Embed Size (px)
Citation preview
1
University of North Carolina at Chapel Hill
Institute for the Environment, Morehead City Field Site
Fall 2015 Capstone Project
Distribution and Effects of Marine Debris along Carteret
County Shorelines -‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
Kelsey Barnhill, Larisa Bennett, Madeleine Denton, Yohana Dierolf, Harrison Dusek, Ryan Leighton, Kayla Pehl, Adam Rok, Anderson
Tran, Abbey Vinson, Patrick Winner
Course Instructors: Dr. Stephen Fegley and Dr. Johanna Rosman
Graduate Research Consultant: Kathleen Onorevole
Field Site Director: Dr. Rachel Noble
This report summarizes work done by a UNC Chapel Hill undergraduate student team. It is not a formal report of the Institute for the Environment, nor is it the work of UNC Chapel Hill faculty.
2
Table of Contents
Background …………………………………………………………………………………….4
General Survey Methods ………………………………………………………………………6
Chapter 1: Spatial Distribution of Marine Debris
1 Introduction…………………………………………………………………………………….7
2 Methods…………………………………………………………………………………….......7
3 Results…………………………………………………………………………………….........8
4 Discussion……………………………………………………………………………………...15
Chapter 2: Organism Utilization of Marine Debris
1 Introduction…………………………………………………………………………………….16
2 Methods………………………………………………………………………………………...17
3 Results………………………………………………………………………………………….19
4 Discussion……………………………………………………………………………………...26
5 Conclusion……………………………………………………………………………………..28
Chapter 3: Bacterial Analysis
1 Introduction…………………………………………………………………………………….29
2 Methods………………………………………………………………………………………...29
3 Results………………………………………………………………………………………….31
4 Discussion……………………………………………………………………………………...36
Chapter 4: Impacts of Exposure on Marine Debris
1 Introduction…………………………………………………………………………………….37
2 Methods………………………………………………………………………………………...38
3 Results………………………………………………………………………………………….40
4 Discussion……………………………………………………………………………………...43
5 Conclusion……………………………………………………………………………………..45
3
Chapter 5: Comparisons of Marine Debris in Carteret County with Other National and International Locations
1 Introduction…………………………………………………………………………………….46
2 Methods………………………………………………………………………………………...47
3 Results………………………………………………………………………………………….47
4 Discussion……………………………………………………………………………………...53
Chapter 6: Synthesis
1 Summary of Main Findings……………………………………………………………………54
2 Future Work……………………………………………………………………………………55
3 Management Recommendations……………………………………………………………….56
Acknowledgements …………………………………………………………………………….58
References ………………………………………………………………………………………59
4
Background Marine debris is “any manufactured or processed solid waste material that enters the
marine environment from any source” (Gregory & Andrady, 2003). Marine debris has become a major environmental concern because of the deleterious effects it can have on marine life, degradation of water quality, hazards to human health, and hindrance to marine activities such as fishing and recreation (Islam and Tanaka, 2004). Marine debris was first identified as a concern in the 1950s and 1960s (Gregory & Andrady, 2003). The prevalence of debris is expected to increase in the future due to new uses of synthetic material, which degrades very slowly. The availability of these products has also increased and will likely continue to expand in the future (Laist, 1987). It would be very difficult to obtain reliable estimates of the amount of debris currently in the marine environment because there are many different avenues that can bring debris into the marine system and some debris has been in the ocean for many years (Derraik, 2002). Marine debris comes from fishing, both commercial (Cawthorn, 1989, DOC, 1990) and recreational (UNESCO, 1994), and careless beachgoers (Wilber, 1987), as well as land-based sources such as construction and littering (Islam and Tanaka, 2004). Whether dumped from industry or littered by the public, this carelessly-handled waste is washed, blown, or discharged into waterways by rain, snowmelt, and wind (Sheavly & Register, 2007).
While the problem of marine debris exists on a global scale, it is addressed most effectively at regional levels and it is therefore important to take into account local trends. For this reason, our study addressed marine debris in Carteret County in North Carolina, USA. Three sites were selected near Beaufort, North Carolina, based on their location and use, in order to compare the effects of human recreational activities on marine debris presence (Figure 1). Shackleford Banks, an island, was chosen as the site that receives the highest frequency of human use. Many people come to sunbathe, fish, and picnic on this island, more so than the other two sites in our study. Carrot Island was selected for its lower visitor traffic but higher nearby boat traffic and closer proximity to the Town of Beaufort, while Treasure Island was selected as a site that is rarely or never frequented by humans and has little boat traffic (Stephen Fegley, personal communication; Nov. 2015). By incorporating sites with varying degrees of human use, we hoped to differentiate between the amount of trash deposited directly by humans and that deposited indirectly by waves and currents.
5
Figure 1. Our three sampling sites (outlined in orange) were positioned at different distances away from Beaufort.
Shackleford Banks sampling site was on the sound side of the island, which experiences strong tides and most likely more human traffic than Treasure Island, but less than Carrot Island. The sampling transect for this site extended to the east of the ferry dock. Treasure Island lies to the South of Carrot Island and to the East of Horse Island. It was the second closest to the highest area of human activity (Beaufort), yet receives less human visitation. We sampled the southwest beach of the island, which was the longest continual strip of beach on the island. Our Carrot Island sampling site lies Northwest of the other two sites and is the closest to Beaufort as well as the most highly visited. The stretch of beach we sampled was is adjacent to Taylor’s creek, a major waterway in Beaufort with significant boat traffic.
The purpose of this study was to take stock of current local marine debris levels and assess its effects on organisms and human health, as well as to place our findings into a larger geographical context. Our study is divided into five focus topics: 1) spatial distribution, 2) debris utilization by organisms, 3) bacterial presence, 4) exposure to the marine environment, and 5) geographical context. Through our surveys and experiments, we aimed to provide an informative review to residents of Carteret County as well as identify areas of focus for management and targeted clean-up of marine debris.
6
General Survey Methods Debris surveys were conducted at Carrot Island, Shackleford Banks, and Treasure Island.
In order to quantify the spatial distribution of debris as well as its variability in our surveys, we created a system comprised of a uniform transect and tidal zone delineations (Figure 2). A 200-m long section of beach at each site was selected based on easy access for our boat and away from locations of previous cleanups. Each 200-m section was divided into 5 contiguous 40-m long zones, labeled A-E to create 5 different replications at each site. GPS coordinates were taken at the beginning and end of each transect line at every site (Figure 3).
Figure 2. An aerial view of part of our sample beach at Shackleford banks showing the delineation of different sampling zones. The exact dimensions of each zone varied at each site and were decided based on natural topography. The 200m line is not to scale, but is included to illustrate how the sampling zones were ordered across the transect.
Figure 3. Coordinates of 200m sampling transect across sites.
Sampling Site Transect Start Transect End Carrot Island 34° 70’90.1”N 76°63’67.3”W 34° 70’93.8”N 76°63’67.3”W
Treasure Island 34° 70’06.9”N 76°63’76”W 34° 70’19.3”N 76°63’88.8”W Shackleford Banks 34° 68’60.1”N 76°64’16.4”W 34°68’64.4”N 76°64’37.3”W
Each sampling site was divided into the general tidal delineations of supratidal, intertidal, and subtidal, with some site-specific variations. Across sites, the supratidal zone extended from the furthest landward point of salt intrusion, estimated by a change in grass community composition from Spartina alterniflora to Spartina patens, moving seaward to the high tide mark, identified by a line of wrack on sandy beach or the grass Juncus roemerianus in marsh areas. The intertidal zone extended from the bottom of the supratidal zone (high water wrack line or J. roemerianus) to the middle of the swash zone at low tide. The subtidal zone extended from the low tide line to the maximum visibility depth. The maximum visibility depth of the subtidal zone was usually 0.6-0.9 meters across sites.
Carrot Island beach was initially divided into intertidal and supratidal zones based on where the high tide line appeared. However, due to the large amount of debris that appeared at the intersection of the two zones, the intertidal was divided into lower intertidal and upper
7
intertidal zones for the other two sites. The upper intertidal zone extended 1.5 meters seaward into the intertidal zone from the supratidal boundary. The widths of the other zones were dictated by environmental boundaries and were measured in each section (A-E). At Shackleford, the intertidal shoreline was much larger (12-17m) and more gradually sloping than any other site and the supratidal zone was much larger than the intertidal zone. By comparison, Treasure Island had a smaller intertidal zone that was mostly marsh rather than sandy beach. Debris was collected at low tide in order to gather material across the full extent of the intertidal zone. Our methods were chosen because they were similar to those used in previously published studies (Silva, 2009). Debris was placed into Ziploc bags and sorted according to tidal zone and replicate number (A-E).
Chapter 1: Spatial Distribution of Marine Debris in Carteret County 1. INTRODUCTION
To better understand how marine debris affects the environment and the
organisms within it, it is imperative to understand what type of debris is most common and where it is located. Synthetic organic polymers, such as plastic and polystyrene, are becoming much more prevalent in human daily life (Derraik 2002). Plastic is a very useful substance due to its unique properties: it is extremely durable and degrades very slowly. It is also lightweight, cheap, and waterproof (Law et al., 2010). These same properties also make plastic a potentially harmful contaminant in the marine environment.
Similar surveys conducted globally have identified plastics and polystyrene as the most and second-most abundant types of debris, respectively (Silva et al., 2015; Kuo and Huang 2014; Ribic et al., 2012). We wanted to know what type of debris was prevalent, as well as the tidal zone and environment where it was found. We were interested in how marine debris was spatially distributed throughout shorelines in the estuarine habitat of Carteret County to see if concentrations varied among areas. We hypothesized that the greatest proportion of debris would be plastic. We also hypothesized that the greatest abundance of debris would be found in areas with the greatest amount of human use.
2. METHODS
After collection, debris was catalogued noting location. In the laboratory, we determined the debris type, size, and mass. We calculated the mass of debris/unit area through weighing on a digital scale each piece of debris found after cleaning off any sand or mud and calculating total weight per area, total number of debris and the abundance of different debris materials at each site.
8
We used ANOVA tests using the variables of location, tidal zone (intertidal and supratidal), and location x tidal zone to determine whether or not the sites and zones had significant differences. To determine which specific sites were significantly different, we used a Tukey-HSD post-hoc test. We did the same analysis with only Treasure Island and Shackleford Banks with the divided intertidal and supratidal zone.
For the comparison between debris type, we used ANOVA tests with the variables of site, type, and site x type for the 5 most common types of debris across all the sites: plastic, Styrofoam, metal, wood, and glass. To determine which specific sites were significantly different, we used a Tukey-HSD post-hoc test. As above, with Treasure Island and Shackleford Banks we analyzed the data with the divided intertidal and supratidal zone.
3. RESULTS
Shackleford Banks and Treasure Island were both found to have significantly higher debris mass per unit area (g/m2) in the supratidal than the intertidal zones, while the opposite pattern was observed at Carrot Island. For the supratidal zone, Treasure Island had the lowest mean mass concentration of debris followed by Carrot Island, with the highest being found at Shackleford Banks (Figure 4). The debris concentration in Carrot Island’s intertidal was only about 19% greater than its supratidal. While all three samples showed significant differences, the other two samples showed greater differences. Treasure Island’s supratidal zone had nearly a 3 times higher concentration than its intertidal; meanwhile, Shackleford Banks had the highest disparity between its two tidal zones, about a threefold increase between zones.
Location (P < 0.0001), tidal zone (P < 0.0001), and the interaction between the two (P < 0.0001) were found to be significant in relation to the mass per area concentrations for the divided zone analysis for Shackleford Banks and Treasure Island. The two divided intertidal zone analyses yielded a significant contrast for Shackleford Banks: a dominant higher mass concentration in the upper intertidal zone, with a drastically smaller lower intertidal zone concentration (Figure 4). Shackleford Banks’ upper intertidal to lower intertidal concentration ratio was 14.5, while Treasure Island’s wasn’t too much lower at 10.8, implying the majority of debris mass was located in the small upper intertidal zone, while the large area of the lower intertidal ended up lowering the overall intertidal mass concentration.
9
Figure 4. Mean mass (in g/m2) of debris in each tidal zone per location ± SE
Location (P = 0.0005), tidal zone (P = 0.027), and the interaction between the two (P = 0.0225) were found to be significant in relation to the abundance per area concentrations. The debris abundance concentration (#/m2) yielded similar trends between each of the 3 locations and their zones (Figure 5). Carrot Island displayed no significant difference in the mean concentration of debris abundance between its two tidal. Shackleford Banks’ supratidal zone was significantly different than every other tidal zone and location, having a 3 times higher concentration than Carrot Island and over 7 times higher concentration than Treasure Island of marine debris abundance.
