Tuesday, 18 February 2020

Searching for suspended and Salp-ingested microplastic debris in the North Pacific, using epifluorescence microscopy.

Marine debris is a worldwide ocean pollution problem, with plastics found in virtually all aquatic environments. The majority of marine debris analyzed to date has been microplastic, plastic particles less than 5 mm in size. However, findings suggest even smaller plastics (less than 333 μm) are both under-sampled due to the inappropriate mesh size of common sampling nets and far more numerous because plastic particles physically degrade over time into progressively smaller pieces. Such small debris can be consumed by, and deleterious to, suspension-feeding marine organisms, including Salps. Salps are pelagic Tunicates that possess the highest per-individual filtration rates among marine zooplankton, ingesting particles from under 1.0 μm to about 1.0 mm in size. They primarily feed in the upper water column, where microplastics are abundant. Once plastics are ingested by zooplankton, they have the potential to bioaccumulate in the food web into larger organisms, along with adsorbed persistent organic pollutants and harmful chemical additives, with unknown physiological consequences.

In a paper published in the journal Limnology and Oceanography Letters on 27 November 2019, Jennifer Brandon of the Scripps Institution of Oceanography at the University of California San Diego, Alexandra Freibott, also of the Scripps Institution of Oceanography, and of the Pacific Northwest Research Station of the United States Forest Service, and Linsey Sala, again of the Scripps Institution of Oceanography, present the results of a study which aimed to isolate, identify, and quantify microplastics 5–333 μm in size, a subgroup of microplastics which they termed mini-microplastics, from surface seawater samples and salp specimens collected from the North Pacific.

Although many zooplankton species consume microplasticc in a laboratory setting, the ecologically significant question lies in whether they are ingesting such particles in situ. Although the measured abundance of surface seawater microplastics is high, it is 1–3 orders of magnitude below model predictions of plastic inputs. In 2014 a considerable influx of both salp tunics and fecal pellets to about 4000 m depth following a bloom of Salpa spp. in the northeast Pacific. Thus, Salps could be a key link explaining the discrepancy between modeled and measured abundances of buoyant plastics, because fast sinking Salp fecal pellets and carcasses may be a vector moving ingested surface microplastics to the deep sea.

A Salp (plural Salps) is a barrel-shaped, planktic Tunicate. It moves by contracting, thus pumping water through its gelatinous body, one of the most efficient examples of jet propulsion in the Animal Kingdom. The Salp strains the pumped water through its internal feeding filters, feeding on phytoplankton. Wikipedia/Oregon Department of Fish and Wildlife.

Isolating, identifying, and quantifying microplastics 5–333 μm in size is a difficult task in the ocean due to their small size and irregularity. Brandon et al. took advantage of the well-documented autofluorescence of many plastics, and modified an epifluorescence microscopy approach normally used to enumerate planktonic microorganisms, to quantify oceanic mini-microplastics in surface seawater and Salp gut contents. Specifically, they asked: What are the distribution and abundances of these mini-microplastics in surface seawater? Are Salps ingesting mini-microplastics in situ? And, does the size distribution of ingested particles reflect that of available plastic particles?

Surface seawater samples and salp specimens used in this analysis came from the following cruises: SEAPLEX (02–21 August 2009), R/V Falkor (21–30 October 2013), SKrillEx I (26–31 July 2014), and SKrillEx II (11–17 June 2015). Surface seawater samples (1–2 m) were collected in metal buckets, immediately filtered onto 5 μm pore polycarbonate filters, and frozen. Brandon et al. sorted Salps from sodium borate-buffered 1.8% formaldehyde preserved plankton samples. These were collected via a 202 μm mesh bongo net at a tow depth of approximately 200 m or a surface-dwelling 333 μm mesh manta net. They tested for airborne plastic contamination during sample collection on a separate cruise in January 2017 by separately filtering both surface seawater samples and ultra-filtered Milli-Q water.

Maps of sampling locations, for testing of microplastics both in surface seawater and in Salp gut contents via epifluorescence microscopy. Surface seawater samples were taken via bucket tow in the open ocean (A) on the R/V Falkor (yellow, California Current; purple, transition region; mint green, North Pacific Subtropical Gyre; grey, SKrillEx sites), in 2013 and the nearshore (B) on SKrillEx I in July 2014 (blue) and SKrillEx II (green) in June 2015. Salp samples were taken via manta tow in the open ocean (A), on SEAPLEX (red), in August 2009, and in the nearshore environment (C), on SKrillEx I in July 2014. In (C) the bright green dots indicate stations with 10 Salps present, dark green indicate less than ten Salps present, and grey indicate no Salps in the sample. Brandon et al. (2019).

