Demographic Differences in the Quantity, Mass, and Anatomical Location of Ingested Plastic in Northern Fulmars ( Fulmarus glacialis ): A Review and Reconsideration of NE Pacific Ocean Samples

On the Washington and Oregon coast of the NE Pacific Ocean from 2008-2015, we found that juveniles represented 83% of Northern Fulmars that were beached. In comparison to older birds, juveniles averaged more mass and pieces of plastic in stomachs. This reflected relatively larger plastic loads in the cranial stomach section, the proventriculus, which we found was associated with relatively large accumulations in the caudal stomach section, the ventriculus. We estimate that the proventriculus could retain almost 10 times as much plastic as the ventriculus and that retention of proventricular plastic largely accounted for the difference in juveniles versus older birds. Our findings contrast with published Atlantic Ocean reports where the proportion of immatures was lower and plastic was retained mostly in ventriculi. The differences in demography and gastric distribution between NE Pacific and Atlantic fulmars may reflect the different sizes of plastic particles that were available. The preponderance of juveniles in NE Pacific Ocean samples and associated relatively large plastic loads overestimate the load for stomach-based sampling is that differences in size of plastic at spatiotemporal scales could affect the utility of stomach samples as bioindicators of oceanic plastic pollution.

Two factors that impinge on the above assumptions and data are the distribution of plastic in sections of the stomach and demographic profiles of samples [1,10,13,21]. In retrospect, the first is significant because legacy protocols for presenting monitoring results pooled loads from the two sections of the stomach: the proventriculus and ventriculus. Pooling was done for expediency [1,2,4] although the stomach sections differ in morphology, function, and physiology [13,21,22]. The proventriculus is thin-walled and expandable with up to 10 times the capacity of the thick-walled ventriculus (see below). Food first enters the proventriculus for storage or chemical digestion before passing into the ventriculus for grinding objects before passage into the intestine. One general finding throughout plastic studies is that if plastic is found, it is primarily in ventriculi [13,17,21,23,24]. The significance of this is that the small size and thick walls of the ventriculus set an upper limit to the amount of plastic that could accumulate while the sphincter between the two stomach sections limits the size of particles that could enter the ventriculus [13,22]. Thus, while providing a standard sampling unit, ventricular samples are limited in the number and size of particles while the expansive proventriculus could hold more and larger pieces [13,21].
Noting that plastic must first pass through the proventriculus to enter the ventriculus, the lower incidence of plastic in proventriculi can only mean that it is cleared more frequently through regurgitation, passage into ventriculi, or both [13,22]. Analyzing plastic from stomach sections separately could provide potentially useful information about temporal differences in the time plastic remains in residence in sections of the stomach [7,13,25]. The proventricular plastic load provides a relatively transient or ephemeral sample of recent ingestion while the ventricular load likely represents residual plastic that is retained for a longer period. In addition, the difference in capacity and relative short-term occurrence of the proventricular load relative to ventricular loads could impart considerable variation in pooled samples.
Noting the constraints on ventricular loads and that adults contain less plastic than immatures [1,9,13,15,26,27], we surmised that the difference in the amount of proventricular plastic in pooled samples accounts for much of the variation in loads and the difference between age groups.
In this paper, we confirm that some juveniles from the NE Pacific Coasts contain more proventricular plastic and associated greater plastic loads than in older birds. We then reconsider our NE Pacific samples in comparison to Atlantic fulmar samples relative to recent research on the availability of the sizes of available plastic.

Study Area and Data
Data from Terepocki et al. [13] examined the dynamics of plastic in two stomach sections while in this paper we examined data from a demographic perspective focusing on differences between juveniles and non-juveniles. The latter consisted of adults and immatures in pre-breeding cohorts (see Results). Plastic was extracted and processed using standard protocol [13,28] and archived at the Slater Museum of Natural History, University of Puget Sound. An additional sample was added to these data from the 2014-2015 time period from fulmars obtained from the same Pacific coast beaches in Oregon and Washington (see [13]). A total of 197 stomach samples were analyzed and 195 were aged (Table 1). In addition to the stomach contents, we also inspected the intestines of the 2014-2015 sample. The intestine was removed, straightened, and bisected longitudinally, and flatten with the lumen exposed in a trough. The trough was placed on a slight incline the contents were washed into a 0.5 mm sieve. The contents were examined under a dissecting scope and processed using standard protocol [13,28].
Along the NE Pacific Coast, juvenile fulmars are easily identified using criteria of no remex or rectrix molt, uniform unworn body plumage, diminutive testes or ovaries with no apparent ova (undifferentiated), and relatively large glandular-walled bursa of Fabricius ( [29]; Figure 12 in [28]). Juveniles were hatched in July [30], and they would be 3-6 months post-fledgling when encountered. Fulmars follow a Simple Basic Molt Strategy [30], and lack of molt, except for some on head and neck, were obvious clues to the juvenile category that we verified with internal data. Immatures had a mix of old and new body feathers, remex or rectrix molt, and thin-walled non-glandular bursas.
The bursa transitions from a glandular thick-walled structure to a membranous thin-walled structure probably during the second year of life, but could persist for a longer period. Adults had similar molt status but lacked a bursa. Gonads of non-breeding adults, which regress in size during the non-breeding period (see [31]) are probably similar to older immatures. Without bursa information, the criteria for designating immatures versus adults would be indefinite (see [1]), and older immatures could be categorized as adults or the reverse. In comparison to other studies (see Discussion) age categorizations might differ dependent on the experience of personnel and age classes might not be comparable if personnel with differing experience made determinations.
Bursas were examined by snipping the ventral lip of the cloaca opening, examining the caudal side of the uroproctodeal fold for an opening, and then inserting a blunt measuring tool to determine depth if an opening was apparent. The bursa was then opened ventrally and the interior was examined and classified as thick glandular wall or thin membranous wall. Width was also measured (see [32]).

