Chapter 2 Results

Figure 1. Study site of project. Twenty humpback whale (Megaptera novaeangliae) fecal samples were collected near the western entrance to the Juan de Fuca Strait, off the southwest coast of Vancouver Island, British Columbia, Canada.

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Table 1. Number of anthropogenic particles (AP) isolated via caustic digestion. The average number of particles in procedural blanks was 1.22 and was subtracted from the particle count of each sample.

Sample ID	Particle Count	Sample Mass (g)	AP/g Dry	AP/g Wet
194f1	12.78	0.47	27.19	6.80
194f2	15.78	1.85	8.53	2.13
199f1	13.78	2.03	6.79	1.70
228f1	5.78	1.17	4.94	1.24
234f1	4.78	1.38	3.46	0.87
234f2	0.78	1.25	0.62	0.16
234f3	11.78	1.31	8.99	2.25
235f1	5.78	1.19	4.86	1.21
235f2	9.78	1.20	8.15	2.04
235f3	3.78	0.71	5.32	1.33
235f4	6.78	2.22	3.05	0.76
235f5	7.78	2.09	3.72	0.93
236f1	7.78	1.28	6.08	1.52
236f2	1.78	1.43	1.24	0.31
255f1	7.78	2.37	3.28	0.82
256f1	5.78	1.62	3.57	0.89
261f2	7.78	0.41	18.98	4.74
263f1	16.78	0.75	22.37	5.59

The total number of anthropogenic particles across all the samples (N=18) is 169.

The dry fecal AP concentration ranges from 0.62 to 27.19 (mean 7.84 ± 7.41 AP/g).

The wet fecal AP concentration ranges from 0.16 to 6.8 (mean 1.96 ± 1.85 AP/g).

The final AP/g is lower when only using counts ≥ limit of quantification (LOQ). This value can be seen at end of the document.

Table 2. Number of anthropogenic particles (AP) isolated via enzymatic digestion. The number of procedural blanks was too low to provide information on the average number of particles per blank. To deal with contamination, particles seen in procedural blanks (clear fibers) were ommitted entirely.

Sample ID	Particle Count	Sample Mass (g)	AP/g Dry	AP/g Wet
194f2	5	0.31	16.13	4.03
199f1	6	0.32	18.75	4.69
228f1	5	0.31	16.13	4.03
234f1	10	0.31	32.26	8.06
234f2	4	0.21	19.05	4.76
234f3	22	0.32	68.75	17.19
235f1	10	0.36	27.78	6.94
235f2	9	0.16	56.25	14.06
235f4	2	0.10	20.00	5.00
235f5	1	0.10	10.00	2.50
236f1	9	0.30	30.00	7.50
236f2	2	0.10	20.00	5.00
255f1	1	0.10	10.00	2.50
256f1	4	0.33	12.12	3.03
261f2	3	0.19	15.79	3.95
263f1	1	0.06	16.67	4.17

The total number of anthropogenic particles across all samples (N=16) is 94.

The dry fecal AP concentration ranges from 10 to 68.75 (mean 24.36 ± 16.37 AP/g).

The wet fecal AP concentration ranges from 2.5 to 17.19 (mean 6.09 ± 4.09 AP/g).

Figure 2. Bar chart of the concentration and polymer composition of anthropogenic particles (AP) isolated from caustically digested humpback whale fecal samples (N=18).

Figure 3. Bar chart of the concentration and polymer composition of anthropogenic particles (AP) isolated from enzymatically digested humpback whale fecal samples (N=15).

FTIR data explained:

Some anthropogenic particles were too small and degraded for FTIR spectroscopy. Therefore, only a subset of particles have a polymer ID. This subset of scanned particles are used for figure 4, 5, and tables 3, 4 below.

• 57.99 % of anthropogenic particles isolated via caustic digestion were analyzed with FTIR (98 out 169 total particles).

• Additionally, 69.15 % of anthropogenic particles isolated via enzymatic digestion were analyzed with FTIR (65 out 94 total particles).

Figure 4. Bar chart depicting the frequncy of occurence for anthropogenic particles isolated from humpback whale feces via caustic and enzymatic digestion.