0"
5"
10"
15"
20"
Shackleford"Banks"
Treasure"Island" Carrot"Island"
Mass$p
er$Area$(g/m
2 )$
Supra:dal"
Inter:dal"
0"
10"
20"
30"
40"
50"
60"
70"
80"
Shackleford"Banks" Treasure"Island"
Mass$p
er$Area$(g/m
2)$
Sampling$Loca6on$
Supra=dal"
Upper"Inter=dal"
Lower"Inter=dal"
10
Figure 5. Mean number of debris items per m2 in each tidal zone for each location.
Shackleford Banks and Treasure Island, which were the last two sites sampled, exhibited higher supratidal concentrations than intertidal; however, only Shackleford Banks’ difference was significant, and when the intertidal zones of both are divided into upper and lower intertidal, the upper intertidal exceeds the lower intertidal concentrations of both. Shackleford Banks’ upper intertidal was significantly greater than its supratidal zone and 35 times higher than its lower intertidal zone, while Treasure Island displayed a 16 time greater concentration in its upper intertidal compared to its lower intertidal.
The greatest frequency of mass of the total debris for each zone and location was always either <0.1 g or 0.1-0.9 g, except for the 60-500 g bin for Shackleford Banks’ lower intertidal. (Figures 6). In the supratidal zones of each location, the <0.1 g weight bin had between 21% and 57% of debris, while the 0.1-0.9 g was almost consistently 20% of total debris, meaning 41% -77% of debris was accounted for in the first two weight bins. The general trend of highly frequent small mass debris continues fairly closely, with the intertidal for each location. 42% of Shackleford Banks’ debris was <0.1 g, and 64% of it was <0.9 g, 22% of Carrot Island’s debris
0"
0.2"
0.4"
0.6"
0.8"
1"
Carrot"Island"
Shackleford"Banks"
Treasure"Island"
Num
ber'o
f'Deb
ris/m
2 '''
Sampling'Loca7on'
Supra=dal"
Inter=dal"
0"
0.2"
0.4"
0.6"
0.8"
1"
Shackleford"Banks"
Treasure"Island"
Num
ber'o
f'Deb
ris/m
2 ''
Sampling'Loca7on'
Supra;dal"
Upper"Inter;dal"
Lower"Inter;dal"
11
was <0.1 g, and 29% of it was <0.9 g, and Treasure Island’s debris had the lowest of the locations at only 9% <0.1 g, but 41% of debris was <0.9 g. The middle bins of each location were generally uniform in frequency and made up little of the total debris. When the intertidal of Shackleford Banks and Treasure Island was divided, the lower intertidal was skewed toward heavier mass frequencies and away from lighter ones. For example, Shackleford Banks’ upper intertidal debris was 50% <0.1 g while the lower intertidal was only 8% <0.1 g and Treasure Island’s upper intertidal was 13% <0.1 g while the lower intertidal was only 2% <0.1 g.
12
Figure 6. Histograms of debris mass at each site, on a log5 scale.
We recorded debris under the categories of plastic, wood, glass, Styrofoam, fabric, metal, cigarette butts, nonnative organic material, concrete or brick, paper, rubber, and metal foil (Figure 7). Carrot Island’s debris consisted of about 50% plastic across both tidal zones. A 6 times higher percentage of glass was found in the intertidal than the supratidal, while cigarette butts were almost twice as common in the supratidal than in the intertidal (13% vs 7%). Styrofoam was also twice as abundant in the supratidal than intertidal (19% vs 10%). Treasure
13
Island had the greatest (insignificant) percentage of plastic than any other site, with intertidal coverage (79% of all debris) being slightly higher than supratidal coverage (64%). The rest of the debris at that site was fairly evenly proportioned, though there were no rubber or fabric in the supratidal and no paper found in the intertidal. Shackleford Banks was Styrofoam dominated, with the supratidal at 64% and the intertidal 52% Styrofoam. Plastic was the second largest percentage in both zones at about 25%, with the rest of the types of debris evenly proportioned and distributed between zones. There were no noticeable changes or patterns in the proportion or distribution of debris when the intertidal was split except for the complete lack of Styrofoam in both locations’ lower intertidal zone.
0"
50"
100"
150"
200"
250"
300"
350"
Supra&dal) Inter&dal)
Num
ber"o
f"Deb
ris"
Zone"
Carrot"Island"
other)
foil)
rubber)
paper)
duct)tape)
organic)
cig.)bu6s)
metal)
fabric)
styrofoam)
0"
100"
200"
300"
400"
500"
600"
700"
800"
Supra&dal) Inter&dal)
Num
ber"o
f"Deb
ris"
Zone"
Shackleford"Banks""
other)
foil)
rubber)
paper)
concrete/brick)
organic)
cig)bu7s)
metal)
fabric)
styrofoam)
14
0"
20"
40"
60"
80"
100"
120"
140"
Supra-dal" Inter-dal"
Num
ber'o
f'Deb
ris'
Zone'
Treasure'Island''
foil"
rubber"
paper"
metal"
Fabric"
styrofoam"
glass"
plas-c"
0"
100"
200"
300"
400"
500"
600"
700"
800"
Lower&Interitdal&Upper&Inter0dal& Supra0dal&
Num
ber"o
f"Deb
ris"
Shackleford"Banks""others&(incl&duct&tape)&foil&
rubber&
paper&
concrete/brick&organic&
cig&bu=s&
metal&
fabric&
15
Figure 7. Zonal distribution of debris type for each zone and location by number.
4. DISCUSSION
Our data suggested there was a relationship between the amount of human activity at a site and the number of debris items per unit area. This suggests that direct human impacts, in the form of local littering, influences the amount of debris found at each site. Our results could be confounded, however, due to a large storm event that occurred before data collection took place at Shackleford Banks and Treasure Island. While Shackleford Banks had the greatest count density of debris, these results may be unrepresentative of the actual amount of debris present, as the beach had been cleaned less than a month earlier. Unfortunately, the exact area that had been cleaned prior as well as the effectiveness of the cleanup was not recorded.
At Shackleford Banks and Treasure Island, the highest density of marine debris was found in the upper intertidal zone. We suggest that this concentration was likely determined by the storm event. With bigger waves occurring, the debris was pushed farther up the shore where it stayed once the storm subsided and the wave activity decreased. The finding that there was more debris in the supratidal than intertidal zones at Shackleford Banks and Treasure Island can be explained by the large size of the lower intertidal, which had the lowest abundance of debris/meter of any of the zones. Debris likely has a short residence time in the lower intertidal zone as any debris light enough to be moved by wave activity would be pushed further up into the upper intertidal zone or transported back into the water.
We found that the majority of marine debris at each site was small, weighing less than 1 gram. This suggests that much of the debris sampled had been subjected to weathering as many appeared to have been broken into smaller pieces and showed evidence of being degraded. Smaller particle size allows the debris to be easily
0"
10"
20"
30"
40"
50"
60"
70"
80"
Lower&Inter*dal& Upper&Inter*dal& Supra*dal&
Num
ber"o
f"Deb
ris"
Zone"
Treasure"Island""
foil&
rubber&
paper&
metal&
Fabric&
styrofoam&
glass&
plas*c&
16
transported by either currents and waves or wind. The source of small debris is difficult to identify due to the various avenues of transport that could have deposited the debris onto our beach sampling sites. The large amount of small debris found increased the total count of debris at each site as well as decreased the debris mass per unit area result, which could be considered data skewing.
Our results for material abundance show the same trend as reported in the literature with regard to the abundance of plastic and Styrofoam, but contradict the literature when it comes to the amount of fishing line present. As suggested in previous studies, the prevalence of plastics in marine debris could be caused by the longevity of the material (Thompson 2004, Carpenter 1972). Fishing line has been reported as a frequent form of debris in many areas around the world; however, it was not found in our sampling (Kuo and Huang 2014, Ribic 2012). This could be explained by the mitigation efforts already in place throughout Carteret County with accessible methods for disposal and the implementation of regular clean-ups on many tourist-heavy beach sites. Similar initiatives could also be effective for limiting the abundance of plastic bags that end up as marine debris, either from littering or being washed up on shore. These could include methods such as on-site recycling at grocery stores or easily accessible disposal containers on the beaches for plastic bags.
Chapter 2: Organism Utilization of Marine Debris
1. INTRODUCTION
Most previous studies of the interaction of organisms with marine debris focus on negative impacts of debris. These include ingestion by marine animals, leaching of harmful chemicals into the oceanic ecosystem, and increased opportunities for harmful invasive species to be introduced in non-native ecosystems (Cadee, 2002, Westerhoff, 2008, Barnes, 2002). One study in the Netherlands showed evidence of seabirds pecking at and ingesting floating Styrofoam and plastic debris (Cadee, 2002). Plastic debris has been shown to leach bisphenol A (BPA) and antimony into seawater (Sajiki and Yonekubo, 2003, Chang et. al., 2005, Westerhoff, 2008). Antimony is a regulated chemical that can cause negative health effects if present in drinking water (Westerhoff, 2008). BPA is a chemical that is currently being researched as a likely carcinogen and endocrine disruptor for a variety of animals (Chang et. al., 2005). As the quantity of plastic found in the marine environment increases, the amount of these harmful chemicals being leached is predicted to escalate.
However, we began our survey unbiased by expectations of negative impacts and focused purely on describing and quantifying the ways in which we observed organisms interacting with and using marine debris. Organisms we observed ranged from macroinvertebrates to
17
microscopic organisms, such as chlorophytes and bacteria found in biofilms. Bacteria are a critical component of the marine food web and marine debris is an ideal substrate for bacterial and algal biofilm growth (Sieburth, 1976). Biofilms can act as a pioneer species, colonizing the debris and laying down a protective mucilaginous layer that enables the subsequent growth of other fouling organisms (Marszalek, 1979). As these materials are carried around the oceans, “hitch-hiking” organisms can end up in waters in which they are not native. Therefore, one problem with marine debris is that it may lead to a potential increase in invasive species (Barnes, 2002, Aliani, and Molcard, 2003, Gregory, 2009). Few studies have examined the potential positive benefits of marine debris as shelter and substrate for native marine organisms.
We expounded upon the spatial distribution study to investigate the ways in which marine debris was used as substrate and shelter for marine organisms. By identifying the ways in which organisms interact with debris and possible benefits it offers, future beach clean-ups in Carteret County can prioritize their cleanup efforts towards collecting first the most problematic debris that does not provide any benefits, such as additional substrate. We posited that the most common beneficial use of marine debris would be organisms using it as shelter.
2. METHODS
Field Sampling:
According to the overall survey methods, debris was found, inventoried, and collected at each site. We excluded data from Carrot Island due to sampling inconsistencies for this location. Debris at Carrot Island was collected just for use by the spatial distribution group rather than examined for evidence of usage and saved for analysis by our group. At the other two sites, the debris that appeared to be used in some way by one or more living organisms was flagged especially for our group to collect. Each member of our team surveyed a different tidal zone. Every flagged piece of debris was photographed with a ruler placed alongside to provide scale. The A-E sector, tidal zone, dimensions of the debris, and description of its use and surrounding habitat were recorded. We noted whether the organism was using the debris to grow on, growing through the debris, using the debris as shelter, or some combination of these conditions. The debris was then collected and stored in a marked Ziploc bag and later turned over to the Spatial Distribution group for further analysis.
18
Quantification of Surface Areal Usage:
Across sites, we found that organisms used debris in a variety of ways. We categorized type of usage as “on”, meaning encrusting or fouling, “through”, meaning an organism (i.e. grasses) had grown through the debris* or “shelter”, meaning that one or more organisms and/or their young were associated with and using the debris as a shelter. “On/Through” is a category for debris in which either both types of use were evident, or the two types were indistinguishable.
For debris photographed across all sites, we compared percent area of usage of each debris item. We did this by using the photographed rulers in the pictures for scale in order to have a reference for how large the debris was. Using the ruler in the image, we overlaid a grid with 1-cm2 cells to estimate both surface area of usage and total surface area of the debris (Figure 8). To calculate percent are of usage, we used the grid to calculate the total area being utilized on an individual piece of debris. That value was then divided by the total surface area of the piece of debris. Area considered “used” was defined as any surface of debris that contained an encrusting or fouling organism. Due to the nature of our measurement, photo analysis was only suitable for “on” or “through” utilization. Other uses of debris by non- colonizing organisms, such as “shelter” utilization by fiddler crabs was quantified by obtaining a count of organisms associated with the debris at the time of the survey.