Statistical Analysis
Data were analyzed using Minitab 17.3.1 [33]. Nonparametric tests were used and medians with quartiles were included in summaries in addition to arithmetic mean ± SE for comparison to other studies. The two-sample signed-rank test referred to as Wilcoxon-Mann-Whitney or Mann-Whitney [34] test in Minitab reports the statistic of Wilcoxon W accounting for ties as appropriate. Probabilities were two-tailed. Summaries and statistics are presented based on all birds including those with no plastic. This is the norm in stomach-based plastic summaries, but underestimates the loads in affected birds (see [35,36]). Data for plastic mass and pieces are presented as arithmetic means and median following the protocol of van Franeker and Meijboom [1].

Results
The distribution of birds per year (Table 1) and month (Table 2) indicates that recovery of age groups differed between time periods and that most were recovered from October through November. Of the three age groups, mass and number of pieces of plastic differed (Table 3) (Kruskall-Wallis 2 H=13.21, p=0.001). Pair-wise comparisons indicated that juveniles differed from immatures and adults, but adults and immature were not significantly different. Adults and immatures were pooled in subsequent analysis. Juveniles comprised 85% (156/195) of aged specimens. Of the juveniles, 87% (136/156) had retained plastic which did not differ from 82% (32/39) of non-juveniles with plastic (Chi-square = 0.668, p = 0.407). Juveniles had more pieces (W 156, 39 =4,178, p < 0.001) and greater total mass (W 156, 39 =4,306, p < 0.001) of plastic than non-juveniles ( Figure 1). Two juveniles from the 2014-2015 period were extreme outliers with 26,251.6 mg and 466 pieces and 20,231.4 mg and 278 pieces (Table 3). However, these did not distort the comparisons since Mann-Whitney-Wilcoxon ranked comparisons were used.   The distribution of plastic in stomachs differed for ages with 51% (79/156) of juveniles retaining proventricular plastic compared to only 28% (11/39) of non-juveniles (Chi-square = 6.319, p = 0.012. In contrast, there was no significant difference between the 87% (136/156) of juveniles and 82% (32/39) of non-juveniles that retained plastic in ventriculi (Chi-square = 1.225, p = 0.268). Only two fulmars, an adult (SDT43) and a juvenile (LUK20) had 15 mg/5 pieces and 20 mg/1 pieces, respectively, in the proventriculus with no plastic in the ventriculus.
Juveniles had greater mass and number of pieces in both proventriculi and ventriculi than nonjuveniles (  = 16,424.00, p < 0.001). These differences result in proventriculus/ventriculus ratios for juveniles of 1.8 for mass and 0.7 for pieces in contrast to non-juvenile ratios of 0.5 for mass and 0.1 for pieces (Table 4). Although large ventricular loads in association with the presence of proventricular plastic had been noted [13], the association with age was not recognized. This association of proventricular loads with larger ventricular loads with age was not apparent in non-juveniles (mass W 21    Intestinal pieces had a median greatest dimension of 3.0 mm (range 1.3-5.9 mm) and weight of 29 mg (range 0.9-117 mg) and included hard plastic, assumed to be polypropylene or polyethylene, extruded foam, and one thread.

Fulmars from the NE Pacific Coast
On the NE Pacific coast, juvenile fulmars represented 83% of the beached samples and averaged five times more plastic by mass than older birds. Juveniles retained more plastic in the expandable proventriculus than ventriculus whereas plastic in older birds was concentrated in the ventriculus. In a comparison of two time periods we also found a difference in the relative distribution of plastic between stomach sections in juveniles. Since proventricular plastic is held for a short term relative to ventricular plastic, variation in proventricular loads induces unknown variability when comparing samples, especially to Atlantic studies that are primarily ventricular plastic. Stomach-based plastic sampling would benefit for further study to elucidate causes for differences in age, gastric distribution, availability of oceanic plastic, and interaction among these factors.

