The presence of plastic pollution in the Australian Fur Seal (Arctocephalus pusillus doriferus) colony of Seal Rocks, Victoria, Australia.

Karl S. Jaeger

AbstractThe presence of plastic pollution in the marine environment is a growing concern for

environmental scientists around the world. As there is little data on the effects of plastic in the World’s marine ecosystem, specifically larger marine vertebrates such as the Australian Fur Seal (Arctocephalus pusillus doriferus) the need for more information to further understand the effects of plastic pollution on the marine ecosystem is of the utmost importance. The aim of this project was to provide a preliminary report on the presence of plastic pollution in the Australian Fur Seal colony of Seal Rocks, Phillip Island, Victoria, Australia.

This project was undertaken in an attempt to increase the data available in regards to the presence of plastic pollution in larger vertebrate mammalian species such as the Australian Fur Seals. 92 seal faecal scat samples were taken from the Australian Fur Seal colony of Seal Rocks in Victoria, Australia. Plastics were isolated from the scat samples by drying, sieving, and separating by floating out plastic material in a saturated NaCl solution. FT-IR analysis was then used to identify what types of plastics were present. Seven scat samples were discarded due to the moisture content being too high. From the 85 scat samples remaining 14 (16%) contained plastic fragments ranging in sizes of approximately 2-15mm in length. (Table A7)(Figure 5-6) FT-IR spectroscopy was then used to confirm the types of plastics present. The plastics indicated were PE, HDPE and Nylon. These plastics are all consistent with waste associated with bottles, plastic packaging and fishing line. In conclusion it was determined that there is a presence of plastic pollution in the Australian Fur Seal colony of Seal Rocks consistent with the findings of current research. (Bravo Rebolledo 2013) (Eriksson 2003) It is in my opinion that these results may warrant further monitoring and research of the effects of the presence of plastic in the marine food web of not only the seal colonies of Victoria, but also across the entire marine ecosystem.

Key Words

Microplastic Bioaccumulation Deleterious

IntroductionSince the observation of microplastic pollution (plastic pieces ≤ 5mm) in the world’s

marine environment in 1972 the effects of these plastics on the marine ecosystem has been of interest to environmental science. (Carpenter 1972) Research into the presence of plastic pollution in the oceans of the world has revealed that 60-80% of accumulated pollution in the marine environment is plastic waste and that the presence of this plastic presents an ever growing threat to the marine environment. (Andrady 2011) (Wright 2013) As the threat of plastic pollution in the marine environment increases, concerns have been raised as to the effects of this plastic on the plant and animal life present in the marine ecosystem once it has entered the Earth’s oceans. (Carson 2013) Studies into the accumulation of plastic pollution in the marine food web have shown that there is an alarming amount of plastic being found in species such as marine birds, seals, turtles and filter feeders such as mussels and zoo plankton species. (Boerger 2010) (Eriksen 2014) (Lavers 2014)

The effects of the bioaccumulation of plastics in the marine food web rather than the direct ingestion of plastics as covered in recent research involving marine birds is of interest as the possible effects of these plastic fragments may present a physical threat to the animals that ingest them. (Eriksson 2003) (Lavers 2014) (Lusher. 2013) Apex predators like the Australian Fur Seal are possibly at a greater risk to the threat of any deleterious effects of plastic pollution due to the bioaccumulation of the plastic fragments in their prey species and any harmful agents that may be associated with the plastic materials. (Wright 2013) (Setala 2014) (Paul-Pont 2010) (Bakir. 2014) It is due to the possible effects of these plastics that the acquisition of up to date data is required to further understand, where this form of pollution can be found and what are the possible effects of its presence in the marine food web. This report will attempt to add further detail to the understanding of the nature of the effects of plastic pollution by answering the question. Is there a presence of plastic pollution in the Australian Fur Seal colony of Seal Rocks, Victoria, Australia?