• For the “through” category of usage, it was not clear whether the debris facilitated or hindered growth. This category was included because it was a notable impact of debris on a natural organism. In many cases with Styrofoam, roots were so bound to the substance as to be inseparable. This suggests that perhaps the slightly degraded Styrofoam offered greater purchase and stability than surrounding sandy sediments.
Figure 8. Debris with surface area grid overlaid on top. This grid was used to calculate percentage area utilized.
19
Biofilm Microscopy: In order to qualify what kinds of organisms were contained in the biofilms we found, we
subsampled representative biofilms from three pieces of debris- a thin plastic bag, a slightly thicker plastic sheet, and an aluminum can. Samples were collected, stained using SYBR Green I (Noble and Fuhrman 1998), and later examined under the microscope. 3. RESULTS Organism usage across debris:
Between the two main collection sites, 52 pieces of marine debris were found being utilized by organisms or vegetation. Plastic pieces made up 46.2% of the debris that was being used, and metal made up 15.4% of material being used (Figure 9). Other debris types being used by organisms were Styrofoam, wood, and rock.
The majority of debris being used by organisms was found in the intertidal zone (Figure 10). Within the intertidal zone, the main debris materials were plastics, with 19 pieces collected in the intertidal zone compared to only 4 pieces collected in the supratidal zone.
13
24
8
4 2 1 Metal
Plas4c
Styrofoam
Wood
Rock
Shell
Figure 9. Numbers of different material types found to be utilized by organisms and vegetation, collected at Treasure Island and Shackelford Banks sites. Numbers in each pie section show the number of that type of debris found (based on the category of material it was).
20
The majority of debris we collected had some type of organism growing on it (Figures 11-12 ).
7 6 1
19
4
4
4
2
1
3
1
0
5
10
15
20
25
30
35
40
Sub4dal Inter4dal Supra4dal
Num
ber o
f Material
Zone
Wood
Shell
Rock
Styrofoam
Plas4c
Metal
0
5
10
15
20
25
30
Metal Plas4c Rock Shell Styrofoam Wood
Occurrence
Material
On/Through
Shelter
Through
On
Figure 10. Spatial distribution of different debris material among different tidal zones. Results shown include data for both Treasure Island and Shackleford Banks. All numbers shown on bars represent the total number of debris pieces found in that zone and within that material category.
Figure 11. Type of utilization of different debris materials. Bars show total number of debris found in that particular material type. Different colors in the bars delineate the different utilizations of that material.
21
For
all debris collected it was also determined what organisms were utilizing that debris. It was found that
biofilms made up the majority of organisms (figure 5); the biofilms were green in
We found that the majority of debris was used by biofilms growing “on” debris (Figure
13). The biofilms were green in color and most likely contained cyanophytes. The cyanophytes utilized the debris by growing on the debris itself. Debris also exhibited remnants of barnacle fouling. Various pieces of hard debris had dead barnacles still attached. These barnacles were identified as Balanus amphitrite. One piece of metal also had the dead remnants of an oyster, identified as Crassostrea virginica. Many of the debris samples were utilized by organisms as a temporary shelter. Uca pugilater (fiddler crabs) utilized different marine debris as shelters as did the Polygyra sp. (terrestrial snail).
1/52 8/52 24/52 11/52 2/52 4/52
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Metal Plas4c Rock Shell Styrofoam Wood
Prop
or5o
n of Occurrence
Material
On/Though
Shelter
Through
On
Figure 12. Fraction of debris items of each material type that were utilized in different ways. Numbers at top of graphs show how many items of each material type were found divided by the total number of debris items that were being utilized by organisms. Percentage values represent amount of each material compared to total debris found.
22
Of the organisms found growing/fouling on debris, the two most abundant were biofilms and barnacles. Of the 52 debris items that were being utilized, 17 were utilized by biofilms as a substrate to grow on. The majority of the biofilms were found on plastics; on average they covered nearly 80% of the plastic debris (Figure 14). Biofilms were also found on Styrofoam in much smaller numbers. Still on average, when found on Styrofoam, they utilized 83% of the surface of the material to grow on. The surfaces of 5 debris samples collected were currently in use or had been utilized at some point as a hard substrate for barnacle attachment. Two pieces of metal and 2 pieces of rock debris were found to have barnacle remnants on them. On average 67% of the metal surface and 25% of the rock surface were utilized by an organism (Figure 14).
5 6 2 1 17 1
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Barnacle Snail Crab Mold Biofilm Oyster
Prop
or5o
n of item
s fou
nd
Organism Type
Sub4dal
Inter4dal
Supra4dal
Figure 13. Fraction of different organisms found utilizing debris in the three collection zones. Numbers at the top of bars represent total number of debris samples that were utilized by each type of organism.
23
Three types of organisms used the collected debris as a refuge or shelter. Six pieces of debris had terrestrial snails utilizing them as cover. The majority of the snails utilizing debris were found on metal debris, with plastic having the second highest abundance of snails (Figure 15). At Treasure Island, there were no snails found near the debris, although a large number of them were observed to be living on the marsh grass behind the supratidal zone. Two pieces of debris at Shackleford Banks were found to have snail egg cases. In both occurrences of egg cases, a terrestrial snail was found near the eggs, also utilizing that piece of debris as shelter.
Two pieces of debris had fiddler crabs utilizing them as shelter. One plastic bag had 7-10 crabs utilizing it as a shelter in the intertidal zone. In the area immediately around the bag there were no fiddler crabs, but approximately 1.5 meters away, towards the water, there were approximately 100 to 200 fiddler crabs utilizing sea grasses and mud as shelter.
2/52
0 0 1/52
2/52
0
14/52 2/52
1/52
0 0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
100.00%
Metal Plas4c Styrofoam Wood Rock
Percen
t Cover
Material
Barnacle
Biofilm
Figure 14. Percent cover of various material types, by the two most prevalent fouling organisms. Numbers at the top of bars show the number of debris samples found of that material type, and the percentage of the total debris collected that it represents.
24
Biofilm identification:
Through microscopy of our three representative biofilm samples, we determined that biofilms observed in the field were mostly comprised of photosynthesizing cells (chlorophytes), and bacterial cells (Figures 16-19).
3/52
2/52
1/52
1/52 1/52
2/52
0
1
2
3
4
5
6
7
8
Metal Plas4c Styrofoam
Organ
ism Cou
nt
Material Type
Snail
Egg
Crab
Figure 15. Number of organisms found using metal, plastic and Styrofoam debris as shelter. Numbers on top of bars represent the proportion of debris samples found to have that organism utilizing that material type.
Figure 16. Photo from the biofilm found on an aluminum can. Yellow and red dots are photosynthetic organisms. Bright green dots are bacterial cells.
25
Figure 17. Photo from the biofilm found on a plastic bag. Larger aggregations of bacterial and algal cells can be seen in the upper left quadrant.
Figure 18. Photo from the biofilm found on a thicker clear plastic sheet. This biofilm appeared green to the naked eye, while the others appeared brown. However this one was significantly dryer, which may account for the lack of algal cells visible through microscopy. The majority of this biofilm is made up of bacteria and detritus.
26
4. DISCUSSION
Plastic was the primary debris material found across all sites. Plastic items were mainly thin, lightweight wrappers and bags. Although metal and Styrofoam debris items were also found in relatively sizeable quantities, we found comparatively larger amounts of plastic in accordance with the trend of human production and usage of plastics (Derraik, 2002). Most plastics, as well as other debris types, were found in the intertidal zone. This is likely because low-density debris washes onto the shore and concentrates in the intertidal zone during periods of high wave action. Consequently, much of the colonized debris found in the intertidal zone may have originated in the water. This is especially evident in debris fouled by organisms such as barnacles, which would have been submerged during initial colonization as barnacles settle and live in the water.
The majority of debris found was utilized by organisms growing on the debris surface. Of all organisms growing on debris, biofilms were found with the greatest frequency. In biofilm samples analyzed by microscopy, we identified photosynthetic organisms- which need significant amounts of sunlight to survive. Most debris with biofilm were lightweight, transparent, buoyant plastics. Since light could pass through the material, these plastics created optimal growing conditions for chlorophytes. Transparent wrappers/bags debris also provided a structure with a large enough surface area for the chlorophytes to take advantage of maximum sunlight exposure. Once the plastics washed up on shore, they possibly acted as a greenhouse allowing chlorophytes to continue growing as long as the plastic remained intermittently wet. Aside from plastics, metal and Styrofoam objects were also found. These materials, along with plastic, provided temporary refuge for organisms in both the supratidal and intertidal
Figure 19. This photo shows an aggregate of bacterial (yellow-green) and photosynthetic (red) cells found in the same sample as figure 10. Across samples, bacteria were often seen in conjunction with photosynthetic cells, likely due to their tendency to concentrate on other particles.
27
zones. We observed that terrestrial snails and fiddler crabs were using debris as shelter, providing them with protection and allowing them to exist in a zone where we did not normally observe them. On Treasure Island, fiddler crabs were naturally living in large abundance closer to the subtidal zone in muddy, wet sediments, whereas terrestrial snails were more abundant on marsh grasses in the supratidal zone. Both of these organisms were found associated with debris outside of zones where larger natural abundances were observed. Although fiddler crabs and terrestrial snails were found directly on or beneath debris, we did not find either of these organisms in the immediate area surrounding the debris. This suggests that the debris is providing them a temporary shelter away from their area of natural occurrence.
We suggest that the comparatively greater strength, rigidity, and structural complexity of these materials attract organisms that use the debris as shelter. The greater strength and rigidity afforded by certain types of marine debris offer a more stable and protective shelter than natural materials such as grass, from predation and environmental stressors (heat, tidal inundation). Most debris samples found were in complex shapes with many open spaces. For example we found debris with snail egg cases attached, suggesting that terrestrial snails use the debris as a refuge to protect themselves and/or their young.
There are both positive and negative aspects of marine debris utilization. The debris could provide a temporary refuge for organisms outside of their natural zone. This might occur if an organism is caught between tidal changes and left exposed to environmental factors and predators. Due to our field observations we believe it is possible that marine debris could provide a refuge for an organism until it could return to its natural zone. This is potentially what happened with the fiddler crabs in the plastic bag we found in the high intertidal zone. It is important to note that these potential positive effects of debris were only observed on an individual basis. We can make no assumptions about the impacts of used debris on the larger ecosystem. It could be that potential benefits to a few organisms are trivial compared to larger environmental effects that we could not observe directly.
Due to a rigid structure and more complex shape, another potential benefit of marine debris is its ability to provide protection for organisms and their offspring. We observed this utilization in the case of the terrestrial snail and its egg cases we found underneath a metal can and, in a separate instance, in a Styrofoam cup. Despite the positive benefit of providing shelter, it is also possible the debris is pulling organisms out of their natural zones. This potential impact was seen with the fiddler crabs we found living under debris further up the shore away from the water and the terrestrial snails observed living further down from the supratidal zone in an area remote from their normal refuge in grasses. In both of these instances, as no other organisms of this type were observed around the debris, it is likely these organisms were separated from their natural habitat.
The biofilms found on much of the plastic debris are often the first layer of colonization before other fouling organisms, such as barnacles, attach to debris (Marszalek, 1979). Once biofilms are present, other fouling species can then colonize substrate while having a protective layer of biofilms between them and the harmful chemicals leached from the plastic material
28
(Marszalek, 1979). This implies that the debris we found with biofilms present could be utilized as a substrate for other fouling organisms. Debris colonization would provide a benefit to fouling organisms on an individual level as they now have a source of additional space to grow and feed on. The mobile nature of the plastic debris could also cause a potential problem when the colonized debris carries organisms to outlying locations. This process potentially could allow non-native species to be transported greater-than-normal distances. This organism transference from one area to another by mobile debris is referred to as “hitch-hiking” (Barnes, 2002). The data collected in this study was not sufficient to determine if the increased presence of biofilms enabled hitch-hiking species, but it is a possibility and should be investigated further.
There are documented issues associated with plastic debris such as chemical leaching that could cause problems for organisms. Plastic debris has been shown to leach chemicals such as BPA and antimony (Sajiki and Yonekubo, 2003, Chang et. al., 2005, Westerhoff, 2008). It is possible that the organisms found in or around the debris are exposed to leaching harmful chemicals. We observed grasses and other vegetation growing through softer plastics and Styrofoam, which could leach chemicals into their root system. Leaching is a known environmental issue with plastic debris, but the long-term effects of leached chemical exposure on fouling organisms utilizing and growing through debris are unknown.