Demographics of Fulmar Plastic Samples
Immature fulmars and other procellariids retain more plastic than adults [1,10,13,17,20,24,26,37,38]. Therefore, a predominance of immatures or adults could bias sample averages higher or lower, respectively, so the inclusion of demographic profiles are recommended for spatiotemporal comparisons [1,10]. The significance of demography was highlighted by a recent analysis of a 17-year data series from the North Sea that indicated the percent of fulmar adults in yearly samples added significantly to a model of a decreasing temporal trend in a proxy for plastic mass in stomachs [6,9,10]. This appears to indicate plastic loads were related to the percent of adults in pooled samples, confirming general conclusions [10].
Several other shorter-term studies are often used to assess the EcoQ% performance in the North Atlantic or Arctic away from the North Sea (see Figure 3 in [10]). In these studies, age was categorized as adult versus immature but sample sizes were too small for comparisons [36,39]. Adult/immature numbers were 49/9 from Iceland [39], 5/35 from Svalbard [36], and 61/9 for the Labrador Sea [7]. Bravo Rebolledo [21] from the Faroe Islands had adequate samples of 151 adults and 96 immatures (or non-adults) but did not statistically test the differences of 0.11 g in adults and 0.18 g in immatures. Bond et al. [40] examined 176 fulmars from Nova Scotia beaches from 2000-2012 and found no age difference but aging criteria or data were not provided. These studies have been used to assess EcoQ% performance by pooling mass for age groups, but inclusion as age as related to plastic loads, as done of recently by van Franeker et al. [10] is recommended (see [1,14,15,41]).
Few studies provided demographic profiles using three ages including juvenile, immature, and adult. Included is our study from the NE Pacific coast that had 83, 5, and 12%, respectively, for three age classes (Table 3). Percentages for the same age classes in North Sea studies through the year 2000 were 24, 24, and 52 ( Table 6 in [1]). As an example of bias from differing demographic profiles, an unweighted arithmetic mean was 0.83 g for the NE Pacific coast sample compared to 0.55 g (Figure 3a in [1]) for the North Sea from 1982 to 2000. If only adults were used, the trend is reversed with 0.22 g for the NE Pacific coast and 0.44 g for the North Sea through 2000 ( Table 6 in [1]). North Sea levels have declined recently [10], but plastic associated with adults was not specified so updated comparisons weren't possible. As an assessment of the amount of plastic in Northern Fulmars as a species or population, the pooled estimate in the NE Pacific sample overestimates loads because of the predominance of juveniles in our samples while the majority of the population or species are adults [30,42]. However, as long as demographic profiles are incorporated into assessments, sampling using stomachs provides some measure of environmental pollution for a region (see reviews [4-6, 9, 10]).
Several reasons have been advanced to account for larger plastic loads in immature birds as compared to adults. One suggestion, specific to recently fledged juveniles, was that larger loads represent plastic from parental transfer [17,20]. This seems negated by our findings that about half the juveniles had no more plastic than older birds and 14% contained no plastic. A related suggestion was that adults would contain smaller plastic loads due to "off-loading" while feeding young via regurgitation [11,26]. Off-loading would likely reduce passage and accumulation of plastic in the ventriculus in some adults, but only those that successfully fed young. This would leave 50% or more [30] of the adults with relatively large plastic loads due to lack of feeding young because of nesting failure. In any case, off-loading or regurgitation would not be confined to the chick feeding periods but probably occurs as needed to clear plastic and other indigestible matter from proventriculi [13]. This leaves a default assumption that adults are more selective foragers than immatures [17,20] or the two groups forage in regions that differ in plastic availability [37].