Methods

Sample collectionSamples were taken from random areas of the colony during two trips to Seal Rocks,

the first in July 2014 and a second in February 2015. Sampling methods for the detection of plastic pollution varied due to environmental conditions. For this project, sampling of the seal’s faecal matter was chosen as this method allowed for ease for collection and more importantly, minimum contact with the seal population. (Eriksson 2003) Each sample was placed into a paper bag, then into a second paper bag. Once this was completed the two bags were placed in a plastic bag and stored in a freezer at -80°C. Paper bags were used to eliminate the variable of the contamination of the samples by other sources of plastic.

Oven Drying.

All handling of faecal material was conducted in a class 2 biological cabinet.

The following method was experimentally determined after initial trialling of a method involving wet faecal scat material. (Claessen 2013) (Hidalgo-Ruz. Gutow 2012) (Hollman 2013) The revised drying method decreased the amount of handling time and also dramatically reduced the amount of liquid waste produced.

The initial seal scat samples were weighed separately using an analytical balance. (Table Appendix A1) This data was recorded to determine the moisture content of the samples. (Initial weight minus dry weight) The seal scat samples were dried in a drying oven at 75°C for 24hrs. Samples were dried on trays lined with tin foil and each scat was placed on a separate square piece of tin foil (10x10cm approx) and placed on the tray. Scats were weighed once dried (dry weight). (Table A1) These results were then statistically analysed to determine mean and standard deviation for weight for initial samples. (Table A2)

SeparationOnce the samples were desiccated (completely void of moisture), the seal scats were

placed into a mortar and crushed gently so as not to damage any possible plastic fragments. Once the seal scat was completely broken up, it was placed into a coarse sieve (1mm) and gently sieved over biological hazard bag. At this point most of the organic material was separated leaving only fragments ≥1mm. Once the seal scat was thoroughly sieved the remaining contents were placed into 250ml beaker containing 150mLs of saturated NaCl solution (900g/Litre) adjusted. (Hollman 2013) (Claessen 2013)

When all the samples were prepared, they were allowed to stand until the heavier material settled to the bottom. (Hidalgo-Ruz. Gutow 2012) Once all heavier material had settled samples were checked for visual signs of separation (plastics floating on surface of NaCl solution and solids at the bottom). (Hidalgo-Ruz. Gutow 2012) Any plastics present were removed using a tea strainer (small sieve) or tweezers, then placed in a Petrie dish (1/sample). (Hidalgo-Ruz. Gutow 2012) The isolated plastics were then stored in Eppendorf tubes filled with 70% Ethanol in a freezer at -80°C. Liquid waste (slurry) was filtered first using funnel and filter papers to separate the solid waste from liquid waste. (As per instruction outlined in Lab RA) Liquid waste was then disposed of via a waste water drain after the addition of Pyroneg (disinfectant), isolated solid waste was autoclaved and incinerated. (As per instruction outlined in Lab RA).

Analysis methodsOnce the plastic fragments were isolated from the scat samples, the pieces were

statistically analysed to determine the mean and standard deviation for size of the fragments isolated. (Table A4-A6) Fourier Transmission Infra Red Spectroscopy (FT-IR) was then used to analyse the viable fragments to confirm the types of materials present. (Hidalgo-Ruz. Gutow 2012). A Spectrum 100 FT-IR was used with a range of 650-4000cmˉ¹, with settings of Energy = 300, Scan =8 and resolution = 4 (default) used. The total number of confirmed plastic

fragments were then statistically analysed to determine the mean and standard deviation for size, number and type of plastics isolated. (Table A7-A8)

Results93 samples were taken from the Australian fur seal colony of Seal Rocks in Victoria,

Australia and analysed for the presence of plastic pollution. (Table A1) Seven samples were discarded due to the moisture content being too high. The remaining scat samples displayed an average mass of 59.53g±54.37g for wet samples and 26.71g±27.07g for dried samples. (Figures 1-2)(Table A2) From the remaining 85 samples 45 plastic like fragments ranging in sizes of approximately 1-15mm in diameter were isolated. (Table A3) Only fragments ≥1mm were kept for analysis, all other material <1mm was discarded. The initial 21 viable samples contained 45 fragments each having an average number of 2.14±1.80 pieces found per scat sample. (Table A3-A6) The initial fragments displayed assorted shapes, with forms such as filament, spherical, triangular and round pieces present. (Figure 3) On examination the plastic fragments clearly showed signs of weathering with clear indications of abrasion and tearing, with sizes in the range of 1-15mm with an average of 5.31mm±3.65mm, with 16% of the initial pieces measuring 5mm and 16% measuring 6mm. (Table A4)(Figure 4)

Figure 1: Distribution of weight of wet scats in initial samples.