5. CONCLUSION
Our study observed a variety of organisms utilizing debris as a substrate for growth, colonization, and shelter. Future research should be conducted on marine debris found in the subtidal zone. Additionally, further research should examine the effects of displacing organisms from their natural habitat to new tidal zones and the ecological implications this has for the larger environment. Research should also investigate the chemical concentrations organisms are exposed to due to plastic leaching and the long-term effects of these compounds, as they may pose a threat to the long-term healthy balance of the marine ecosystem. Based on the dominance of plastics we found and their known problematic environmental effects, we recommend that management strategies should be put into place to prevent plastics from entering the marine environment. Other management strategies should be developed as more information about the environmental effects of plastics, chemical leaching, and hitch-hiking organisms as well as of other types of anthropogenic marine debris is obtained.
29
Chapter 3: Bacterial Analysis
1. INTRODUCTION
Many macro-organisms like crabs and snails were found to utilize marine debris for shelter. However, bacteria also used this marine debris as substrate for colonization. Most previous studies on marine debris have focused on plastic and its effects only on marine life (Derraik, 2002). However, marine debris may also have important impacts on human health. Impacts on human health are recognized to include physical harm, such as cuts, as well as water quality issues related to harmful bacteria from the debris (Sheavly & Register 2007). About 5 million deaths are caused by waterborne illness worldwide each year, over 50% due to gastrointestinal illnesses (Myounggon, 2015). Overall, in the United States, there are about 7 million cases of gastrointestinal illnesses, resulting in approximately 12,000 deaths annually (Myounggon, 2015).
The bacteria that cause many of these illnesses attach to a surface after a free-swimming form of growth (Petrova & Sauer 2012). Escherichia coli and Vibrio vulnificus are two bacteria found in marine habitats that are known to cause illness and occasionally mortality. E. coli causes severe diarrhea in its victims. Over 13,000 people were affected by E. coli from 2007-2010 in the European Union (Messens, et al 2015). V. vulnificus can be contracted through eating contaminated seafood, such as the commonly-consumed oyster. It can cause similar gastrointestinal symptoms as E. coli, including fever, chills, diarrhea, and abdominal pain. V. vulnificus also has a high mortality rate in individuals with weakened liver function, often associated with heavy drinking habits (Hong, 2015). V. vulnificus can also lead to wound infections that can cause death within 48 hours. There is a 25% mortality rate for V. vulnificus wound infections (Chiang & Chuang 2003).
E. coli and V. vulnificus pose clear dangers to human health as they can cause severe illness and mortality in affected persons. This study analyzed the frequency with which Escherichia coli and Vibrio vulnificus attach to marine debris locally. If these bacteria are associating with marine debris, beach trash could expose humans to high concentrations of bacteria, and thus pose a greater health threat than has been previously recognized.
2. METHODS
Lab Preparation:
Prior to sample collection, we prepared and poured alkaline peptone water (APW) into whirl-pak bags. We used APW to enrich the marine debris in order to facilitate and accelerate bacteria growth (Cruickshank, 1968). We prepared CHROMagar plates for Vibrio vulnificus detection (CHROMagar, 2012). We prepared IDEXX bottles for coliform and Escherichia coli detection by pouring 90 mL of a mixture of water and Colilert 18 into each IDEXX bottle (IDEXX, 2012).
30
Field Sampling:
Overall Procedure
Survey and collection of samples occurred in mid-fall 2015. We filled 12 Whirl-pak bags with APW. We collected 2 pieces of natural debris and 2 pieces of anthropogenic debris from each tidal zone, (supratidal, intertidal and subtidal), totaling 12 pieces from each site. We stored the debris pieces in Ziploc bags and later transferred the pieces to the whirl-pak bags at the site. Ziploc bags were not sterilized, but a new pair of rubber gloves were used to handle each piece of debris and the bags to decrease chance of cross-contamination. We took debris samples small enough in size to fit into the whirl-pak bags. We took sediment samples near each piece of debris in 50 mL plastic tubes by sticking the tubes into the ground and then storing the samples in the tubes. We stored the debris and sediment samples in a dry cooler for protection and transportation back to the laboratory.
Site-Specific Procedures
Carrot Island
We filled each of the 12 whirl-pak bags with 100 mL of APW. We used twelve, 10-mL syringes with their tips cut off to take 8-cc sediment samples adjacent to each piece of debris. We used twelve 50-mL centrifuge tubes for storage of the syringes.
Shackleford
We filled 12 whirl-pak bags with 75 mL of APW. We used twelve, 50 mL centrifuge tubes to collect ~35-40-cc sediment samples adjacent to each piece of debris.
Treasure Island
We filled 12 whirl-pak bags with 75 mL of alkaline peptone water. We used twelve, 50 mL centrifuge tubes and stuck them into the ground for ~20-30 cc coring sediment samples where each debris piece was found.
Laboratory Analyses:
V. vulnificus Analysis
Immediately after returning from each field site, we incubated the whirl-pak bags containing APW and debris samples for approximately 1 hour at 35°C. We set up a vacuum filtration device in order to filter organisms from the slurry/broth onto gridded membrane paper. We weighed 0.5 g of the top layer of each sediment sample and combined this with 50 mL of water for creating the slurry. This created a standard of 1 g per 100 mL of water. For each sediment sample, we filtered 35 mL of slurry. For each debris piece, we filtered 50 mL of APW. We placed the membrane papers onto CHROMagar plates, which we inverted and placed in an incubator for ~18-24 hours at 37 °C. We removed the plates after the allotted time and the target colony numbers were counted. We then doubled the actual colony numbers to get to the standard of 100 mL since 50 mL of the APW was actually filtered.
31
Coliform & E. coli Analysis
We placed 10 mL of either sediment slurry or APW in which the debris was soaked, in an IDEXX jar containing 90 mL of water and media, keeping the 100 mL standard. We shook each bottle by gentle inversion and poured the contents into separate IDEXX trays. The trays were sealed using a heat sealer and placed in an incubator at 35 °C for ~18-24 hours. We removed the trays after the allotted time and coliform wells were identified. We then used UV light for E. coli detection. We used an IDEXX MPN generator to determine the most probable number of colonies based on the number of wells in the IDEXX tray that were yellow (for coliforms) and yellow and glowing (for E. coli) under UV light based in a volume of 100 mL (the standardized volume).
3. RESULTS
Carrot Island samples, of both sediment and debris, had slightly more E. coli than those from Shackleford Banks (Figure 20). Both contained significantly higher average abundances of E. coli than did Treasure Island (P = 0.0008) (Fig. 1) as determined by a three-way ANOVA.
A
A
B
0
0.5
1
1.5
2
2.5
3
Carrot Island Shackleford Treasure Island
Log MPN
per 100 m
L
Site
Figure 20. Average E. coli concentration per sample, both sediment and debris, on each sample site. Different letters show significant differences between sites. Error bars indicate standard error.
32
There were significant differences in E. coli abundance among different tidal zones (Figure 21). The samples from the subtidal and intertidal zones had significantly larger average E. coli concentrations than the samples from the supratidal zone across all the sites (P < 0.0001) (Figure 21).
A A
B
0
0.5
1
1.5
2
2.5
3
Sub4dal Inter4dal Supra4dal
Log MPN
per 100 m
L
Tidal Zone
Figure 21. Average E. coli concentration per unit sample from each tidal zone, including both sediment samples and debris samples. Different letters show significant differences between zones. Error bars indicate standard error.
33
The debris contained significantly higher average abundances of E. coli than the sediment surrounding it (P < 0.0001) (Figure 22).
Average E. coli abundance on debris in the subtidal and intertidal zones was significantly higher than the abundance on sediment and debris in the supratidal zone (P < 0.0001) (Figure 23). Additionally, the abundance of E. coli on sediment at Treasure Island was significantly lower than any other site and sample type, followed by Shackleford Banks sediment (P < 0.0001) (Figure 24).
A
B
0
0.5
1
1.5
2
2.5
3
Debris Sediment
Log MPN
per 100 m
L
Sample Type
Figure 22. Average E. coli concentration per unit sample on debris and surrounding sediments. Different letters show significant differences between sample types. Error bars indicate standard error.
34
A
A
C BC
B BC
0
0.5
1
1.5
2
2.5
3
3.5
4
Sub4dal Inter4dal Supra4dal
Log MPN
per 100 m
L
Tidal Zone
Debris
Sediment
AB
A
AB
A
B
C
Debris
Sediment
Figure 23. Average E. coli concentration for debris and sediment samples across three tidal zones. Letters above bars indicate significance. Error bars indicate standard error.
Figure 24. Average E. coli concentration when interacting between sample type and site. Letters above bars indicate different levels of significance. Error bars indicate standard error.
35
There was no significant difference in Vibrio vulnificus concentration across site, zone, or sample type (debris or sediment). However, the interaction between tidal zone and sample type revealed a significant difference in that intertidal debris had higher abundances than subtidal debris (P = 0.003). The ANOVA also found a pattern in V. vulnificus abundance between different tidal zones. Outlier data may have caused this pattern and despite not being significant (P = 0.21), the intertidal zone seems to show more V. vulnificus on average (Figure 25).
0
10
20
30
40
50
60
Sub4dal Inter4dal Supra4dal
CFU per 100mL
Tidal Zone
Figure 25. Average V. vulnificus per sample in different tidal zones. Error bars indicate standard error.
36
Anthropogenic debris tended to have more V. vulnificus present than natural debris, although the difference was not significant (P = 0.18) (Figure 26).
4. DISCUSSION
Treasure Island had a much lower abundance of Escherichia coli compared to the other two sites (Figure 1). This difference was greatest in the supratidal zone. This is probably due to the bacteria experiencing desiccation in the supratidal zone. This zone would rarely be completely inundated since it is above the mean high-tide level and would only be inundated in the highest of tides and in storm events (Figure 2, Billi and Potts, 2000; Bridge and Demicco 2008). The comparison between debris and sediment indicated that on average E. coli favors attaching to the debris more than the sediment that surrounds it in the environment (Figure 3). This is likely due to biofilm growth on marine debris like plastics, which allow bacteria to thrive (Lobelle and Cunliffe 2011; Donlan 2002). E. coli abundance in sediment on Treasure Island was the lowest of any site and sample type (Figure 5). This may be due to a different sediment type in Treasure Island because it was a marsh environment and had thick mud compared to Carrot Island and Shackleford, which both had finer sand. While there were significant differences between E. coli abundance, no significant differences were observed in Vibrio vulnificus among different sites, tidal zones and debris types. It seemed that there was a pattern that V. vulnificus was more abundant in the intertidal zone compared to the subtidal and supratidal zones on average, but more data and testing is needed to qualify this result (Figure 6). This pattern may be due to the intertidal zone being well oxygenated, as vulnificus accounts for a portion of aerobic
0
5
10
15
20
25
30
35
40
45
Anthropogenic Natural
CFU per 100mL
Debris Origin
Figure 26. Average V. vulnificus per sample on natural and anthropogenic debris. Error bars indicate standard error.
37
bacteria in marine environments (Morris 2014). It seemed that V. vulnificus preferred anthropogenic to natural debris, but further testing is needed to verify this result (Figure 26).
The results of this study have implications for the health of people that volunteer to clean up beaches. However, only the pathogenic types of both organisms have health implications. Further testing is needed to determine if the types of bacteria found are actually pathogenic. Our results indicate that both of these organisms are not only present in the marine environment, but they attach to marine debris of anthropogenic and natural origin (Chiang and Chuang, 2003; Messens et al., 2010). However, this could be harmful to recreational beachgoers because of the chance of infection by V. vulnificus if cut by a piece of marine debris on the beach, such as a nail or shell. This also means that volunteers that clean up beaches must protect themselves by wearing gloves as they pick up debris from beaches. While the numbers of both types of bacteria that were found from this study are low enough that they do not pose a threat to human health, preventative measures should still be taken such as wearing protective gloves and walking carefully to avoid injury and serious health complications later. Our results, while not statistically significant for V. vulnificus, show a possible pattern that could be supported by a larger and more variable sample size. Further experimentation and analysis is needed in order to verify V. vulnificus’s association with marine debris in order to deem marine debris a possible health hazard with this bacteria. For E. coli, it is possible some of the IDEXX methods resulted in false-positives due to faulty equipment, which could also have compromised and skewed the data. This means that statistically significant data that was found during E. coli analysis may not actually be significant, meaning further testing and improvement of the methods is needed for better, true results.