Figure 5. Pie chart of polymer distributions between caustic and enzymatic digestions.

Table 3. Top 5 most common scanned anthropogenic particles types isolated via caustic digestion.

Colour	Polymer ID	Morphology	Count	Percentage (of scanned particles)
Clear	Cellulose	Fiber	15	15.3
Clear	Polyester	Fiber	13	13.3
Clear	Polyethylene	Fiber	8	8.2
Black	Polyester	Fiber	5	5.1
Clear	polyamide	Fiber	5	5.1

Table 4. Top 5 most common scanned anthropogenic particles types isolated via enzymatic digestion.

Colour	Polymer ID	Morphology	Count	Percentage (of scanned particles)
Blue	Cellulose	Fiber	21	32.3
Black	Cellulose	Fiber	16	24.6
Black	PTFE	Fiber	5	7.7
Red	Cellulose	Fiber	5	7.7
Black	Polyester	Fiber	3	4.6

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Raw data from caustic digestion:

Figure 6. Examples of anthropogenic particles isolated from humpback whale feces via caustic digestion. A: Polyester fiber from sample 194F1, B: Polypropylene fiber from sample 194F2, C: Polyamide fiber from sample 194F2, D: Cellulose fiber from sample 236F1, E: Cellulose fiber from sample 194F1, F: Polyethylene fragment from sample 199F1, G: Acrylic fragment from sample 255F1, H: Polyethylene fiber from sample 199F1, I: Polyethylene fragment from sample 261F2.

Figure 7. Principle component spectrum of polyethylene fiber isolated from sample 261F2 via caustic digestion.

Table 5. Length and width of 169 isolated anthropogenic particles.
Measurement	Mean	Min	Max
Length (μm)	1564.12	35.85	21949.35
Width (μm)	59.29	3.16	1238.82

Table 6. Morphology of 169 isolated anthropogenic particles.
Morph	Count	Percent
Fiber	143	84.62
Fragment	21	12.43
Film	4	2.37
Sphere	1	0.59

Table 7. Colour of 169 isolated anthropogenic particles.
Colour	Count	Percent
Clear	78	46.15
Black	29	17.16
Blue	15	8.88
Yellow	15	8.88
Red	12	7.10
Green	11	6.51
Grey	7	4.14
Brown	1	0.59
Pink	1	0.59

Table 8. Polymer ID of 98 scanned anthropogenic particles.
Polymer	Count	Percent
Polyester	27	27.55
Cellulose	25	25.51
Polyethylene	23	23.47
Polyamide	11	11.22
Polypropylene	7	7.14
PTFE	4	4.08
Acrylic	1	1.02

Raw data from enzymatic digestion:

Figure 8. Examples of anthropogenic particles isolated from humpback whale feces via enzymatic digestion. A: Polyester fiber from sample 199F1, B: Polypropylene fiber from sample 199F1, C: PTFE fiber from sample 234F2, D: Polyurethane fiber from sample 235F1, E: Cellulose fiber from sample 234F1, F: PTFE fiber from sample 234F3, G:Polypropyelene fiber from sample 235F4, H: Cellulose fiber from sample 234F3, I: Cellulose fiber from sample 263F1.

Figure 9. Principle component spectrum of polypropylene fiber isolated from sample 199F1 via enzymatic digestion.

Table 9. Length and width of 94 isolated anthropogenic particles.
Measurement	Mean	Min	Max
Length (μm)	841.53	3.16	5367.65
Width (μm)	29.67	1.42	510.24

Table 10. Morphology of 94 isolated anthropogenic particles.
Morph	Count	Percent
Fiber	88	93.62
Fragment	4	4.26
Film	2	2.13

Table 11. Colour of 94 isolated anthropogenic particles.
Colour	Count	Percent
Black	44	46.81
Blue	26	27.66
Grey	7	7.45
Red	7	7.45
Green	3	3.19
Orange	3	3.19
Brown	2	2.13
Clear	2	2.13

Table 12. Polymer ID of 65 scanned anthropogenic particles.
Polymer	Count	Percent
Cellulose	47	72.31
PTFE	7	10.77
Polyester	6	9.23
Polyamide	2	3.08
Polypropylene	2	3.08
Polyurethane	1	1.54

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Important step:

We must decide which fecal microplastic concentration is more accurate (caustic versus enzymatic) for use in the stochastic simulation model.