Figure 2: Distribution of weight of dry scats in initial samples.

Figure 3: Examples of confirmed plastics. Samples 1b and 8b.

2%

12%

12%

12%

16%16%

2%

7%

5%5%

2%5% 2% 2%

% Distribution of size in initial samples

1mm 2mm 3mm 4mm 5mm 6mm 7mm8mm 9mm 10mm 11mm 12mm 13mm 15mm

Figure 4: Percent distribution of isolated fragment size in initial seal scat samples.

FT-IR spectroscopy was then used to determine the types of plastics present. The total number of fragments confirmed to be plastic was 18 pieces isolated from 14 out the initial 85 samples (16%). (Figure 6) (Table A7) The plastic fragments confirmed by FT-IR analysis had a size range of 2-12mm in length with an average of 6.06mm±2.53mm (Table A8) with 28% of the confirmed pieces measuring 6mm. (Figure 5) The isolated plastics displayed colouring of brown/grey, clear and white (Table A7) with the types plastics identified as Polyethylene (PE) 61%, High density Polyethylene (HDPE) 11% and Nylon 28%. (Table A7)(Figure 7) On examining the plastic fragments the pieces clearly showed signs of weathering with clear indications of abrasion and tearing consistent with server exposure. (Eriksson 2003) (Figure 3)

6%6%

17%

17%28%

11%

6%6%

6%

% distrubition of size of in samples containing confirmed fragments

2mm 3mm 4mm 5mm 6mm8mm 9mm 10mm 12mm

Figure 5: Percent distribution of confirmed plastic fragment sizes.

84%

16%

Percetage of samples containing plastic fragments

Samples containing no plastic Samples containing plastic

Figure 6: Percentage of confirmed samples containing plastic.

61%11%

28%

Types of plastic found in samples containing viable fragments

PE HDPE Nylon

Figure 7: Percentage distribution of types of plastics isolated from seal scat samples.

DiscussionSeal Rocks is located approximately 2km off the south-west coast of Phillip Island,

Victoria and is the home to the one of the largest Australian Fur Seal colony in Australia. With a feeding radius of <200km within the Bass Strait region, (Figure 8) with some feeding trips recorded up to 1208km away from the colony, (Kirkwood. 2011) the seals have a large area in which they can come into contact with the plastic pollution. (Carson 2013) From the 85 samples collected from the colony 16% were shown to contain plastic fragments. Analysis of the plastic material isolated from the scat samples revealed the fragments had a mean size of 6.06mm±2.53mm, with a minimum of 2.00mm and a Q1-Q3 range of 4-8mm. (Table A8) The types of materials isolated were consistent with common plastic waste associated with marine pollution found in recent studies. (Eriksen 2014) The sizes of plastic fragments isolated from the Seal Rocks colony are consistent with research conducted on the seal colony of Macquarie Island found in sub-Antarctic waters of the coast of Australia. (Eriksson 2003) The research conducted by Cecilia Eriksson and Harry Burton on the presence of plastic pollution in the Macquarie Island seals revealed a similar size range of 2-5mm fragments being isolated with a mean size of 4.1mm with statistical outliners of up to 30mm. (Eriksson 2003) In 2013 a study involving harbour seals that had beached themselves and perished due to Phocine Distemper Virus (PDV) on the Dutch island Texel, were autopsied and 11% of the stomachs examined revealed the presence of plastic particles of varying sizes. As in the Eriksson paper, size (in this instance) is a good indication that the plastic pieces were not ingested by the seals directly as a part of their regular feeding habits, as has been documented in studies on marine birds and fish. (Lavers 2014) (Tanaka 2013) (Hirai 2011) (Fendall 2009) (Bravo Rebolledo 2013)