Chapter 4: Impacts of Exposure on Marine Debris
1. INTRODUCTION
The previous two chapters looked at the colonization and utilization of marine debris by both macro- and micro-organisms. However, during their studies it was unclear how long each piece of found debris had been exposed to the environment. This begged the question of how long it took before debris newly introduced into the environment was colonized. Our Capstone subgroup conducted an exposure experiment to determine how new debris is affected by tides, weather, and organisms in different tidal zones, and primarily focused on the colonization of organisms. We examined the exposure debris encounters in the marine environment in order to determine the rate at which different aspects, such as deterioration and use of substrate by organisms, affect new debris. Our goal in this experiment was to see what happens to debris after a month of exposure to natural physical processes and organisms, such as degradation and colonization. Few studies have been conducted on different degradation rates of marine debris and on different colonization rates of organisms on marine debris. The lack of studies in these two areas encouraged us to look into this topic because we wanted to see how different materials
38
fare in the marine environment. There is also a lack of many studies conducted on short-term exposure of debris to the natural environment; most studies occur over a longer time span, such as a year (e.g. Pomerat and Weiss 1946), and for this reason we thought a short-term experiment would be beneficial to the scientific community. We expected, particularly on our subtidal debris, to find fouling organisms, such as barnacles, bryozoans, hydroids, mollusks and polychaete worms, because they are the most abundant type of organisms that live on marine debris (Barnes 2002).
2. METHODS
We conducted our exposure experiment at two different sites: Piver’s Island in Beaufort, North Carolina and behind the UNC Chapel Hill’s Institute of Marine Science facility in Morehead City, NC, about five miles away from Piver’s Island. At each site we placed debris attached to rope in three tidal zones: subtidal, intertidal and supratidal. We employed four categories of debris: glass, aluminum, plastic and polyethylene. In our experiment we used a glass bottle, a soda can, a plastic bottle (made mostly from polyethylene and polypropylene) and a plastic grocery bag (made mostly from polyethylene and polypropylene) for each of our debris types. Since plastic is the most abundant debris material found in the ocean (Derraik 2002) and comes in different forms that have different properties, we decided to use two plastic items in our experiment, a hard and soft plastic item, to represent this abundance and diversity. All of the debris we used was new and clean and had not been exposed to weather or encountered physical disturbances or organisms prior to the start of the experiment. Prior to starting our experiment we recorded mass measurements of each piece of debris by weighing them in order to compare them with the debris’ mass at the end of the experiment.
At each site’s intertidal and supratidal zones we placed 2 ropes perpendicular to the beach on which we attached one piece of each of the four types of debris along the entire length of the rope (Figure 27). The ropes in the intertidal zone were attached to land via orange stakes that were approximately 0.3 m in length, placed at both ends of the rope to ensure the rope would not move during the duration of the experiment. The ropes in the supratidal zone were placed on land above the tide line and were staked to the ground at both ends to ensure they would not move. The supratidal ropes were marked with a “do not disturb” sign in an attempt to minimize human interference. At the subtidal zone for both sites, 2 pieces of rope were submerged in the water to approximately 2 m in depth and were weighted with a concrete weight in order to keep the rope attached to the bottom; the weights were 2 m deep, with the debris at progressively shallower depths in the water column depending on where it was located on the rope. The weights were all approximately 18 kg and were either rectangular or cylindrical in shape and had a metal ring on top which we tied the rope to. The rope also had a cone-shaped yellow buoy at the top to ensure that we could find the debris later when we collected it; the buoy was labeled as property of IMS and included a phone. The buoys also indicated that they were part of a scientific experiment and not to disturb them.
The rope we used was made out of nylon and was approximately half an inch in diameter; each piece of rope was approximately 1.2 m in length. We replicated the experiment at all tidal zones for each site, giving a total of six pieces of rope per site, to ensure that we had enough data
39
replicates. The debris was attached to the ropes at equal distances apart to ensure that they had limited contact with the other pieces of debris. The debris were placed about 20 cm apart from each other, with approximately 20 cm separating the end pieces of debris from the buoy at the top of the rope and the weight at the bottom of the rope. The debris was attached to the rope using cable ties that were 10 cm in length. We randomized the order of the debris attached to the rope in each rope. We used a total of 48 pieces of debris, 12 of each type of material, in our experiment.
The subtidal moorings were collected 35 days after they were first put out into the subtidal zone. Due to weather and tidal conditions, the intertidal and supratidal moorings were collected one day after the subtidal moorings, so they were out for 36 days. When we retrieved the moorings, the rope with the debris attached were placed in individual buckets to keep them from contaminating each other and to keep them from getting contaminated by the boat (subtidal) and ground (intertidal and supratidal). After the mooring lines were collected from the field, the mass for each piece of debris was recorded and compared to its original mass prior to exposure. We also recorded the type and abundance of organisms and sediment that was on the debris and conducted t-tests on our data to determine whether significant differences occurred between the different types of debris.
Figure 27. Image of intertidal debris at Piver’s Island which shows the layout of our debris lines. The glass bottle (at bottom of picture) corresponds to the farthest distance from the water.
40
3. RESULTS
Organismal colonization of debris:
We recovered all 12 debris lines after 35-36 days of being in the field. One piece of debris, a plastic bottle in the subtidal zone on Piver’s Island, was lost during the experiment, and the majority of one glass bottle was lost from the intertidal zone at IMS; the remaining 46 pieces of debris were intact and were still attached to the ropes at the end of our experiment. We observed 9 different types of organisms on our debris from the 3 tidal zones (Figures 28 and 29). The most abundant organisms we found were barnacles and bryozoans, most of which were found on the subtidal debris (Figures 28 and 29). The other 7 organisms were found in trace amounts on the debris but were too scarce to be included in comparative analysis. They included amphipods, snails, hydroids, serpulid polychaetes, flatworms, hydrozoans, and tubeworms; empty worm tubes were also found on some of the debris (Figure 29).
In both study sites, barnacle abundance was greatest on the glass bottle compared with the rest of the debris materials (Figure 28). Slightly more than half of all the barnacles found on the subtidal debris were on the glass bottles, with roughly one-third of the total barnacles found on the aluminum cans (Figure 28). Very few barnacles were found growing on the plastic grocery bags (Figure 28). Encrusting bryozoan distribution was generally even among the objects, with the metal cans having a slightly higher abundance than the other items in the subtidal zone (Figure 29). 92% of barnacles colonized the materials placed in the subtidal zone while only 8% of the barnacles were found on materials placed in the intertidal zone at both study site locations.
Within the subtidal zone, barnacles were seen to have the highest abundance on metallic cans and glass bottles at the Piver’s Island study site (Figure 28). A t-test showed a significant difference between subtidal and intertidal barnacle growth (p=0.006), so barnacles grow more favorably in the subtidal zone versus the intertidal zone. A t-test showed no significant difference between the number of barnacles on the aluminum can and the glass bottle (p=0.645). There were significantly more barnacles on the glass bottle than both the plastic bottle and the plastic bag in the subtidal zone (p=0.003 and p=0.004, respectively). A t-test showed that the difference in the number of barnacles in the subtidal zone was not statistically different between the aluminum can and the plastic bottle (p=0.145). There was also no significant difference in barnacle growth in the subtidal zone between the aluminum can and the plastic bag (p=0.129).
The amount of debris with living organisms increased the farther away from land the debris were located (e.g. the subtidal zone had more organisms than the supratidal zone) and increased drastically from the intertidal zone to the subtidal zone; the reverse can be seen in the amount of debris found without live organisms (Figure 30). Overall the amount of debris with organisms is similar to the amount of debris without organisms when combined for all three zones; however, within each zone the organismal colonization is much more variable. 45% of the debris we used in our experiment contained living organisms at the end of our experiment, so the debris with and without live organisms was almost equal.
41
Figure 28. Amount of barnacles found on debris lines in the subtidal zone at both study sites. The amount of barnacles represents only a population size in terms of barnacle abundance and does not take the size of the barnacles into consideration.
42
Figure 30. The percent of debris found with live organisms on them. 45% of debris contained live organisms. All of the pieces of debris in the subtidal zone contained live organisms, whereas the debris in the other two zones varied in whether or not they contained live organisms.
Figure 29. Amount and type of live organisms found on debris, excluding barnacles.
43
Debris mass alteration over time:
Results regarding mass alterations were highly variable among the three tidal zones. In the supratidal zone, aluminum cans and glass bottles both lost mass, while the plastic bags and plastic bottles gained mass. The objects in the intertidal zone all gained mass except for the broken glass bottle, which was not included in our analysis since the glass bottle was not intact. In the subtidal zone, all items increased in mass (Figure 31). The supratidal zone had very little fouling and sediment on the debris which is likely the reason for the minimal change in mass the debris experienced. The intertidal debris increased in mass mostly from sediment and organisms that were on the debris, particularly the plastic bag and the plastic bottle since those items collected the most sediment.
4. DISCUSSION
Our results indicate that the majority of the organisms found on our debris lived in the subtidal zone instead of the intertidal and supratidal zones; this could have occurred for several different reasons. The supratidal zone gained the least amount of mass during the experiment, with the intertidal zone and then the subtidal zone gaining the most mass (Figure 31). No marine organisms were found in the supratidal zone because it is a terrestrial environment and is therefore not a conducive habitat for marine species. The only way marine organisms would have been found in the supratidal zone would have been after a flooding event that had carried organisms past the typical high tide line. Few marine organisms were found in the intertidal zone because it is a semi-terrestrial environment and thus its inhabitants must be able to survive through varying conditions and a changing environment. The intertidal zone experienced the
Figure 31. Compares the average mass changes in all types of debris for each of the three zones. The standard errors are included for each of the tidal zones.
44
most variance of debris mass out of the three zones, which is most likely attributed to the fact that the debris was constantly being affected by tides and currents and therefore experienced the most change in environment compared with the other two tidal zones. Since few organisms can withstand the difficulties and stress of living in the intertidal zone, the only way that our debris could have come into contact with more organisms in this zone would have been if a flood or other weather event had caused the tides to remain high for an extended period of time, allowing for more marine organisms to use our debris as substrate. The subtidal debris had the most marine organisms because it was the only full marine environment that we examined in our study. We would have expected our subtidal debris to have come into less contact with marine organisms if the time our experiment had been conducted had not coincided with a larval season of invertebrates, since invertebrates made up the majority of the organisms found on our subtidal debris. The subtidal debris had the largest increase in mass most likely due to the large amount of barnacles and other organisms that used the subtidal debris as substrate. Mud and other sediment that was stuck beneath the organisms could also have contributed to this mass gain on the subtidal debris.
It is estimated that the amount of anthropogenic debris in the ocean has doubled the reproduction of fauna in the subtropics and has tripled the reproduction of fauna at high latitudes (Barnes 2002), which could increase the amount of organisms that colonize on marine debris. Temperature could also have affected the amount of organisms that we found on our debris; the lower the water temperature is, the less marine fauna that is found in the area (Barnes 2002). Since our experiment occurred during the time period when the water and air temperature in Bogue Sound was dropping due to changing seasons, the local invertebrate populations may have been smaller than they typically are in the summer, resulting in fewer organisms found on our debris. Besides temperature, the amount of organisms found in the subtidal zone could have partially been because the debris was floating in the water column instead of sitting on the ocean floor. If the debris had been sitting on the ocean floor there could have potentially been more organisms that used the debris as a habitat, such as benthic invertebrates and other benthic plants that do not disperse high into the water column. Debris located on soft benthic areas stimulates invertebrates to settle and colonize (Katsanevakis et al. 2007; Renchen and Pittman in Clark et al. 2012), and this potentially causes marine animals to congregate in areas where there is debris (NOAA Ingestion 2014). Although our design did not include debris sitting on the sediment, the weight likely increased the number of invertebrates compared to free-floating debris. Although our subtidal debris was located in the water column and not on the ocean floor, it nonetheless could have attracted more invertebrates than it would have if the debris had been floating in the water column without a weight at the bottom.
Mass differences in our debris could have occurred due to several different reasons. Debris could have gained mass due to sediment, substrate, and organisms living on the debris. We expected our debris to increase in mass from the beginning to the end of the experiment due to colonizing organisms, but this trend was obviously not consistent for all of our debris types. The debris could have lost mass due to degradation or from ingestion by animals. Degradation occurs over a long time span (O’Brine and Thompson 2010), so if our debris did undergo degradation then it would have been very minimal due to our short experiment time. It could also
45
be possible that some of the small invertebrates and colonizing organisms found on our subtidal debris could have ingested small particles of plastic that eroded off of our plastic debris.