Gastric Distribution of Plastic in Fulmars
Our NE Pacific fulmar sample appears to be unique in having a large proportion of plastic in proventriculi, especially in some juveniles. One other study examined the distribution of plastic in stomach sections separately using categories of adult versus non-adult from the Faroe Islands (North Atlantic) fulmars [21]. The percent of birds with proventricular plastic did not differ by age (adults 33% or 50 of 151, non-adult 42% or 40 of 96) ( Table 4 in [21]) (Chi-square=1.85, p=0.173). For a direct comparison, we categorized our data in the same way (adult versus non-adult) and significantly fewer adults (27% or 6 of 22) contained proventricular plastic than non-adults (54% or 84 of 155) (Chi-square=9.98, p=0.015). A reason for a greater incidence of proventricular plastic in NE Pacific fulmars could be that pieces were larger averaging 8.3 mm greatest dimension and 65.6 mg [13] as compared to 6.95 mm and 20 mg for the Faroe Island samples (Table 4, Figure 5 in [21]). Ventricular sizes were more similar averaging 5.5 mm and 25.3 mg [13] and 5.65 mm and 11.8 mg (Table 4, Figure 6 in [21]), which is an estimate of the approximate size that can squeeze through the proventricular-ventricular constriction [13,14,21]. Smaller pieces in Bravo Rebolledo [21] could pass into the ventriculus, while the larger pieces in NE Pacific fulmars may be too large to pass into the ventriculus thus accounting for large proventricular loads in NE Pacific fulmars..
As noted in the Introduction, a concentration of plastic in ventriculi indicates that proventriculi were cleared of plastic either through regurgitation or passage into the ventriculi. Although this is an obvious point, implications differ depending if plastic loads are used as bioindicators or are used to assess biological significance. For bioindicator use, capacity estimates based on maxima are with 2.6 g for ventriculi and almost 10 times more at 24.4 g for proventriculi (Table 3). In the context of these maxima, plastic loads are far below what a fulmar could hold (Table 3), especially given the amount of environmental plastic available [12,43,44]. Presumably, as the ventricular maximum nears or if pieces are too large to pass into the ventriculus, plastic accumulates in the proventriculus or is regurgitated. The difference in the accumulation of plastic in proventriculi in comparing the 2011-2012 to 2014-2015 time periods is unexplained but could reflect latency to regurgitate, size or age of particles, distance from pollution sources, foraging differences, or a combination of these ( [45], also see Section 4.4, below). Proventricular plastic probably is recently acquired and retained for a short time relative to ventricular plastic [7,13,25]. Unlike our NE Pacific samples, the Atlantic samples are mostly ventricular [1,2,7,10,21] so direct comparisons of pooled samples between the two oceans seems ill-advised.

Changes in Oceanic Plastic and Gastrointestinal Dynamics
Changes include a shift in the composition of stomach plastic from uniform pellets to irregularly shaped user particles in the 1990s to 2000s [3,47,48]. This shift was also evident in oceanic plankton tow-net sampling [44,[49][50][51]. In addition, as the mass of oceanic plastic ages, the number of pieces increases due to fragmentation with a corresponding reduction in size and increased accumulation of pieces less than 10 mm greatest dimension. Size varies with proximity to pollution sources, currents, exposure, temperature, and mixing [12,44,49,51]. Unlike the pellet-to-user plastic shift, this change has not been considered in fulmar or other seabird studies. Most studies appear to assume that plastic size is constant and that stomach samples reflect changes in the availability of the amount of oceanic plastic (e.g., [6,10]).
The significance of the shift in the size of user particles to stomach-based sampling is speculative. If fulmars selected particles in a certain size range, then stomach loads would sample changes in availability within the acceptable size range. If they ingest what was available, and particle size was reduced in time or regionally, then smaller particles might pass directly through the proventriculus into the ventriculus. If small enough, particles could pass immediately into the intestine and out of the birds. In planktonic sampling, the predominant particle size in the NW Atlantic is <10 mm greatest dimension having changed from 10 mm to 5 mm through the 2000s [49] and continues to fragment [44]. Further north, in the Arctic Sea, the predominant size was 1-2 mm greatest dimension [51]. Based on the size of intestinal plastic ( [13,21]; this study) these small particles could pass through the gastrointestinal tracts. Since retained plastic is used for biomonitoring, the possibility that particle size is not constant in time or space should be considered along with demographics and gastric distribution of plastic. Extending this to differences in Pacific versus Atlantic, Atlantic plastic may be smaller pieces that are easily regurgitated or passed through into the ventriculus and intestine. This might account for the concentration of plastic in the ventriculus in Atlantic samples in contrast to samples from the NE Pacific coast.

Conclusions
Upon reconsideration, the NE Pacific Ocean plastic samples from fulmars do not appear to be directly comparable to those from Atlantic Ocean studies. This is due to a preponderance of juveniles along the NE Pacific coast that retained relatively large plastic loads, particularly in proventriculi. Our review demonstrates that the amount of plastic retained in samples can vary because of the expansive capacity of the proventriculus versus the limited capacity of the ventriculus. Unlike ventricular plastic, retention of proventricular plastic is relatively short-term before regurgitation or passage into the ventriculus. The NE Pacific coast samples may differ from the Atlantic in that proventricular pieces were larger than those in the Atlantic suggesting that environmental plastic pieces are larger in the NE Pacific, Pacific fulmars are selecting larger pieces, or both. Because of the preponderance of juveniles from the NE Pacific, loads for the population, region, and species are biased upward as are the use of pooled values in comparison to other regions. Based on a review of oceanic plastic, plastic input is either stable or increasing, while weathering of existing plastic is fragmenting and increasing the relative proportion of smaller pieces. This along with demographics and gastrointestinal dynamics could influence the utility of stomach plastic as bioindicators and change biological impacts.