Based on the literature, the caustic method of digesting organic material is harsh on certain kinds of anthropogenic particles, especially nylon and PTFE (Turri et al., 2024; Karami et al., 2016). Regenerated cotton, although not plastic, is readily degraded by caustic reagents (i.e., nitric acid and hydrogen peroxide) (Pfieffer & Fischer, 2020). Therefore, it is reasonable to assume the caustic data will underestimate the true fecal anthropogenic particle concentration.

That said, approximately 5x more sample mass was used with the caustic digestion compared to the enzymatic method. As consequence of more sample mass, the total number of anthropogenic particles is greater in the caustic versus enzymatic data (169 versus 94). Further, the caustic data have a greater diversity of polymer IDs. This can be attributed to the heterogeneous distribution of anthropogenic particles in biological samples with larger samples encapsulating a greater diversity of potential particle variants (Cross et al., 2025).

Enzymes like proteinase K have little to no effect on anthropogenic particles (Cole et al., 2014), potentially providing a more accurate picture of AP loads in whale feces. However, the digestive efficacy of Proteinase K is low compared to nitric acid and hydrogen peroxide, so less sample mass can be processed. The question remains whether a low sample mass with no anthropogenic particle degredation (i.e., enzymatic digestion) is better than a high sample mass with moderate anthropogenic particle degredation (i.e., caustic digestion).

In either digestion method, modified cellulose is quite abundant. This makes sense.

“Most fibers floating in the world’s oceans are not plastic but dyed cellulose.” https://pmc.ncbi.nlm.nih.gov/articles/PMC7274779/

Aside from the digestion methodologies themselves, the caustic and enzymatic experiments deal with contamination in different ways. In both datasets, APs which match the polymer ID, colour, and morphology of particles found in field and laboratory controls were removed from analyses. However, the laboratory procedural blanks were treated differently between digestion methods. With the caustic method, there were enough procedural blanks (n=9) to subtract an averaged count value (average #AP/blank) from each of the fecal samples. Further, the average #AP/blank was used to calculate a limit of detection (LOD; mean + 3 X SD) and limit of quantification (LOQ; mean + 10 X SD) for the caustic digestion study (Bråte et al., 2022). Using samples which satsify a particle count ≥ LOQ, the dry fecal microplastic concentration ranges from 0.62 to 27.19 AP/g (mean 7.84 ± 7.41). Further, the wet fecal microplastic concentration ranges from 0.16 to 6.8 AP/g (mean 1.96 ± 1.85). In comparison, the enzymatic method had too few procedural blanks (n=2) to accurately subtract an average # AP/PB from each fecal sample. Instead, procedural blanks were treated in the same way as all other controls by omitting fecal MPs which match the characteristics of particles found in procedural blanks (clear fibers).

Ultimately, the average dry concentration of anthropogenic particles isolated via caustic digestion is 7.84 ± 7.41 MP/g and the average dry concentration of anthropogenic particles isolated via enzymatic digestion is 24.36 ± 16.37 MP/g.

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Wilcoxon Rank Sum Test

A Shaprio-Wilk test was used to assess normality of AP/sample data. The data was not normal so a Wilcoxon Rank Sum test was used to differentiate between the anthropogenic particle abundance in controls and samples.

Figure 10: Comparison of anthropogenic particle abundance (AP/sample) between samples and controls. The points represent the mean particle count of each sample type. Horizontal bars indicate group means ± standard deviation. A Mann–Whitney U test revealed a significant difference between groups. ****(P<<0.05).

For the caustic digestion, the AP concentration in the sample data was significantly greater than the controls and therefore reliable for reporting. The difference was not statistically significant (p = 0.02861) with the enzymatic digestions

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Supplementary document

Click to expand contamination data

A total of 85 anthropogenic particles were isolated across a variety of controls. Here is the breakdown:

Table 13. Number of anthropogenic particles isolated across different controls.