Brandon et al. sorted, measured, and identified Salp species and life history stage from preserved specimens from each sampling location, located in three open ocean regions: North Pacific Subtropical Gyre, California Current, the transition region, and a nearshore region. Life history stage was designated as blastozooid, the sexual chain-forming generation, or oozooid, the asexual solitary generation. Salp guts were dissected; however, any existing mucous nets and gill bars were not analyzed to avoid artifacts of net feeding.

Traditional epifluorescence microscopy techniques add fluorochromes to stain the DNA and proteins of plankton so that identifying features appear under different reflected wavelengths of light. Because Brandon et al.'s target was identification of plastics, not living organisms, they did not add any fluorochromes. Brandon et al. left prepared slides at room temperature for at least 24 hours to diminish chlorophyll a autofluorescence of plankton before visualization. This ensured the most fluorescent particles on microscopy images were likely microplastics, bacteria, or transparent exopolymeric particles. Brandon et al.tested multiple plastic and nonplastic reference materials, such as cotton and wool, under the four light excitation channels of our microscope to determine their autofluorescence. Filtered surface seawater samples and Salp gut contents were prepared for microscopy using an all-glass filtration apparatus.

Brandon et al. created a decision tree to determine if a particle was plastic. Generally, plastics appeared as long, thin fibers or flat fragments with sharp edges. Plastic particles fluoresced uniformly and did not have inner striations, coloration patterns, or features suggestive of biological particles, such as spines, nuclei, or organelles. Not all plastics fluoresce, so this was not used as a diagnostic feature Particles that were invisible under transmitted light but fluoresced under another light channel were determined to be transparent exopolymeric particles. Particles identified as likely diatom frustules, including chain-formers and pennates like Pseudo-nitzschia, were not counted as plastics. When in doubt, particles were not counted as plastic, so Brandon et al.'s estimates are conservative and most likely underestimate total mini-microplastic abundance.

Decision tree for enumerating plastic microdebris on slides, used to determine which particles were plastic and which were biota or other detritus. Brandon et al. (2019).

Particles were categorized as short or long fibers and fragments. The lengths, widths, areas, and fluorescence were recorded for every fragment and short fiber (under 300 μm) in automated images. Long fibers (at least 300 μm) were enumerated in separate, manual visual transects at lesser magnification to eliminate the possibility of double-counting single large fibers that were not visualized in their totality in automated images. Fibers under 200 μm in length were not counted in manual transects; however, there may be some overlap between the short fibers counted in automated images and long fibers counted in manual transects, due to the 200–300 μm overlap. Brandon et al. recorded long fiber color, length, and width with an ocular micrometer.

Brandon et al. analyzed plastic particles in filtered salp gut contents via epifluorescence microscopy. Because Salp gut walls and ingested biogenic material can fluoresce, fluorescence was considered a secondary characteristic of ingested plastic over particle shape and reflectivity under transmitted light. However, fluorescence was checked to visualize inner striations or patterns characteristic of diatom chains. When in doubt, particles were not counted as plastic. The thick gut walls of Salps and ingested biogenic material most likely occluded some plastic, so our data underestimate total plastic ingestion.

To calculate salp ingestion rates of plastic, mini-microplastic counts were divided by gut clearance times for each species identified, which ranged from 2.5 to 6.25 hours.

Brandon et al. found different patterns of fluorescence between plastic and biological materials, and when in doubt, particles were not counted as plastic. Using a controlled test, they determined that the vast majority of mini-microplastic materials in these filtered seawater samples were not from contamination during processing.

Transmitted light and epifluorescence images of microplastics from surface seawater, including a plastic fragment (A), thick and thin short plastic fibers (B), a long fiber and transparent exopolymer particles (C). Column (1) transmitted light; Column (2) Excitation 450-490 nm, Emission greater than 515 nm; Column (3) Excitation 340-380 nm, Emission 435-485 nm; Column (4) Excitation 465-495 nm, Emission 635-685 nm. Brandon et al. (2019).

Brandon et al. detected no significant spatial heterogeneity in seawater plastic concentrations across the Falkor transect (at 12 h intervals) for three open ocean regions: North Pacific Subtropical Gyre, California Current, the transition region. Nearshore samples from SKrillEx I and II were collected at approximate 15 km intervals, and showed no significant spatial heterogeneity possibly due to small sample sizes. Mean open ocean mini-microplastic concentrations compared to nearshore demonstrated significant heterogeneity between regions. Nearshore mini-microplastic concentrations differed from all other regions

Open ocean mini-microplastic concentrations were on the order of 10-100 per litre for short fibers and fragments with lower long fiber concentrations (1-10 per litre). In contrast, the fluorescent long fibers were 3.5–6.5 times more abundant than mean concentrations of nearshore short fibers and fragments on SKrillEx I, and almost eight times more abundant on SKrillEx II.