Figure 8: Map of location and feeing area. (Kirkwood 2008)

This raises the question of the origin of the fragments. The ingestion of plastic by marine animals is well documented, with studies on the ingestion of plastic pollution in all its forms by marine animals revealing that plastic pollution has found its way into the marine food web of all marine inhabitants. (Avio 2015) (Lavers 2014) (Eriksson 2003) The seal’s diet consists of a variety of species such as Cephalopods including Arrow Squid, Nototodarus gouldi and Calamari and fish species Redbait (Emmelichtys nitidus), Barracouta (Thyrsites atun), Red Cod (Pseudophycis bachus), Jack Mackerel (Trachurus declivis) and Leatherjackets (Family Monocanthidae). (Kirkwood 2008) (Kirkwood 2008) (Kirkwood. 2011) Studies have shown the accumulation of plastic pollution in pelagic species not unlike those consumed by the seals of Seal Rocks begins with the smaller organisms at the lower end of the food web that these fish regularly feed on. (Bravo Rebolledo 2013) (Baulch 2014) (Boerger 2010) (Setala 2014) The plastic found in these species and the species of prey within the food web is consumed accidently, this has been well documented in studies involving fish, marine bird species and plankton. (Lavers 2014) (Hirai 2011) (Setala 2014) Plastic fragments are mistaken for food and then passed along the food chain, finally making their way to apex predators such as seals and marine birds. (Bravo Rebolledo 2013) (Lavers 2014)

A number of factors may influence the availability of plastic and the preferences marine animals exhibit towards them. For example, the size and type of materials the fragments are made of can influence the bioavailability of this pollution in the marine ecosystem. The relative density of the plastic can affect the buoyancy of the plastic in seawater allowing the material to be spread throughout the water column. (Table 1) (Lobelle

2011) Other factors that can affect the bioavailability of plastic in the marine environment is the absorption of hydrophobic chemicals to the surface of the plastic.

This process can change the relative density of materials in the aquatic environment by adding extra material to the particle. (Rochman. 2014) Recent studies have also shown that the accumulation of microbial colonies on the surfaces of plastic and the subsequent formation of biofilm, may contribute to the change of density of these materials and it is this process of changing density that exposes this pollution to the pelagic strata inhabitants. (Lobelle 2011) The data collected from this project when compared with recent studies is consistent with the indications that the seals are not consuming the plastic as a part of their regular feeding habits, but are consuming the plastic that has been accumulating in their prey species as it passes along the marine food web. (Andrady 2011) (Bravo Rebolledo 2013) (Eriksson 2003) (Setala 2014)

ConclusionPlastic pollution in the marine environment is now of great concern as it presents a

threat not only to marine habitat but to the global ecosystem. (Eriksen 2014) It was the aim of this report to determine if there was a presence of plastic pollution in the Australian Fur seal colony of Seal Rocks, Victoria and to quantify the amount of plastic present. The data collected through the analysis of the scat samples taken from the Seal Rocks colony when compared with current research in regards to plastic pollution in marine animals, shows significant indication that there is presence of plastic pollution within the colony of Seal Rocks.

Current research into the effects of plastic pollution clearly show that plastic pollution in all its forms presents a possible threat to the marine environment. As detailed in this report it is clear there is a presence of plastic pollution in the food web of the Seal Rocks colony. The extent of plastic pollution can been seen in the data collected by current research and has been reported to be effecting the marine ecosystem in many areas. It is only through further education of the global governments and their communities that we can begin to develop a better understanding of the increasing problems created by plastic pollution. (Kalogerakis 2015) (Eriksen 2014) Initiatives like the original Marine Pollution (MARPOL) convention and recently the Marine Strategy Framework Directive (MSFD) will make aware the increasing need to develop further research programs to begin to control and understand the increasing effects of plastic pollution in the marine ecosystem, if not in general the world’s ecosystem. (Kalogerakis 2015)

AcknowledgmentsThank you to the staff of the Phillip Island Nature Parks (PINP), with special thanks to Dr Peter Dann and Dr Rebecca Macintosh (PINP). Thank you to the staff of RMIT university, again with special thanks to Associate Professor Mark Osborn (RMIT).