Control Type	Particle Count	Percent
Seawater blank (n=18)	50	58.8
Field air control (n=12)	5	5.9
Fecal aliquot air control (n=12)	5	5.9
Lyphophilizer air control (n=2)	9	10.6
Caustic procedural blank (n=9)	6	7.1
Enzymatic procedural blank (n=2)	10	11.8

Table 14. Particles isolated from caustic procedural blanks. PB ID Morphology Colour Polymer ID 24344fpb No Detect No Detect No detect 24351fpb No Detect No Detect No detect 24352fpb Fiber Clear NA 24352fpb Fiber Red NA 24353fpb No Detect No Detect No detect 25006fpb Fiber Black NA 25007fpb Fiber Yellow NA 25013fpb No Detect No Detect No detect 25014fpb No Detect No Detect No detect 25020fpb Fiber Clear NA 25020fpb Fiber Clear NA

LOD and LOQ calculated from caustic procedural blanks are 3.26 and 9.33, respectively.

Table 15. Particles isolated from enzymatic procedural blanks. PB ID Morphology Colour Polymer ID PB1 Fiber Clear NA PB1 Fiber Clear Polyester PB1 Fiber Clear Cellulose PB1 Fiber Clear Cellulose PB1 Fiber Clear Cellulose PB1 Fiber Clear Cellulose PB1 Fiber Clear NA PB2 Fiber Clear Cellulose PB2 Fiber Clear Cellulose PB2 Fiber Clear Cellulose

Enzymatic LOD and LOQ are both 0 since clear fibers were omitted from analyses.

Figure 11: Polymer & morphology of particles in seawater blanks. 68 % of anthropogenic particles isolated in seawater procedural blanks were analyzed with FTIR (34 out 50 total particles).

Figure 12: Polymer & morphology of particles in field air controls. 60 % of anthropogenic particles isolated in field air controls were analyzed with FTIR (3 out 5 total particles).

Figure 13: Polymer & morphology of particles in laboratory air controls. 64.29 % of anthropogenic particles isolated in laboratory air controls were analyzed with FTIR (9 out 14 total particles).

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Table 16. Colour of particles isolated from seawater blanks.
Colour	Count	Percent
Clear	42	84
Black	3	6
Blue	3	6
Green	1	2
Red	1	2

Table 17. Colour of particles isolated from field air controls.
Colour	Count	Percent
Blue	4	80
Black	1	20

Table 18. Colour of particles isolated from lab air controls.
Colour	Count	Percent
Black	9	64.29
Blue	3	21.43
Clear	2	14.29

Click to expand supernatant data

Table 19. Putative anthropogenic particles isolated from the supernatant of processed samples.

Sample ID	Particle Count	Sample Volume (ml)
194f1sn	8	5
194f2sn	2	160
199f1sn	0	185
228f1sn	1	80
234f1sn	0	20
234f2sn	0	30
234f3sn	11	200
235f1sn	4	80
235f2sn	0	250
235f3sn	2	160
235f4sn	2	40
235f5sn	1	130
236f1sn	2	80
236f2sn	0	10
255f1sn	0	140
256f1sn	0	130
258f1sn	0	40
261f1sn	0	40
261f2sn	1	40
263f1sn	0	10

Figure 14. Bar chart showing the count and polymer composition of anthropogenic particles (AP) isolated from humpback whale fecal supernatant.

Table 20. Colour of particles isolated from supernatants.
Colour	Count	Percent
Black	16	47.06
Blue	10	29.41
Red	3	8.82
White	3	8.82
Brown	1	2.94
Green	1	2.94

Particle colour comparison between supernatants and samples

Figure 15. Stacked bar graph showing colour proportion of anthropogenic particles (AP) isolated from caustic digestions and associated supernatants.

Figure 16. Stacked bar graph showing colour proportion of anthropogenic particles (AP) isolated from enzymatic digestions and associated supernatants.

Click to expand seawater data

After excluding particles matching those seen in controls (procedural blanks and channel controls), there was a total of 44 APs found in 314.21 liters of seawater among nine samples. Each sample was an average of 31.421 liters.

AP length range was 26.837733, 4014.218224 µm (mean 1001.5233 µm) and width was 1.265143, 1528.677931 µm (mean 50.5874292 µm).

