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README.md

Title

Linking marine microorganism to human health

1.Introduction

Sea Spray Aerosol (SSA) is generated by the bubble bursting of whitecap waves occurring in the sea surface microlayer (SSML), i.e. on the top layer of 1~1000 µm of seawater (Michaud et al., 2018). It contains mixed salts, organic materials, and marine biogenic. With 71% ocean coverage of the Earth, SSA represents as a major atmospheric aerosol particle type, and an essential source of human inhalable particles in the coastal area. Since the coastal zone (~100 km to the coastline) supports a population that is three times denser than the global average (Crossland et al. 2005), characterization of the concentration of bioactive ingredients in SSA, the aging and spreading of SSA particles, and the human health effects of bioactive ingredients in SSA are vital for understanding and evaluating the human health consequences of SSA.

We have designed the following long-term sampling survey along with lab simulation experiments to (1) reveal the formation mechanism of human health-related compounds in SSA, (2) investigate the aging and transmission pattern of SSA bioactive particles, and (3) hopefully predict the SSA-Human-Health-Index through conventional monitoring methods.

2.Knowledge Gaps

2.1.Sources of the bioactive ingredients in the SSML vary in regions and seasons.

Phytoplankton is considered to be a major source of bioactive ingredients in SSML. Phytoplankton releases organic matter into SSML by secreting extracellular polymeric substances or toxins (Pereira et al., 2009), and can also add organic matter into SSML by cell lysis after death (Lei et al., 2012). Some other microorganisms, i.e. virus and yeast have been found living in the SSML (Chang et al., 2016), which are also potential ingredients in SSA (Aller et al., 2005).

Yet there are limited reports about the microorganism composition affecting the SSML bio-active ingredients composition and concentration during normal condition. Most studies focused on extream ecological events like algae blooming. In order to link marine microorganism to human health at a daily aspect, find out the sources of the bioactive ingredients in the SSML at a normal condition is needed. Since the community of algae varies in regions and seasons, a seasonal variation in SSML composition is expected, and should also be considered.

Illustration for Knowledge Gap 1

2.2.The detection threshold of bioactive ingredients in SSA can’t meet our requirement.

Because of the enrichment effect (O’Dowd et al., 2004), the biogenic ingredients in the seawater can be concentrated dozens or even thousands of times. Still, the particle of SSA with a size range from 0.01~2.5 μm is too small to make full detection and quantification of the ingredients. Prediction the concentrations of bio-active ingredients in SSA by the enrichment factors and the concentrations of bio-active ingredients in SSML can be a way around (Prather et al., 2013; Jayarathne et al., 2016). Enrichment rate of SSA compounds have been studied in recent decades, most studies focus on the enrichment rate of chemical categories of compounds in SSA (Cochran et al., 2017; Bertram et al., 2018), rather than the enrichment rate of bioactive ingredients related to human health. There are limited study cases investigated the enrichment factor of organic matters in SSA, but the enrichment factors can jump from 60 to 1200 for the same chemical (Jayarathne et al., 2016). A deeper investigation into the enrichment factors, especially that of bioactive ingredients in SSA is necessary.

Illustration for Knowledge Gap 2

2.3.Aging and spreading pattern of SSA has not been well studied, especially on bioactive ingredients.

Freshly emitted SSA particles will go through several processes (aging) before can be inhaled by humans, including sedimentation, evaporation, and degradation. The sedimentation and evaporation of SSA particles are relatively well studied (Fu et al., 2013; Freud et al., 2017). While the degradation processes of bioactive ingredients in SSA are still unknown, mediated by solar radiation and oxidants in the atmosphere, oxidation and degradation of bioactive ingredients are expected during the spread of SSA particles.

Evidence supporting the long-distance spread of SSA have been reported: particles are detected 100 km away from the coast in Scottish mainland (Smith et al., 1993) and 320 km away from the Gulf of Mexico in Alabama (Bondy et al., 2017). But no study has been done on the spreading pattern of SSA with an emphasis on bioactive ingredients.

2.4.Positive human health effects of SSA are still not clear.

Studies about the health risks of SSA majorly focused on the negative health effects caused by extreme ecology events, such as brevetoxins generated during harmful algal blooms, e.g. red tide (Fleming et al., 2009). Yet, limited studies have been done on the potential impacts of SSA on human health in the coastal area. A recent study reported inductions of apoptosis were observed when human lung cancer cells exposed to natural SSA (Asselman et al., 2019). Although the mechanism of positive SSA exposure effects is still unknown, and we don’t know which compound(s) is (/are) effective in SSA, the research result indicates that there are potential positive health effects at an environmentally relevant concentration of SSA. The expose dose of bioactive ingredients in SSA to human lung cell can be vital to the health effect, but no research has drawn a clear curve for the health effect against distance to the coastline of bioactive ingredients in SSA.