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Appendix (results)Table A1: Scat samples, initial data

Sample # Foil (g)Foil + Scat Wet

(g) Wet Weight (g)Foil + Scat Dry

(g) Dry Weight (g)

1 3.55 56.48 52.93 38.65 35.1

2 2.68 55.6 52.92 23.01 20.33

3 3.63 39.81 36.18 15.56 11.93

4 2.73 56.9 54.17 23.02 20.29

5 4.31 54.81 50.5 24.69 20.38

6 4.37 31.19 26.82 13.73 9.36

7 4.42 116.39 111.97 7.97 3.55

8 4.51 79.3 74.79 50.97 46.46

9 4.81 270 265.19 120 115.19

10 4.44 190 185.56 130 125.56

11 4.42 93.24 88.82 31.22 26.8

12 4.3 57.7 53.4 27.69 23.39

13 4.4 98.78 94.38 59.23 54.83

14 4.41 53.78 49.37 18.86 14.45

15 4.5 38.78 34.28 24.58 20.08

16 4.46 38.24 33.78 15.27 10.81

17 4.36 179.83 175.47 54.45 50.09

18 4.36 35.91 31.55 23.27 18.91

19 4.26 137.91 133.65 115.41 111.15

20 4.63 40.21 35.58 16.15 11.52

21 4.02 125 120.98 75 70.98

22 4.37 106.41 102.04 27.28 22.91

23 4.5 115.21 110.71 32.35 27.85

24 4.69 66.45 61.76 29.31 24.62

25 3.92 63.37 59.45 37.1 33.18

26 4.37 89.56 85.19 25.78 21.41

27 4.11 37.54 33.43 26.03 21.92

28 4.6 78.4 73.8 55.17 50.57

29 4.41 34.81 30.4 17.75 13.34

30 4.53 33.72 29.19 11.27 6.74

31 4.53 37.56 33.03 18.93 14.4

32 4.41 257 252.59 106.63 102.22

33 3.95 34.27 30.32 12.69 8.74

34 3.78 155 151.22 65.02 61.24

35 4.77 26.1 21.33 13.96 9.19

36 3.98 42.01 38.03 17.86 13.88

37 4.81 20.81 16 11.44 6.63

38 3.66 95.13 91.47 53.83 50.17

39 4 125 121 80.5 76.5

40 3.07 46.75 43.68 22.39 19.32

41 3.94 34.9 30.96 13.89 9.95

42 4.12 115 110.88 42.3 38.18

43 3.93 57.77 53.84 38.69 34.76

44 3.81 16.26 12.45 10.47 6.66

45 3.88 41.14 37.26 22.31 18.43

46 4.01 150 145.99 54.38 50.37

47 3.82 32.44 28.62 12.84 9.02

48 3.89 180 176.11 118.53 114.64

49 3.78 85.27 81.49 47.04 43.26

50 3.77 66.08 62.31 42.11 38.34

51 3.9 31.9 28 25.84 21.94

52 3.87 47.93 44.06 18.06 14.19

53 3.92 25.98 22.06 16.4 12.48

54 3.99 49.51 45.52 28.05 24.06

55 4.02 113.5 109.48 52.01 47.99

56 3.8 30.27 26.47 23.79 19.99

57 3.98 27.49 23.51 13.09 9.11

58 0 0 0 0 0

59 3.83 33.93 30.1 12.95 9.12

60 4.03 33.1 29.07 23.76 19.73

61 3.99 42.27 38.28 18.62 14.63

62 3.74 19.26 15.52 7.74 4

63 3.85 17.89 14.04 15.43 11.58

64 3.98 59.48 55.5 36.03 32.05

65 0 0 0 0 0

66 4.01 52.98 48.97 38.76 34.75

67 3.72 32.73 29.01 21.83 18.11

68 3.9 19.55 15.65 10.22 6.32

69 3.92 17.8 13.88 11.7 7.78

70 4.14 69.63 65.49 44.09 39.95

71 3.88 104.64 100.76 56.63 52.75

72 3.95 11.78 7.83 6.96 3.01

73 4.1 91.66 87.56 36.2 32.1

74 0 0 0 0 0

75 3.98 34.52 30.54 19.78 15.8

76 0 0 0 0 0

77 3.76 25.2 21.44 13.64 9.88

78 0 0 0 0 0

79 3.87 112.77 108.9 56.54 52.67

80 0 0 0 0 0