Almost every open ocean fragment and short fiber was below 333 μm in length and would have been missed by previous studies using larger mesh nets. Long fibers were usually over 333 μm, but thin enough to easily slip through 333 μm mesh. The minimum lengths of fragments and short fibers were between 14 and 50 μm for all locations, approaching the 5-μm pore size of the filters. For long fibers, both surface area and length were significantly different among regions, with significantly shorter fibers in the transition region. Similarly, in the nearshore samples (SKrillEx I and II), every measured fragment and short fiber length was under 333 μm

Individual particle surface area ranged from 0.0 003 to 0.71 mm², compared to earleir studies using a 333 μm net, which detected particles 0.01–565 mm². These earlier studies found plastic particle lengths ranging from 0.34 to 65.7 mm, while Brandon et al found lengths from 0.01 to 16.27 mm (including long fibers). Ultimately, the most pronounced difference between Brandon et al.'s findings and those of earlier studies was not the size range of particles, but rather their concentrations. Mini-microplastics in this study were five orders of magnitude more abundant than the over 333 μm microplastics of earlier studies. However, when concentration was multiplied by surface area Brandon et al. found that the over 333 μm microplastics had significantly higher areal concentrations than the under 333 μm mini-microplastics.

Salps have a very interesting life cycle, known as alternation of generations.  Salps have two different life stages: a solitary asexual stage and a colonial sexual stage.  The solitary stage of Salps (also referred to as the oozooid stage) has a special structure called a stolon.  This stolon develops into chains of the colonial stage.  When a chain of the aggregate stage has grown large enough within the solitary organism the chain will be released.  The chain is the sexual stage of the Salp (also referred to as the blastozooid stage).  In many species, the colonial stage (chain) looks very different from the solitary stage, and in fact when scientists first discovered Salps they often thought that the colonial and solitary stages of the same species were actually different species.  Chains of the colonial stage can extend for several meters in the water, and include hundreds of individuals.  The chains start off as females, and are fertilized by the sperm from older chains.  Once the eggs within a chain are fertilized, an embryo (of the solitary stage) will grow within each individual of the colonial stage.  The colonial stage will then give live birth to the solitary (asexual) stage so that the process can repeat itself.  As the chains matures it will switch from a female to a male. FSU Zooplankton Ecology and Biogeochemistry Lab.

Every single Salp gut analyzed contained plastic. Blastozooids had higher ingestion rates than oozooids. In general, nearshore Salps were larger than open ocean Salps  The California Current Salps were the smallest and had the lowest plastic ingestion rates. Excepting the North Pacific Subtropical Gyre and transition region oozooids, there was no detectable relationship between body length and plastic ingestion rate for dissected Salps. Although Brandon et al found regional differences in mini-microplastic concentrations in the water column, there was no significant effect of region on Salp plastic ingestion rate. Fibers made up 91% of the total ingested particles. The surface area and lengths of fibers and fragments differed significantly between most regions.

Brandon et al. compared the size of ingested mini-microplastics with that of ambient mini-microplastics in surface seawater, both from their data and from earlier studies Most of the net-collected particles from earlier studies fell within the size range of potential Salp food particles. At all sample locations, the average size of particles consumed by Salps was significantly smaller than the size of ambient seawater plastic.

Brandon et al. successfully used epifluorescence microscopy to identify mini-microplastic particles in natural seawater samples and Salp gut contents. This method required careful judgment and expertise to distinguish biotic from plastic materials. Furthermore, autofluorescence of Salp gut walls and biogenic materials made ingested plastic fluorescence only a secondary identification characteristic. This method allowed us to distinguish plastic from nonplastic particles and fluorescent from nonfluorescent plastic, but not to identify specific plastic types. Isolating plastic-type autofluorescence patterns under specific emission wavelengths may permit such differentiation in future work. However, our ultimate goal was to use standard epifluorescence microscopy techniques to differentiate plastics from nonplastic particles in order to obtain accurate bulk measurements of plastics under 333 μm, which the method accomplished.

This study may be one of the first to estimate the abundance of the smallest mini-microplastics in surface seawater, which are consistently under-sampled. Brandon et al. found a mean plastic concentration across all locations of 8277 particles per litre (8 277 000 particles per m³). Their particle concentrations averaged 5–7 orders ofmagnitude higher than previous studies. This highlights the previously unquantified significance ofmini-microplastics inmarine debris counts.

Nearshore samples had higher plastic concentrations than open ocean samples. This agrees with published findings that have recorded similar spikes in plastic concentrations nearshore, close to populated areas, with a decline in plastic moving offshore. The difference in plastic concentrations between SKrillEx I and II may be explained by annual differences in rainfall and watershed input to these nearshore waters.