3.Research Questions

  • Who is producing the organic ingredients in SSML (phytoplankton)? Any condition and variation?
  • Can we predict the concentration of bioactive ingredients in SSA based on enrichment factors and SSML composition?
  • How are the aging and spreading pattern of bioactive ingredients in SSA?
  • What’s the effective distance (to the coastline) of bioactive ingredients in SSA.

4.Research Design

4.1.Relationship between Bioactive Ingredients Concentration and Marine microorganisms

4.1.1.Workflow

Workflow for Research 1

4.1.2.Field Sampling

Field sampling will be carried out once a month. The sampling site should be fixed and located in open seas with less human disturbance. The environment factors of the sample site should be recorded according to Protocl 5.1.

Each time collecting SSML samples and ~10m seawater samples. SSML sample collection will use Protocol 5.2.1, seawater sample collection will use Protocol 5.2.2.

4.1.3.Sample Processing and Analysis

Chemical composition of SSML samples will be analysised using Protocol 5.3.1. Ecological composition of the sampling site will be analysised using Protocol 5.3.2. Selection of environment factors are based on other research results (Breuer et al., 2016), taxonomic groups are determined by 95% identity threshold.

The dataset of this research will containe the folllowing variables:

Category Variables
Environment Factors T{air, water}, pH, C{O2, PO4, NH4, NO2, NO3}
Ecological Composition of Seawater A_Group{1:10}}
Chemical Composition of SSML C_{Chlorophyll a, DHA, EPA, Ergosterol, Tryptanthrin, Violacein} (Need further discussion, and selected based on the profiles of exsisting species in seawater)

*A: Abundance (cells/L); C: Concentration (mg/L); T: Temperature.

4.1.4.Expected Outcome

  • The seasonal variation pattern in the composition of marine phytoplankton community.
  • Causal relationship between the concentration of bioactive ingredents and the abudance of phytoplankton taxonomic groups under different environment conditions.

4.2.Enrichment Factors of Bioactive Ingredients in SSA

4.2.1.workflow

Workflow for Research 2

4.2.2.Field Sampling

Field sampling will be carried out once a month. The sampling site should be fixed and located in coastline with less human disturbance. The environment factors of the sample site should be recorded according to Protocl 5.1.

Each time collecting SSML samples and SSA samples. SSML sample collection will use Protocol 5.2.1, SSA sample collection will use Protocol 5.2.2.

4.2.3.Sample Processing and Analysis

Chemical composition of SSML samples will be analysised using Protocol 5.3.1. Chemical composition of SSA samples will be analysised using Protocol 5.5.

The dataset of this research will containe the folllowing variables:

Category Variables
Environment Factors T_{air, water}, pH, Distance}
Chemical Composition of SSML C_{Chlorophyll a, DHA, EPA, Ergosterol, Tryptanthrin, Violacein} (Need further discussion, and selected based on the profiles of exsisting species in seawater)
Chemical Composition of SSA C_{Chlorophyll a, DHA, EPA, Ergosterol, Tryptanthrin, Violacein}

*C: Concentration (mg/L); T: Temperature.

4.2.4.Expected Outcome

  • Enrichment factors of bioactive ingredient in SSA under different environment conditions.
  • Variation patterns of enrichment factor against distance to coastline.

4.3.Aging and Spreading Pattern of Bioactive Ingredients in SSA

4.3.1.workflow

Workflow for Research 3

4.3.2.Field Sampling

Field sampling will be carried out once a month. The sampling transections should be fixed and located in coastline with less human disturbance. There will be three transections during each sampling: 0 m, 10 m, 100 m, 10 km. The environment factors of the sample site should be recorded according to Protocl 5.1.

4.3.3.Sample Processing and Analysis

Chemical composition of SSA samples will be analysised using Protocol 5.5.

The dataset of this research will containe the folllowing variables:

Category Variables
Environment Factors All parameters in Protocl 5.1
Chemical Composition of SSA C_{Chlorophyll a, DHA, EPA, Ergosterol, Tryptanthrin, Violacein}

*C: Concentration (mg/L); T: Temperature.