Throughout the course of our experiment, our debris experienced changes due to biological and physical processes. One of the intertidal ropes at IMS became unattached at the top of the beach because the stake came out and was thus lying parallel to the beach when we retrieved it; when we put the rope out at the beginning of our experiment it was lying perpendicular to the beach. This change in rope position and dislodgment of the top stake was probably caused by tidal cycles (Uhrin and Schellinger 2011). The tides could have been the cause of the breaking of the glass bottle in the intertidal zone at IMS. Our debris appeared to have undergone slight degradation while it was in the field; some of this was probably caused by physical processes like wind and waves and also could have been partially due to invertebrates through ingestion (NOAA Ingestion 2014).
Biofilms could have been on our debris in small quantities, but due to time constraints we were not able to thoroughly examine how much biofilm was actually present on our debris. Biofilms act as a mediator of the cyprid larval attachment process for many different species of barnacles (Wieczorek et al. 1995; Thompson et al. 1998; Thiyagarajan et al. 2006). Cyprids can distinguish between different biofilms and prefer to attach to biofilms that are similar to their adult habitat (Qian et al. 2003; Thiyagarajan et al. 2006). It was difficult to determine whether any of our debris had biofilms on them, but if they did have biofilms then it can be assumed that the type of biofilms that accumulated on our debris were types were similar to the adult habitat of barnacles.
Surface wettability is thought to affect the attractiveness of substrate to larvae, with material that has a high wettability being the most attractive to certain types of larvae (Hung et al. 2008). Hung et al. (2008) found that cyprids, regardless of age, prefer to attach to glass than polystyrene because glass has a higher wettability and absorb less water. Our results correlate with this finding because our glass bottles had the highest amount of barnacles on them (Figure 28). Our debris may have also been affected by the priority effect because early settlers on the debris could enhance or impede ensuing settlement (Kohler et al. 1999). For example, the filamentous structure of hydrozoans attracts blue mussels and biofilms, which may consequently affect the amount of barnacles that settle on the substrate (e.g. Maki et al. 1992; Peterson and Stevenson 1989), and high amounts of barnacles tends to attract additional cyprids, which would then cause more barnacles to grow on our debris (Knight-Jones and Crisp 1953).
5. CONCLUSION
The location of debris in the marine environment can play a large role in whether or not the debris becomes utilized and becomes used in the marine community, or whether it remains as a nuisance and hazard for marine life. Based on this study, we can conclude that even though debris in the marine environment can be hazardous to marine life through entanglement and ingestion (NOAA Entanglement 2014; NOAA Ingestion 2014), it can also benefit species that require substrate to attach to, such as barnacles and bryozoans that were found on our subtidal debris, and can provide habitat if the debris is large enough. It can be suggested that the subtidal
46
zone is both the best and worst place for marine debris out of the three tidal zones because the subtidal zone is the zone where organisms can take advantage of debris, but it is also the zone where debris can cause the most negative impacts on marine life. Also, since our experiment was conducted in such a short time span, we were not able to get clear results on how quickly debris becomes affected by physical and biological factors, such as degradation and colonization, in the marine environment. This suggests that marine debris is not greatly affected by these factors in the short term and must be in the system longer than a month.
Chapter 5: Comparisons of Marine Debris in Carteret County with Other National and International Locations 1. INTRODUCTION
Marine debris is often in the media due to the different problems that it causes, such as ingestion of plastic bags by marine animals. Beach cleanups have been going on for decades to battle the growing problem. The information from beach cleanups is often used to study marine debris because traveling into the ocean to collect data about quantities of debris is not always practical and it is more efficient to use beach cleanups to estimate marine debris in the water (Walker et al, 1997). A large problem with marine debris is that, unlike debris on land, marine debris can be carried great distances until it is in uninhabited areas such as in the middle of the Atlantic Ocean and even sometimes travels across oceans. Conducting regular cleanups as well as monitoring marine debris around the world is necessary to address the issue of marine debris (Garrity & Levings, 1993).
There are more than a hundred published surveys of beach debris, but comparing them is very difficult because there is no standard method of collection or recording the debris. The surveys covered different sized areas and sometimes small debris was not recorded. When debris was recorded, sometimes it was categorized by function and other times by weight. Many studies recorded all debris from the ocean edge to the highest strandline, but some studies only covered specific sections of the beach. Most studies did not look for buried debris despite hypotheses that it may account for large amounts of total debris on beaches (Ryan et al., 2009). It can be very difficult to make quantitative comparisons between studies due to the lack of universal standardization. This study uses data from beach clean ups that have been standardized as much as possible to remove the effects of differences in collection and reporting methods. Standardized data is used to draw comparisons between the debris found in Carteret County and the debris found elsewhere in the world and to determine if there are correlations between amounts of debris and either population or tourism.
47
2. METHODS
Since 1985, The Ocean Conservancy has conducted a yearly cleanup of marine debris around the world. Their data are accessible to the public. I used the data for 2013 and 2014 and compared it to data from local cleanups in Carteret County that used the same methods and data sheets as the Ocean Conservancy. The Ocean Conservancy provides data from clean ups in close to a hundred different countries. In order for comparison to be possible, data from countries that did not participate in the Ocean Conservancy cleanup for both years was not used, as well as any countries with incomplete data for either year. From the countries that were left, I used a random number generator to choose 50 countries.
The Ocean Conservancy provides data sheets with specific categories to each cleanup crew to obtain standardized information across all of the areas cleaned. The categories that were used for this analysis are total people working the cleanup, total items collected, total miles covered by the cleanup, and total pounds of debris collected during the cleanup. It was not possible to directly compare the total number of items of debris collected, because the distances covered by the cleanups differed. To compare the data, I used the other categories to standardize the total number of items collected by dividing the total items found by the total miles covered to give me items per mile.
The first portion of the analysis compared Carteret County percentages with the total percentages for the 50 countries selected earlier. The first part of this study address two research questions: 1) Whether there is a relationship between amount of tourism to a country and amount of marine debris and 2) Whether population affects the amount of marine debris. To answer these questions I found 2013 and 2014 population data for each of the studied countries (CIA, 2015). Although tourism is more difficult to quantify, I used international tourism receipts as an indicator (World Bank, 2015). Both of these data sets were compared to the data that we had received from the Ocean Conservancy to search for correlations in the data.
In addition, I wanted to determine the difference between the amount of debris found in Carteret County that collected in North Carolina overall as well as compare Carteret County to the rest of the world.
The Ocean Conservancy data classified debris into different categories. I compared the percentages of the different kinds of debris using a T-test to see if there was a significant difference between Carteret County and the rest of North Carolina.
3. RESULTS
The top 6 types of debris found in both Carteret County and the world were the same. These categories were: cigarette butts, food wrappers, plastic bottles, bottle caps, plastic grocery bags, and beverage cans. We compared the percentages of these categories of debris with each other. Every other type of debris not in these categories was placed in the category of “other” (Figure 32). Nearly 40% of the debris items cleaned from the beaches of Carteret County in 2013 were cigarette butts (Figure 32), compared to 15% worldwide. Plastic bottles made up about 4% of
48
debris cleaned from Carteret County and about 7% of marine debris worldwide (Figure 32). Both Carteret County and the worldwide data showed that approximately 2% of the debris collected consisted of plastic grocery bags.
Figure 32. Comparison of the percentages of debris types in Carteret County vs the world totals in 2013.
Carteret County was compared to North Carolina and was found to have a density of debris that was 7 times that of North Carolina (Figure 33). The difference between the two when a T-test is run is statistically significant (p=0.007 for 2013 and 2014).
0.393
0.050 0.043 0.028 0.018 0.035
0.434
Cateret County Debris Types 2013
Cigare[e Bu[s Food Wrappers
Plas4c bo[les Bo[le caps (plas4c)
Plas4c grocery bags Beverage cans
Other
0.15
0.123
0.069
0.062
0.032 0.025
0.539
Total World Debris Types 2013
Cigare[e Bu[s Food Wrappers
Plas4c Bo[les Bo[le Caps (plas4c)
Plas4c grocery bags Beverage cans
Other
49
Figure 33. Comparison between amounts of debris collected during cleanups in Carteret County and debris collected in beach cleanups across North Carolina overall during 2013 and 2014.
When compared to the other states on the Eastern coast of the United States, North Carolina had the lowest debris items per mile in 2013 and second lowest in 2014 (Figure 34). We then took these numbers and assumed that they were total rate and then multiplied them by the total miles of shoreline for that state in order to rescale the data to account for the length of shoreline per state. The results are plotted as total assumed debris (Figure 35).
Figure 14. Comparison of debris collected in cleanups in the states on the East Coast of the United States in a) 2013 and b) 2014
0 500
1000 1500 2000 2500 3000 3500 4000
Conn
ec4cut
Delaware
Florida
Georgia
Maine
Maryland
Massachuse[
s New
Ham
pshire
New
Jersey
New
York
North Carolina
Penn
sylvania
Rhod
e Island
South Carolina
Virginia
Items p
er M
ile
a) 2013
0 500
1000 1500 2000 2500 3000
Conn
ec4cut
Delaware
Florida
Georgia
Maine
Maryland
Massachuse[
s New
Ham
pshire
New
Jersey
New
York
North Carolina
Penn
sylvania
Rhod
e Island
South Carolina
Virginia
Items p
er M
ile
State
b) 2014
0
100
200
300
400
500
600
700
800
2013 2014
Items p
er M
ile
North Carolina Carteret County
50
1 10 100
1,000 10,000
100,000 1,000,000 10,000,000
100,000,000
Conn
ec4cut
Delaware
Florida
Georgia
Maine
Maryland
Massachuse[
s New
Ham
pshire
New
Jersey
New
York
North Carolina
Penn
sylvania
Rhod
e Island
South Carolina
Virginia
Debris (Item
s/mile)
a) 2013
Figure 35. Comparison of total assumed debris in each state on the East Coast based on The numbers from Figure 3 were assumed to be a total rate and then multiplied by total miles of coastline to get total assumed debris for that year.
1
10
100
1000
10000
100000
1000000
10000000
Conn
ec4cut
Delaware
Florida
Georgia
Maine
Maryland
Massachuse[
s New
Ham
pshire
New
Jersey
New
York
North Carolina
Penn
sylvania
Rhod
e Island
South Carolina
Virginia
Debris (item
s/mile)
b) 2014
51
When Carteret County was compared with 50 countries chosen randomly, it was equivalent to the 42nd of those countries in both 2013 and 2014 in items/mile (Figure 36).
1
10
100
1000
10000
100000
Croa4a
Gh
ana
Singapore
Turkey
Mozam
biqu
e Indo
nesia
Ph
ilipp
ines
Dominican Rep
ublic
Sint M
aarten
, Dutch W
est
Jamaica
Sri Lanka
Japan
Malaysia
Ge
rmany
Costa Rica
Spain
Greece
Chile
Australia
Egypt
Thailand
Mexico
Argen4
na
Uruguay
Norway
Nicaragua
Ecuado
r Malta
Repu
blic of K
orea
Belize
U.S. V
irgin Island
s Maldives
Saint K
i[s a
nd Nevis
Puerto Rico
China
Hong Kon
g South Africa
India
Saint V
incent and
the
United Arab Emira
tes
Sloven
ia
Carteret Cou
nty
Canada
Bahamas
Saud
i Arabia
Italy
United States
Saint Lucia
Banglade
sh
Portugal
Items p
er M
ile
a) 2013
1
10
100
1000
10000
100000
China
Dominican Rep
ublic
Mozam
biqu
e Ho
ng Kon
g Singapore
Ghana
Spain
Philipp
ines
Saud
i Arabia
Portugal
Sri Lanka
Nicaragua
Turkey
Jamaica
Germ
any
Indo
nesia
Sint M
aarten
, Dutch
Maldives
Thailand
United Arab Emira
tes
Saint Lucia
U.S. V
irgin Island
s Greece
Chile
India
Mexico
Repu
blic of K
orea
Belize
Ecuado
r Pu
erto Rico
Malaysia
Sloven
ia
Saint K
i[s a
nd Nevis
Costa Rica
Uruguay
Norway
Japan
Bahamas
South Africa
Canada
Argen4
na
Carteret Cou
nty
United States
Australia
Croa4a
Saint V
incent and
the
Malta
Egypt
Banglade
sh
Items p
er M
ile
b) 2014
Figure 36. Quantity of Marine Debris collected in cleanups in different countries around the world in a) 2013 and b) 2014.
52
There was no significant trend between density of debris and total population of the selected countries (Figure 37).
