylum: Annelida Phylum: Annelida

tube worm Ericthonius rubricornis Chorilia longipes Phylum: Annelida Phylum: Arthropoda Phylum: Arthropoda

Glebocarcinus oregonensis Schizoporella sp. & Spirobis sp Lichenopora sp. Phylum: Arthropoda Phylum: Bryozoa, Annelida Phylum: Bryozoa

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Crisia sp. Corella willmeriana Styela gibbsii Phylum: Bryozoa Phylum: Chordata Phylum: Chordata

Chelysoma productum Urticina crassicornis Strongylocentrotus sp Phylum: Chordata Phylum: Cnidaria Phylum: Echinodermata

Pododesmus macrochisma Delectopecten vancouverensis Trichotropis cancellata Phylum: Mollusca Phylum: Mollusca Phylum: Mollusca

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Hiatella arctica Chlamys rubida Strongylocentrotus sp. Phylum: Mollusca Phylum: Mollusca Phylum: Mollusca

Crepidula sp. Modiolus modiolus Neptunea lyrata Phylum: Mollusca Phylum: Mollusca Phylum: Mollusca

Tetrastemma nigrifrons flat worm vase sponge Phylum: Nemertea Phylum: Platyhelminthes Phylum: Porifera

C-3