4.3.4.Expected Outcome

  • Aging and Spreading pattern of bioactive ingredient in SSA.
  • The concentration of bioactive ingredient in SSA can drop below detection threshold when the distance to coastline reaches 100 m. An simulation system will be needed for further investigation.

4.4.Health Efficiency Curve of Bioactive Ingredients in SSA Against Distance to Coastline

4.4.1.workflow

4.4.2.Field Sampling

4.4.3.Sample Processing and Analysis

4.4.4.Expected Outcome

4.5.Comprehensive Simulation System for SSA Research

4.5.1.workflow

Workflow for Comprehensive Research

4.5.2.Phytoplankton Species Selection

  • Define the POLYGON of Belgian part of the North Sea region with shp file from https://geo.be/.
  • Get the species list during the recent 20 years within the POLYGON from Ocean Biogeographic Information System with their API.
  • Shorten the list by limit the residence depth of species to ~10 m based on the report about vertical distribution of algae (Park et al., 2001) and the limitation of sample access.
  • Phylum of Chlorophyta, Cryptophyta, Cyanobacteria, Discomitochondria, Euglenozoa are selected based on the ability of photosynthesis.
  • Further actions needed for selection of around 10 species sorted by the dominance in different seasons and the potential bio-active products.

4.5.3.Bioactive Chemical Selection

5.Protocols

5.1.Site information

Site information listed below should be recorded each time before sampling processes. Sheet-1 gives an example of site information parameters that should be filled in and measured on-site.

5.1.1.General information

For each field sampling, the following information should be recorded:

Parameter Description
SiteInfoID The unique ID for the sampling site information, one ID for each sampling operation (can have multiple samples in one operation).
Date Date for sampling operation.
TimeBegin Time when the sampling operation begins.
TimeEnd Time when the sampling operation end.
Participants Names of the participant in this sampling operation.

Take pictures of the sampling site and the surroundings if possible.

5.1.2. Site location

Geographic coordinates of sampling site should be measured using GPS equipment. Recommended App is My GPS Coordinates (iOS | Android). GPS signal accuracy should be within 100m, otherwise a professional GPS device is needed. The coordinate data should be accurate to 3 decimal places. The distance to coastline should also be in-situ measured and recorded if possible, otherwise, use R script to calculate afterwards.

Parameter Unit Accuracy Description
Latitude °N 3 decimal places Latitude of sampling site
Longitude °E 3 decimal places Longitude of sampling site
D2Coast km 1 decimal places Distance form sampling site to coastline

5.1.3.Wind speed and direction

Wind speed and direction should be measured with WeatherFlow WINDmeter. Measurement should be performed three times: before, during, and after the sampling process. Both average speed and gust speed should be recorded. See HERE for the device user guide.

Figure 5.1.3 Wind speed and direction measurement

Parameter Unit Accuracy Description
WGSpeed1 km per hour 1 decimal place Gust wind speed before sampling
WASpeed1 km per hour 1 decimal place Average wind speed before sampling
WDirection1 ° 1 decimal place Wind direction before sampling
WGSpeed2 km per hour 1 decimal place Gust wind speed during sampling
WASpeed2 km per hour 1 decimal place Average wind speed during sampling
WDirection2 ° 1 decimal place Wind direction during sampling
WGSpeed3 km per hour 1 decimal place Gust wind speed after sampling
WASpeed3 km per hour 1 decimal place Average wind speed after sampling
WDirection3 ° 1 decimal place Wind direction after sampling

5.1.4.Temperature, relative humidity and air pressure

Temperature (dry bulb temperature), relative humidity and air pressure should be measured with WeatherFlow WINDmeter or other professional equipment for measurement.

Parameter Unit Accuracy Description
Temperature °C 1 decimal place Dry bulb temperature in Celsius
Humidity % 1 decimal place Relative humidity using dry and wet bulb thermometer
AirPressure inHg 2 decimal place Atmospheric pressure

5.2.Water Sampling

This part is adapted and modified from Guide to best practices to study the ocean’s surface and GB 17378.3-2007 Marine Monitoring Code Part 3: Sample Collection, Storage and Transportation

5.2.1.Sea surface microlayer (SSML) sampling

5.2.1.1.Mesh screen sampling method

This sampling method is preferred when the wind and waves are not strong. Otherwise, please use the glass plate sampling method.