There was no relationship found between income generated by tourism and the amount of debris (Figure 38).
R² = 0.01392 1
10
100
1000
10000
100000
1 100 10,000 1,000,000 100,000,000 10,000,000,000
Debris (Item
s/Mile)
Popula4on
Figure 38. Amount of debris collected in the yearly cleanup versus income generated by tourism. The y-axis is presented with units of Items/person/mile. The x-axis is shown in American dollars.
Figure 37. A graph examining the possible relationship between population of a country and the amount of marine debris found during its cleanup. No clear relationship can be observed.
53
4. DISCUSSION
When I compared Carteret County to the 50 randomly selected countries, Carteret County had a higher number of items per mile than 8 countries including Canada and the United States. This along with the large difference between Carteret County and North Carolina at large, suggests that for some reason, Carteret County has a larger amount of marine debris than might be predicted. However, North Carolina as a whole, has a much lower amount of debris than most of the rest of the east coast of the United States. This could be due to many different factors, but this analysis did not provide enough information to answer that question.
Another pertinent question was whether the distribution of marine debris in Carteret County was roughly equal to the global distribution. When the two distributions were compared, the percentages of the different types of debris remained about the same except for cigarette butts. The global percentages of cigarette butts found were only 15% in comparison to 40% in Carteret County. The only other notable difference between the two distributions was that only 5% of the debris collected in Carteret County was made up of food wrappers, while the worldwide percentage was around 12%. This suggests that while Carteret County is doing better than the world at large at reducing food wrappers from reaching the water, it is doing rather poorly at keeping cigarette butts from ending up as marine debris. There has been lots of public attention drawn to reducing plastic marine debris because of its impact on marine organisms, but not nearly as much attention has been drawn to the effects of cigarette butts being left on beaches and in the oceans. Based on the numbers shown earlier in the results with nearly 40% of debris that was collected in Carteret County in 2013 being cigarette butts and only 2% being plastic grocery bags, more emphasis should be placed on keeping cigarette butts out of the environment than plastic grocery bags. Much is known about the effects of the chemicals within cigarette butts on humans, but not much is known about their effect on marine ecosystem health. More research needs to be put into this topic, but also, policies need to be implemented in order to greatly reduce the amount of cigarette butts that end up as marine debris.
The comparisons that were drawn between population and marine debris as well as between tourism income and marine debris are very tentative. The countries that participated in the cleanups are likely more committed to cleanups or have greater resources to devote to cleanups than the countries that did not participate which could affect the results of these comparisons.
54
Chapter 6: Synthesis 1. SUMMARY OF MAIN FINDINGS
Globally, the majority of the human population is concentrated in close proximity to various bodies of water such as lakes, rivers and oceans. Consequently, as the human population has exponentially increased, so has its load in waste and trash. Some of this trash ends up in the bodies of water, especially the ocean and has only increased as human activity has increased. However, Carteret County has less marine debris on its shorelines compared to a lot of the rest of the world. Through our survey of marine debris in Carteret County, we have determined the extent and type of debris present, its utilization and colonization by organisms, as well as potential ramifications for human health across three sites of varying intensity of human activity. We found that across two of three sites, Carrot Island and Treasure Island, plastic debris was the most prevalent while Styrofoam was most abundant on Shackleford Banks (Chapter 1, Figure 7). However, the amount of debris found in these locations may have been influenced by storm events that occurred during our sampling. For example, Shackleford Banks had the greatest count density of debris, with a majority of the debris coming from the upper intertidal zone, as with Treasure Island. This is likely due to the debris being pushed up the beach during high water levels. The majority of this debris found was also small in size, with most pieces being less than 1g in mass. This is likely due to the weathering process to which the debris was subjected, which would degrade and break down larger pieces of debris into the smaller pieces.
Of debris found that was being used by organisms, plastic was also the most commonly colonized by biofilms (Chapter 2, Figures 4 and 5). These fouling micro-organisms were found almost exclusively on lightweight flexible plastic, such as food wrappers. Since biofilms are known to act as first colonizers (Marszalek, 1979) we expected these types of plastic debris to be colonized first and therefore the most colonized in our controlled exposure experiment. However, colonization was highest on hard substrate, especially glass bottles, and was primarily comprised of communities of barnacles and bryozoans rather than biofilms. Barnacles were the most abundant colonizers and were found mostly on glass. Bryozoans were the second most abundant, yet showed no real substrate preference (Chapter 4, Figure 29). There were also several other species found that were not in appreciable abundances, but indicate that a variety of different organism types can settle and grow on debris (Chapter 4, Figure 29). This diversity and settlement by larger fouling organisms suggests that the time period of fouling and colonization by various organisms may be much shorter than we originally thought, but is still likely to be highly variable and substrate specific. The marine debris that was placed in the subtidal zone was the most heavily settled by organisms compared to the intertidal and supratidal debris placements (Chapter 4, Figure 30). This suggests that most colonization of debris in the marine environment occurs in the subtidal zone. This relates back to the debris that was found during our spatial surveys in the intertidal and supratidal zones that had colonization. It is likely that that debris was originally colonized in the subtidal zone and was washed up into the other zones by storms.
The debris usage survey noted many similar findings, though colonization was mainly by biofilms on plastic surfaces. The association between the two was so common, that we suggest that there may be a mechanism at work by which lightweight flexible plastics enable and promote the growth of biofilms and chlorophytes (Chapter 2, Figures 11, 12, and 13). This could
55
potentially be due to the fact that they float and therefore allow access to light while still being in the water. Our study also determined that marine debris is used by organisms in ways other than direct colonization. Macro-organisms such as fiddler crabs and snails appeared to be using some debris as temporary shelter for themselves and/or their young (Chapter 2, Figure 13). We suggest that this is likely due to the higher rigidity of debris in comparison with natural shelter, such as marsh grass. While it may seem that a temporary shelter would be beneficial to the individual organism in the short term, long term effects and broader environmental ramifications could differ significantly.
Usage of marine debris can vary greatly depending on the amount of time that the debris is in the system (Chapter 4). As mentioned above, we found that marine debris is used by a host of encrusting and individual organisms, such as barnacles, bryozoans, or fiddler crabs (Chapters 2 and 3). However, with a closer look, there are also microbes that have colonized marine debris as well. The bacteria Escherichia coli and Vibrio vulnificus were found at all three sites of Carrot Island, Shackleford Banks and Treasure Island on marine debris. E. coli and V. vulnificus were both found in highest concentrations on debris that was located in the intertidal and subtidal zones compared to the supratidal zones. This is likely due to the bacteria suffering desiccation in the supratidal zone (Billi and Potts, 2000; Bridge and Demicco 2008). Also, it seemed that E. coli favored attaching more to the debris compared to the sediment that surrounded the debris, and this is likely due to the potential biofilm growth on the debris which can attract and harbor large amounts of bacteria (Lobelle and Cunliffe 2011; Donlan 2002).
The bacteria that were found at these sites are not necessarily hazardous to human health, but some strains of V. vulnificus can cause septic shock and necrosis of wound infections while E. coli presence in a marine system is used as an indicator for water quality and can be an indicator for the presence of other deadly pathogens (Centers for Disease Control and Prevention, 2013; Centers for Disease Control and Prevention and American Water Works Association, 2013). The bacteria were found in low concentrations at the three sites, so pose little harm to humans. However, for beach cleanup volunteers, it is recommended that they wear gloves when picking up and disposing of debris in order to reduce the chance of infection. 2. FUTURE WORK
During our survey of debris usage, we uncovered the possibility of organisms being moved out of their natural environment due to marine debris. One such example was us finding barnacles, which are usually found in subtidal zones, attached to debris found in the upper intertidal and supratidal. Though we were not able to observe or investigate this directly, it is another possibility that should be further explored along with the ways in which this displacement could affect the organism's life cycle. Another area of research should be the extent to which hitch-hiking organisms are being transported on marine debris – how often this happens and how far they go. In terms of microorganisms, further studies that use a larger sample size will be needed to determine whether V. vulnificus is significantly more likely to attach to anthropogenic debris rather than natural debris. The exposure experiment should be run in different seasons and for different lengths of time in order to get a more complete picture of how quickly marine debris is colonized and by what organisms. Checking on the state of debris colonization at smaller more regular intervals would be more beneficial to determine in what order colonization occurs and more exact time frames for this process for particular species. An additional step for this kind of experiment would be to assess the balance between the
56
accumulation of organisms and the degradation of the available substrate. Studies on the residence time of debris, taking into account multiple locations as the debris is transported through tides and currents, would be another prime subject of further research. The last piece of recommended future research would be to study non-amenity beaches and how debris gets onto these types of beaches since direct human use of them is rare.
3. MANAGEMENT RECOMMENDATIONS
Given the large amount of debris quantified in our project, management intervention to reduce marine debris is required at multiple levels: educating the public on the extent of marine debris and their role in exacerbating the problem, creating a system of incentives and disincentives to reduce introduction of marine debris to the environment, and intelligently legislating to minimize effort and resources while maximizing removal of debris. Marine debris affects everyone living in a coastal area, especially those looking to recreate or fish in the water or on the beaches (Cuomo et al. 1988). As such, marine debris should be a concern to everyone living in a coastal area. Many people do not realize how widespread debris is in the marshes and beaches around them and how they are contributing to this problem, deliberately or accidentally. Raising awareness of the types and frequencies of material most commonly found, how humans facilitate the entrance of it into the marine system, and the harm it has on organisms is an essential step in helping people consider their role in generating, but also, reducing and cleaning up, marine debris. Educating the younger generation will be of particular importance if we are to make any progress against this problem. One way of facilitating such outreach could be an extension of an existing program at the Pine Knolls Shore Aquarium- an interactive turtle care exhibit. A similar program centering around marine debris could be created for children, showing different types and examples of marine debris in the environment, as well impacts on popular macrofauna. A play station for children in which they can see the effects of debris firsthand, such as a turtle model with a plastic bag in its stomach, would reach and impact children and possibly adults as well. One of the largest social issues surrounding marine debris is a sense of disconnect between the environment and the consequences of human actions. A program such as this would bring people into contact with this issue and might motivate them to care more about what goes into the environment and be more likely to reduce their own personal contribution to marine debris. Education is perhaps the most powerful tool for persuading people to change or develop a certain mindset and be willing transform it into behavior. Combined with a system of incentives, education would benefit those in a management position trying to improve the state of marine debris pollution in their jurisdiction. A system of incentives and disincentives in Carteret County would encourage a voluntary behavioral change in the residents and visitors of Carteret County without the forced elimination of materials or practices. For example, to discourage the use of plastic bags without an outright ban, consumers could be allowed to choose to bring their own reusable bag or pay a plastic bag fee for which they are only reimbursed once the bag is brought back to the store’s recycling center. Alternatively, the fee may be paid without the possibility of reimbursement. This fee would need to be large enough to inspire a behavior change. An accompanying public awareness campaign would show the benefits of making the switch away from plastic to reusable bags. The success in this method of marine debris prevention results not only in less of it, but makes people aware of the potential impact they can unknowingly have on the ecosystem around them. On the other hand, a positive incentive can be implemented within Carteret County to reduce the
57
introduction of marine debris to the environment. For example, encouraging the placement of trash cans around business areas or areas of high traffic, such as the beach or boat docks where the easiest option for trash disposal is littering. This method of reducing marine debris targets, like the disincentive method, the source of the problem, by easing the effort people must make to properly dispose of their trash. It is essential to reduce the input of marine debris if the current situation is to improve at all, as mitigating its presence is made harder with an endless and non-decreasing stream of marine debris. In specific relation to Carteret County and Morehead City (renowned for its fisheries), the placement of waste and recycling bins, especially for fishing waste, such as line, hooks, and nets, at popular public docks, piers, and mooring stations would offer a place for fishermen and boaters in general to dispose of their fishing gear conveniently. While these bins are very successful in collecting fishing trash and many such receptacles are already in use, the problem lies in the lack of timely removal of wastes and upkeep of the bins (Keith Rittmaster, personal communication, October, 2014). The most probable solution to maintaining these bins would be assigning this duty to a position already held by someone working on the docks, since removal by volunteers is often unreliable and may allow waste buildup. Overflow in waste bins prevents further proper disposal, and can wash debris into local waterways (Keith Rittmaster, personal communication, October, 2014). Once the input of marine debris is reduced and the public is aware and ready to act, current approaches to cleaning up the environment should be modified in light of our findings. Firstly, clean-up efforts, if limited in resources, should be focused on the zones of highest debris concentration, such as the upper intertidal or supratidal zones of shorelines. This way, more debris can be removed with less effort. In addition, we recommend increased focus of future clean-up efforts on non-amenity beaches, as there is already a focus on maintaining trash-free amenity beaches that attract tourists. Beaches and marshes like the ones in our study, are non-amenity beaches, but still have relatively large amounts of marine debris. These beaches are often richer in wildlife; therefore, organisms are more likely to interact with debris in these areas. Especially around our sample area, Beaufort and Morehead City, there is seasonal high-density boat traffic to these islands, excluding Treasure Island. Carrot Island and Shackleford Banks receive relatively large numbers of boaters who moor and traverse onto the island, and leave behind debris. Beaches in areas close to development or frequented by summer boaters should receive more attention during summer and fall clean-up efforts, given that we found the largest amount of debris on Carrot Island and Shackleford Banks.