Table 21: Morphologies of anthropogenic particles found in subsurface seawater samples

Morphology	Count	Percent (%)
Fiber	33	75.0
Fragment	7	15.9
Film	4	9.1

Table 22: Colour of anthropogenic particles found in subsurface seawater samples

Colour	Count	Percent (%)
Red	17	38.6
Blue	15	34.1
Clear	5	11.4
Black	4	9.1
Green	1	2.3
Grey	1	2.3
Yellow	1	2.3

Table 23: Polymer IDs of anthropogenic particles found in subsurface seawater samples (Only 32 of 44 or 70% of particles scanned)

Polymer ID	Count	Percent (%)
Cellulose	20	62.5
Polyester	4	12.5
Poly(vinyl alcohol)	2	6.2
Polyamide	2	6.2
Polysulfone	2	6.2
PTFE	1	3.1
Polypropylene	1	3.1

Table 24: Number of anthropogenic particles and corresponding sample volumes of samples filtered by the Ascension. Particles seen in procedural blanks and controls were ommitted. MP per M³ < 0.0005 (very low), < 0.005 (Low), < 1 (Medium), < 10 (High), > 10 (very high).

Sample ID	Count	Volume (l)	Depth (m)	Concentration (AP/M3)	Category
0715-25m	0	15.033	25.4220	0.00000	Very Low
0715-50m	3	20.042	52.1500	149.68566	Very High
0716-100m	10	24.529	99.9300	407.68070	Very High
0716-50m	10	40.297	50.7970	248.15743	Very High
0719-50m	1	40.158	46.2677	24.90164	Very High
0911-5m	10	40.247	5.1460	248.46572	Very High
0914-25m	4	40.074	25.4000	99.81534	Very High
0914-50m	1	40.338	50.4000	24.79052	Very High
0916-50m	4	13.377	50.4000	299.02071	Very High
0917-25m	1	40.115	25.6900	24.92833	Very High

Figure 17: Anthropogenic particle concentration (AP/M3) across multiple depths (5, 25, 50, and 100 m). Each yellow dot represents a sample (n=10) collected by the Ascension

Figure 18: Bar chart of the number of anthropogenic particles and polymer composition of subsurface seawater samples collected with the Ascension

Table 25: Total number of anthropogenic particles (APs) detected in each procedural blank sample

Sample ID	AP Count
asens-pb	2

Table 26: Total number of anthropogenic particles (APs) detected in each control sample

Sample ID	AP Count
0916-cntr-7	9
0911-cntrl	4
0916-cntr-4	3
0914-cntr-7	2

Table 27: Colours of anthropogenic particles found in control samples from the Ascension dataset

Colour	Count	Percent (%)
Clear	15	83.3
Black	2	11.1
Blue	1	5.6

Table 28: Morphologies of anthropogenic particles found in control samples from the Ascension dataset

Morphology	Count	Percent (%)
Fiber	17	94.4
Fragment	1	5.6

Table 28: Polymer types of anthropogenic particles found in control samples from the Ascension dataset (only 44.4% of APs from this control were scanned)

Polymer Category	Count	Percent (%)
Cellulose	7	87.5
Polyester	1	12.5

Table 29: Colours of anthropogenic particles found in procedural blanks from the Ascension dataset

Colour	Count	Percent (%)
Clear	1	50
Grey	1	50

Table 30: Morphologies of anthropogenic particles found in procedural blanks from the Ascension dataset

Morphology	Count	Percent (%)
Fiber	2	100

Table 31: Polymer types of anthropogenic particles found in procedural blanks from the Ascension dataset (100% of APs scanned)

Polymer Category	Count	Percent (%)
Cellulose	1	50
PTFE	1	50

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The data from the subsurface seawater samples are highly suspect. The sample volume is too small to capture the expected particle abundance in the water column. Based on literature, the AP/m³ in the Northeast Pacific is likely somewhat low, < 15 particles per m³ (Choy et al., 2019).

Figure 19: Plot of sampling volume needed to detect microplastics versus expected microplastic concentrations in sewater. The blue line shows the volume needed for a laboratory with a MDA of 7.