81 3.95 215 211.05 40.26 36.31

82 3.85 26.4 22.55 11.63 7.78

83 3.75 7.73 3.98 4.44 0.69

84 3.85 62.58 58.73 22.06 18.21

85 3.75 15.08 11.33 5.38 1.63

86 3.89 76.23 72.34 31.29 27.4

87 3.93 65.43 61.5 29.09 25.16

88 3.81 56.08 52.27 19.75 15.94

89 4.08 48.92 44.84 18.77 14.69

90 0 0 0 0 0

91 0 0 0 0 0

92 3.93 77.24 73.31 49.72 45.79

93 3.85 29.4 25.55 10.91 7.06

Table A2: Descriptive Statistics of wet and dry seal scat samples

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Descriptive Statistics: Wet Weight (g), Dry Weight (g)

Variable N N* Mean SE Mean StDev Minimum Q1 Median Q3Wet Weight (g) 93 3 59.53 5.64 54.37 0.00 26.01 44.06 83.34Dry Weight (g) 93 0 26.71 2.81 27.07 0.00 9.11 19.32 35.70

Variable MaximumWet Weight (g) 265.19Dry Weight (g) 125.56

Table A3: Number of fragments isolated from scat samples.

Sample# # of fragments Sample#2 # of fragments21 8 25 22 2 32 13 1 33 14 1 34 05 3 36 16 1 38 08 3 40 1

11 2 47 312 0 48 018 1 52 121 2 58 122 2 59 023 6 78 2

Table A4: Size of plastic fragments isolated from scat samples.

Sample# Sample size (mm) Sample#2 Sample size (mm)21a 3 21a 81b 4 21b 41c 2 22a 151d 2 22b 81e 2 23a 91f 2 23b 61g 3 23c 41h 2 23d 52a 3 25a 52b 5 25b 33 1 32 44 6 33 6

5a 12 34 05b 9 36 85c 6 38 06 7 40 3

8a 5 47a 138b 10 47b 118c 10 47c 6

11a 5 48 011b 4 52 612 0 58 1218 5 59 0

78a 678b 5

Table A5: Descriptive statistics of number of fragments.

Descriptive Statistics: No. of frags, Sample size (mm), Sample size (mm)2

Variable N N* Mean SE Mean StDev Minimum Q1 Median Q3No. of frags 21 0 2.143 0.392 1.797 1.000 1.000 2.000 2.500

Variable MaximumNo. of frags 8.000

Table A6: Descriptive statistics of size of fragments isolated.

Descriptive Statistics: size mm 2

Variable N N* Mean SE Mean StDev Minimum Q1 Median Q3size mm 2 48 0 5.313 0.526 3.645 0.000 3.000 5.000 7.750

Variable Maximumsize mm 2 15.000

Table A7: FT-IR results, confirmed plastics isolated from scat samples.

Sample # Type of plastic Size (mm) Colour

1b PE 4Grey

2a PE 2

Brownish/yellow

2b PE 8White

4 PE 6White

8b PE 10White/Clear

18 PE 5Grey

21a PE 8White

23a HDPE 9White/Clear

23b Nylon 6Brown/Grey

23c Nylon 4Brown/grey

23d Nylon 5Brown/Grey

25a PE 5White/Grey

32 PE 4White

33 PE 6Grey

40 Nylon 3Clear

47c Nylon 6White/Clear

58 HDPE 12Clear

78a PE 6Solid Clear

Table A8: Descriptive statistics of fragment size in confirmed samples.

Descriptive Statistics: type of plastic Size (mm)

Variable N N* Mean SE Mean StDev Minimum Q1 Mediantype of plastic Size (mm) 18 0 6.056 0.597 2.532 2.000 4.000 6.000

Variable Q3 Maximumtype of plastic Size (mm) 8.000 12.000