58
Acknowledgements The Morehead City Field Site students of 2015 would like to thank our mentors Johanna Rosman, Stephen Fegley, and Kathleen Onorevole for their time, effort, and valuable guidance throughout this capstone experience. We would also like to thank Captain Joe Purifoy for his knowledge of the surrounding area in Carteret County and for taking the time to take us to our various field sites. We would like to thank the entire Noble Lab for their help and guidance in the design of the experiment of the bacterial portion of the project. We would like to thank Dee Edwards-Smith for the Big Sweep - Carteret County data and for the educational talk on the potential negative impacts of marine debris. We would also like to thank Paula Gillikin for providing advice on potential field sites and allowing us access to the Rachel Carson Reserve in order for us to conduct our field studies. We would like to thank the Ocean Conservancy for the geographical and statistical data used in the National and International comparisons portion of this project. We would also like to thank the Cape Lookout National Seashore in allowing us access to Shackleford Banks. Lastly, we would like to thank the Institute of Marine Sciences and the Institute for the Environment Morehead City Field Site for funding and allowing us to partake in this valuable experience.
59
References
Aliani, S., & Molcard, A. (2003). Hitch-hiking on floating marine debris: macrobenthic species in the Western Mediterranean Sea. Hydrobiologia, 503 (1), 59 – 67.
Andrady, A. (2003). Plastics in the Marine Environment. In Plastics and the Environment. Research Triangle Park, NC: Wiley-Interscience.
Barnes, D.K.A. (2002). Biodiversity: Invasions by marine life on plastic debris. Nature, 416, 808–809. Billi, D., & Potts, M. (2000). Life without water: responses of prokaryotes to desiccation. In K.B.
Storey, & J.M. Storey (Eds.), Environmental Stressors and Gene Responses (181– 192). Amsterdam: Elsevier.
Bridge, J., & Demicco, R. (2008). Earth Surface Processes, Landforms and Sediment Deposits. Cambridge, UK: Cambridge University Press.
Cadee, G. C. (2002). Seabirds and floating plastic debris. Marine Pollution Bulletin, 44 (11), 1294 – 1295.
Cawthorn, M. (1989). Impacts of marine debris on wildlife in New Zealand coastal waters. In Proceedings of Marine Debris in New Zealand’s Coastal Waters Workshop, 9 March 1989, Wellington, New Zealand (5–6). Wellington, New Zealand: Department of Conservation.
Centers for Disease Control and Prevention, & American Water Works Association. (2013). Frequently Asked Questions About Coliforms and Drinking Water. Centers for Disease Control and Prevention.
Chang, C. et. al. (2005). Determining leaching of bisphenol A from plastic containers by solid-phase microextraction and gas chromatography–mass spectrometry. Analytica Chimica Acta, 539 (1 & 2), 41 – 47.
Chiang, S., & Chuang, Y. (2003). Vibrio vulnificus infection: clinical manifestations, pathogenesis, and antimicrobial therapy. Journal of Microbiology, Immunology, and Infection, 36 (2), 81-88.
CHROMagar. (2012). CHROMAGAR. Retrieved December 6, 2015, from http://www.chromagar.com/food-water-chromagar-vibrio-focus-on-vibrio-species-23.html#.VmSaivmDFBd
Clark, R., Pittman, S. J., Battista, T. A., & Caldow, C. (2012). Survey and impact assessment of derelict fish traps in St. Thomas and St. John, U.S. Virgin Islands. Silver Spring, MD: U.S. Department of Commerce.
“Country Comparison: Population.” World Factbook. Central Intelligence Agency, 2015. Web. 7 Nov. 2015.
Cruickshank, R. (1968). Medical Microbiology. 11th ed. London, UK: Livingstone Ltd. Cuomo, V., Pane, V., & Contesso, P. (1988). Marine litter: Where does the problem lie? Progress in
Oceanography, 21 (2), 177-180. Derraik, J. (2002). The pollution of the marine environment by plastic debris: a review. Marine
Pollution Bulletin, 44, 842-852. DOC––Department of Conservation. (1990). Marine Debris. Wellington, New Zealand. Donlan, R. (2012). Biolfims: Microbial Life on Surfaces. Emerging Infectious Diseases, 8(9). Garrity, S., & Levings, S. (1993). Marine debris along the Caribbean coast of Panama. Marine Pollution
Bulletin, 26(6), 317-324.
Gregory, M.R. (2009). Environmental Implications of Plastic Debris in Marine Settings—Entanglement, Ingestion, Smothering, Hangers-On, Hitch-Hiking and Alien Invasions. Philosophical Transactions: Biological Sciences, 364 (1526), 2013 – 2025.
60
Hung, O.S., Thiyagarjan, V., & Qian, P.Y. (2008). Preferential attachment of barnacle larvae to natural multi-species biofilms: does surface wettability matter? Journal of Experimental Marine Biology and Ecology, 361, 36-41.
IDEXX. (2012). Colilert®-18. Retrieved December 6, 2015, from https://www.idexx.com/water/products/colilert-18.html
"International Tourism, Receipts (current US$)." International Tourism, Receipts (current US$). World Bank, 2015. Web. 7 Nov. 2015.
Katsanevakis, S., Verriopoulos, G., Nicolaidou, A., & Thessalou-Legaki, M. (2007). Effect of marine litter on the benthic megafauna of coastal soft bottoms: A manipulative field experiment. Marine Pollution Bulletin, 54, 771–778.
Knight-Jones, E.W., & Crisp, D.J. (1953). Gregariousness in barnacles in relation to the fouling of ships and to the anti-fouling research. Nature (Lond), 171, 1109-1110.
Köhler, J., Hansen, P.D., & Wahl, M. (1999). Colonization patterns at the substratum-water interface: how does surface microtopography influence recruiement patterns of sessile organisms? Biofouling, 14, 237-248.
Kuo, F. & Huang, H. (2014). Strategy for mitigation of marine debris: Analysis of sources and composition of marine debris in northern Taiwan. Marine Pollution Bulletin, 83, 70-78.
Lobelle, D. and Cunliffe, M. (2011). Early Microbial Biofilm Formation on Marine Plastic Debris. Marine Pollution Bulletin, 62, 197-200. http://dx.doi.org/10.1016/j.marpolbul.2010.10.013
Maki, J.S., Rittschof, D., & Mitchell, D. (1992). Inhibition of larval barnacle attachment to bacterial films: an investigation of physical properties. Microbial Ecology, 23, 97-106.
Marszalek, D. S. et. al. (1979). Influence of Substrate Composition on Marine Microfouling. Applied and Environmental Microbiology, 38 (5), 987 – 995.
Messens, W., Bolton, D., Frankel, G., Liebana, E., McLauchlin, J., Morabito, S., & Threlfall, E. J. (2015). Defining pathogenic verocytotoxin-producing escherichia coli (VTEC) from cases of human infection in the european union, 2007-2010. Epidemiology and Infection, 143 (8), 1652-1661. doi:http://dx.doi.org/10.1017/S095026881400137X
Morris, J. (2014). Vibrio vulnificus infections. Retrieved December 7, 2015, from http://www.uptodate.com/contents/vibrio-vulnificus-infections
National Oceanic and Atmospheric Administration (NOAA). (2014). Entanglement: Entanglement of marine species in marine debris with an emphasis on species in the United States. National Oceanic and Atmospheric Administration.
National Oceanic and Atmospheric Administration (NOAA). (2014). Ingestion: Occurrence and Health Effects of Anthropogenic Debris Ingested by Marine Organisms. National Oceanic and Atmospheric Administration.
Noble, R.T. & Fuhrman, J.A. (1998). Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria. Aquatic Microbial Ecology, 14, 113-118.
O’Brine, T. & Thompson, R.C. (2010). Degradation of plastic carrier bags in the marine environment. Marine Pollution Bulletin, 60, 2279-2283.
Peterson, C.G., & Stevenson, R.J. (1989). Substratum conditioning and diatom colonization in different current regimes. J Phycology, 25, 790-793.
Petrova, O.E., & Sauer, K. (2012). Sticky situations: key components that control bacterial surface attachment. J Bacteriol, 194, 2413–2425. http://dx.doi .org/10.1128/JB.00003-12.
Pomerat, C.M., & Weiss, C.M. (1946). The influence of texture and composition of surface on the attachment of sedentary marine organisms. The Biological Bulletin, 91, 57-65.
61
Qian, P.Y., Thiyagarjan, V., Lau, S.C.K., & Cheung, S.C.K. (2003). Relationship between bacterial community profile in biofilm and attachment of the acorn barnacle Balanus Amphitrite. Aquatic Microbial Ecology, 33, 225-237.
Ribic, C., Sheavly, S., & Klavitter, J. (2012). Baseline for beached marine debris on Sand Island, Midway Atoll. Marine Pollution Bulletin, 64, 1726-1729.
Ryan, P., Moore, C., Van Franeker, J., & Moloney, C. (2009). Monitoring The Abundance Of Plastic Debris In The Marine Environment. Philosophical Transactions of the Royal Society B: Biological Sciences, 364 (1526), 1999-2012.
Sajiki, J. & J. Yonekubo. (2003). Leaching of bisphenol A (BPA) to seawater from polycarbonate plastic and its degradation by reactive oxygen species. Chemosphere, 51 (1), 55 – 62.
Sheavly, S. & Register, K. (2007). Marine debris and plastics: environmental concerns, sources, impacts and solutions. Journal of Polymers and the Environment, 15 (4), 301–305.
Sieburth, J. M. (1976). Bacterial Substrates and Productivity in Marine Ecosystems. Annual Review of Ecology and Systematics, 7, 259 – 285.
Silva, M., Araújo, F., Castro, R., & Sales, A. (2015). Spatial–temporal analysis of marine debris on beaches of Niterói, RJ, Brazil: Itaipu and Itacoatiara. Marine Pollution Bulletin, 92, 233-236.
Thiyagarajan, V., Lau, S.C.K., Cheung, S.C.K., & Qian, P.Y. (2006). Cypris habitat selection facilitated by microbial biofilms influences the vertical distribution of subtidal barnacle Balanus trigonus. Microbial Ecology, 51, 431-440.
Thompson, R.C., Norton, T.A., & Hawkins, S.J. (1998). The influence of epilithic microbial films on the settlement of Semibalanus balanoids cyprids – a comparison between laboratory and field experiments. Hydroboiologia, 375, 203-216.
Uhrin, A. V., & Schellinger, J. (2011). Marine debris impacts to a tidal fringing-marsh in North Carolina. Marine Pollution Bulletin, 62, 2605-2610.
UNESCO. (1994). Marine Debris: Solid Waste Management Action Plan for the Wider Caribbean. IOC Technical Series, 41, Paris.
Walker, T., Reid, K., Arnould, J., & Croxall, J. (1997). Marine debris surveys at Bird Island, South Georgia 1990–1995. Marine Pollution Bulletin, 34 (1), 61-65.
Westerhoff, P. et. al. (2008). Antimony leaching from polyethylene terephthalate (PET) plastic used for bottled drinking water. Water Research, 42 (3), 551 – 556.
Wieczorek, S.K., Clare, A.S., & Todd, C.D. (1995). Inhibitory and facilitatory effects of microbial films on settlement of Balanus Amphitrite Amphitrite larvae. Marine Ecology Progress Series, 119, 221-228.
Wilber, R.J. (1987). Plastic in the North Atlantic. Oceanus, 30, 61–68.
![WATERWAY RISK ASSESSMENT WATERWAYS MANAGEMENT PLANNING TODAY TO BE READY FOR TOMORROW PAWSA [WATERWAY NAME]](https://img.pdfslide.tips/doc/110x75/56649efb5503460f94c0d4ff/waterway-risk-assessment-waterways-management-planning-today-to-be-ready-for.jpg)