Equipment

  • Screen sampling device (mesh size 1 mm2, Lechtenfeld et al., 2013)
  • Polyethylene vessels
  • Funnel

Figure 5.2.1.1-1 Mesh screen sampling device

Sampling procedures

  • Rinse the mesh screen, vessels and funnels with acetone and air-dried.
  • Put the rinsed screen into the seawater at an angle, once it is submerged completely in the water, it should be kept horizontal and wait for 30 seconds.
  • Slowly lift the screen vertically out of the seawater.
  • Tilt the screen at an angle, so that the SSML sample can drop into the funnel and finally collected in the vessels.
  • Mark the vessels.

Figure 5.2.1.1-2 Mesh screen sampling procedures (modified from Cunliffe et al., 2014)

5.2.1.2.Glass panel sampling method

Equipment

  • Glass panel sampler
  • Polyethylene vessels
  • Funnel
  • Silicone wiper

Sampling procedures

  • Rinse the glass plate, vessels and funnels with acetone and air-dried.
  • Insert the glass plate vertically into the seawater with the handle and slowly lift it out, keep it vertical during the procedure.
  • Use the wiper to scrape off the liquid adhered to the glass plate, collect it in the vessel via funnel.
  • Mark the vessels.

Figure 5.2.1.2 Glass panel sampling procedures (from Cunliffe et al., 2014)

a)glass plate sampler with integral PVC handle showing sample collection (courtesy of Manuela van Pinxteren, TROPOS, Germany).
b) squeegeeing a simple plate sampler held with a clean plastic clamp.
c) squeegeeing a glass plate sampler using “clean hands/dirty hands” technique (courtesy of Manuela van Pinxteren, TROPOS, Germany).
d) a glass plate and sample recovery device containing integral Teflon wiper and funnel, based on the design of Hardy et al. (1985).

5.2.2.Surface seawater sampling

Equipment

  • Vertical water sampler with build-in thermometer
  • Polyethylene vessels
  • Funnel

Sampling procedures

  • Rinse the water sampler, vessels, and funnels with surface seawater at the sample site.
  • Close the water outlet valve of the sampler.
  • Slowly lower the sampler vertically with the rope connected to it, the sampler bottom valve will open automatically when lowered, and seawater flows into the sampler.
  • Continue to lower it untill the top of the sampler completely immersed in sea water, close the upper opening manually and pull the sampler up.
  • Read the water temperature and write it down.
  • Open the water outlet valve of the sampler to collect the water sampler in the vessels via funnel.
  • Mark the vessels.

5.3.Water Sample Analysis

5.3.1.Chemical Composition of Water Sample

5.3.2.Ecological Composition of Water Sample

5.3.2.1.Shotgun Metagenome Analysis

5.3.2.2.Using Cell Counting Chamber

5.4.SSA Samping

Equipment

  • Constant flow vacuum pumps
  • Filters (type to be discussed)
  • Filter holders
  • PVC tubes
  • Ziplock bags
  • Anti-oxidation bags

Filter holder preparation

  • Each time 5 filter holders will be prepared, 4 for actural sampling and 1 for blank (carried with other three and).
  • Wash the filter holders and underdrain discs (Figure) with deionized water and then rinse with acetone. Remove residual acetone using purified high-pressure air.
  • Use clean tweezer to place a filter on the underdrain discs fitted in the filter holder, note that the front of the filter is facing outward (to the air inlet).
  • Make a small mark on the filter edge, align the mark on the filter with the valve on the outside of the filter holder, and close the filter firmly.
  • Put the prepated filter holder into ziplock bag for latter usage.

Sampling procedures

Field Setups:

  • Setting up the stand on a solid foundation, no block in the upwind direction.
  • Mark the filter holders as either sample or blank.
  • Clip the prepared filter holders on the stand, at a hight around 170 cm. Make sure the air inlet facing the upwind direction, and the valve on the outside of the filter holder is up right.
  • Connect each filter holder to a vacuum pump with PVC tubes.
  • If a diesel generator is used, it needs to be placed in the downwind and as far away from the sampling area as possible.

Schematic diagram of sampling devices, inspired by Sam Baelus’s Master’s dissertation and Emmanuel

SSA sample collection:

  • Open the air sampling pump with a flow rate of 10 L·min-1 for about 200 minutes, till 2000 L air flow collected (calculated by pump build-in counter).
  • Keep the upwind clear without people and make notes if there is anyting wrong.
  • After stoping the pump, remove the tubes on the filter holds and put it into ziplock bag with anti-oxidation bags.

5.5.SSA Samples Analysis

References