Many estimates of macro- and microdebris, including modeled debris trajectories agree that the highest concentrations of open ocean marine debris occur in convergence zones of subtropical gyres. However, Brandon et al. did not detect a significant increase in mini-microplastic concentration in the North Pacific Subtropical Gyre and their open ocean samples were not significantly different across regions. Many possible sinks of mini-microplastics could account for this. Plastic below 5 μm in size presumably degrade beyond the detection limit of Brandon et al.'s method. Plastics can also be biofouled and sink out of surface water, or ingested and removed from the water. As plastic accumulates in the North Pacific Subtropical Gyre and breaks down into progressively smaller pieces, Brandon et al.'s data suggest that plastic under 333 μm is removed from the gyre through biofouling, ingestion, or degradation at the same rate it is being supplied. In the nearshore zone, however, mini-microplastics, especially long fibers (in the 200 μm–17 mm range), likely have a higher rate of input than loss. All of these sources and sinks require further research to be better parameterized.

Earlier studies sampled almost no particles smaller than 0.333 mm × 0.333 mm (0.11 mm²), due to the mesh size of the sample collection net, while Brandon et al. detected many particles below that limit (minimum size 0.000 3 mm²). Their results show the majority of plastic concentrations occur between under 333 μm and 0.11 mm². Although the mini-microplastics they measured were more numerically abundant, they did not comprise the majority of the plastic surface area in the water. Organisms that colonize surface substrates in the ocean are more likely to find surface area on micro- and macroplastics rather than mini-microplastics, despite the numerical abundance of mini-microplastics.

This is the first record of Salp ingestion of microplastic in situ. Every Salp dissected had plastic in its gut, regardless of species, life history stage, or region of the ocean sampled. Salp gut clearance times are on the order of 2–7 hours, so Brandon et al. are confident that by only analyzing the gut, they avoided artifacts of net feeding or other contamination. Airborne contamination is a major concern in modern microplastic work, especially when samples are dominated by fibers, as in this study (91% of the Salp-ingested particles). However, our processes of seawater filtration, slide preparation, and salp dissection limited contamination. Compared to filtered control samples, most of the plastics in Brandon et al.'s surface seawater samples were not contamination.

Brandon et al. detected no regional differences in plastic ingestion by Salps, excluding the much lower values of the California Current Salps. This finding is likely attributable to the very small body size of those salps. The California Current Salps had the lowest ingestion rate of any region, whereas for surface seawater, concentrations of mini-microplastics in the nearshore environment were significantly higher than the entire open ocean. Overall, however, both Salp ingestion and surface seawater plastic concentrations had limited regional differences.

Salps are predominantly generalist suspension feeders with ingestion based primarily on particle size, typically from less than 1 μm to 1 mm. All seawater mini-microplastic measured, and almost all the plastic in earlier studies, fall within their possible ingestion range. Yet, the Salps sampled by Brandon et al. ate significantly smaller pieces of plastic than were available in the ambient surface water. This may be explained by the fact that Salps can efficiently collect down to submicron particles and feed throughout a greater area of the water column than the surface, where larger,more buoyant plastic is retained.

Salps are of ecological importance due to several factors: their notoriously rapid growth and opportunistic reproductive rates that can lead to extremely high population densities or 'blooms', higher filtration rates per individual than any other zooplankton grazer, and production of dense fecal pellets that can result in high vertical fluxes of this material to deeper depths. The large fecal pellets of Salps have proven to possess rapid sinking and slow decomposition rates such that they can reach the deep ocean relatively intact, transporting organic carbon and potential microplastics with them. Brandon et al.'s evidence for the widespread and universal consumption of microplastics by Salps leads Brandon et al. to believe that Salps may be an important vector of marine debris transport from the surface ocean to deep-sea communities. The transport of microplastics via Salps may be critical to incorporate into microplastic export calculations as an overlooked output from surface waters.

See also...

https://sciencythoughts.blogspot.com/2019/12/assessing-impact-of-plastic-waste-on.htmlhttps://sciencythoughts.blogspot.com/2019/09/cetacean-sightings-within-great-pacific.html
https://sciencythoughts.blogspot.com/2019/01/mercury-and-selenium-levels-in.htmlhttps://sciencythoughts.blogspot.com/2015/10/microplastics-in-deep-sea-marine.html
https://sciencythoughts.blogspot.com/2014/12/counting-floating-plastics-in-worlds.htmlhttps://sciencythoughts.blogspot.com/2014/05/marine-litter-on-european-seafloor.html
Follow Sciency Thoughts on Facebook.