Sample curation and submission for analysis

Sedimentology

Sedimentology samples can be transported as refrigerated freight in a fully sealed, rigid container (e.g. esky) to Geoscience Australia for laboratory measurement. Alternatively, researchers may transport samples to their own labs if performing analyses in-house or through laboratories accredited by the National Association of Testing Authorities (NATA). Regardless of where the sample is analysed, data should still be submitted to the national seabed sediments collection data repository on the AusSeabed Marine Data Discovery portal (https://portal.ga.gov.au/persona/marine) (Section 9.7.2). Analytical methods include wet sieve separation into mud, sand and gravel fractions, laser granulometry of mud and sand fractions, and acid digestion of carbonate content for the bulk or mud and coarse fractions. Other methods are also available for those with their own expertise and equipment (e.g. calcimeter method in Kennedy and Woods (2013)).

When lodging samples, the following metadata are recommended prior to receipt of sediment samples:

  • Survey metadata including: survey name, survey number, survey vessel, start and end date of survey, latitude and longitude of survey bounding area, name of chief investigator;
  • Sample location for every sample listed in decimal degrees to at least five decimal places;
  • Sample water depth for every sample listed;
  • Sample ID follows a standard naming convention (see example attached);
  • Sample bags are labelled clearly with the sample ID (as above); and
  • Sample condition as when collected (i.e. wet, disaggregated, excess water drained).

Biogeochemistry

Geochemical analysis of sediment samples should be conducted by the organisation undertaking the survey. Alternatively, sample analysis should be outsourced to Geoscience Australia (Loss on Ignition analysis, as described below) or NATO-accredited commercial laboratories or collaborators (chl-_a _analysis).

Total organic matter content

Total organic matter content of marine sediments is determined by Loss on Ignition (LOI). Note that LOI is not the same as total organic carbon (TOC) (Schumacher 2002). Parameters such as temperature and combustion time vary among individual researchers, and there is no universally adopted standard. Here we choose parameters based on a compromise appropriate to a diverse range of environments (Heiri et al. 2001, Wang et al. 2011). We strongly recommend that researchers use these guidelines to ensure data from different surveys can be compared. The general recommended steps for LOI to contribute to a national standardised dataset are:

  1. Record wet weight of the sample.
  2. Homogenise the wet sample (1-2 g dry weight).
  3. Place sample into a pre-weighed crucible.
  4. Oven dry for at least 24 h at 105°; longer times may be needed in fine sediments.
  5. Reweigh crucible and dry sediment.
  6. Place crucible in muffle furnace and combust at 550°C for 4 h.
  7. Weigh crucible and combusted sediment.

The water content is the difference between the wet and dry sediment weights and is expressed as a percentage of the initial sediment weight. The total organic matter content is obtained as the difference between the dry and combusted sediment weights and is expressed as a percentage of the sediment dry weight.

Chlorophyll - a & phaeophytin

Chlorophyll-_a _is the principal pigment in plants and is a biomass indicator of aquatic micro-algae which support food webs in the sea, and phaeopigments (e.g. phaeophytin) are the degraded non-photosynthetic products of chlorophyll (e.g. Bax et al. 2001). The ratio between them indicates the “freshness” of the organic matter. Note that samples can be freeze-dried first and this may increase extraction efficiency but also increases the risk of chlorophyll degradation over time. For the purposes of this field manual, we recommend using wet material; this will ensure comparability among datasets. The general steps for chl-_a _analysis are:

  1. Place approx. 5 g wet sediment into centrifuge tube.
  2. Add 10 mL acetone (90% saturated with MgCO3).
  3. Mix rigorously (with glass rod or vortex mixer).
  4. Place in an ultrasonic bath for 30 minutes under dark conditions (Note: other methods can be used, e.g. shaker).
  5. Centrifuge sample (>1500 g for 5 minutes) and decant extract.
  6. Use a spectrophotometer to measure absorbance at 665 and 750 nm.
  7. Acidify extract with 2 drops of 0.1 N HCl, mix and rest for 60 s.
  8. Measure absorbance again at 665 and 750 nm.
  9. Calculate the concentrations of corrected chl-_a_ and phaeophytin using the equations of Lorenzen (1967).
Redox

Redox measurements are provided onboard with a probe and thus no post-survey procedures are required, other than to calculate redox potential and QC data.

Biology

  1. All animals from a given grab or box core should be sorted into separate small containers based on phylum or class to facilitate taxonomic identifications (arthropod, annelid, mollusc, echinoderm, other). This can be done onboard if time permits, but consideration must be given to working under a microscope on a moving vessel. Sorting can usually be done by a non-expert, with only a few groups posing potential challenges (Figure 9.4). Containers should be filled with ethanol or formalin (as per Pre-Survey Preparations) and labelled appropriately with solvent-proof paper.
  2. In order to test for potential bias due to differences in sorting efficiency among people, randomly selected samples should be re-sorted by a different person. Removal of 95% or more of the organisms during the sorting process is acceptable; otherwise, re-sorting may be necessary (Simpson et al. 2005) [recommended when multiple people are involved in Step 1]
  3. Within each sorted phylum, identify organisms to a taxonomic resolution that enables data production in a timely manner, and then count individuals. Identifications can be done by the organisation that collected the samples, museum taxonomists, geneticists, or external private consultants. Care must be taken to ensure consistent nomenclature is used for undescribed or unnamed species (e.g. defined operational taxonomic units, OTUs).

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Figure 9.4: A brief description of taxa that can be challenging to identify but are often encountered when sorting organisms from elutriated sediment samples.

a) Scaphopods (molluscs). These are curved shells with a larger and smaller hole on each end.
b) Aplacophorans (molluscs). These are often confused with worms but are actually molluscs covered with spicules that can make them look furry.
c) Foraminiferans (protists). Forams with tests (i.e. shells) can be mistaken for gastropod shells and can be particularly common in deep-sea sediments and beach sand. This field manual does not target forams so their inclusion in sample processing is not required (image from Wikimedia).
d) Crinoids (echinoderms). The small animals or their dropped arms can superficially resemble polychaete worms.
e) Hermit crabs (crustaceans). These can be mistakenly sorted as gastropods because the crab has retreated into its shell and is barely visible.
f) Ophiuroid arms (echinoderms). These can often be confused with polychaetes, but you’ll never see a head. There is no need to save ophiuroid arms unless the central disk is present.
g) Ostracods (crustaceans). Ostracods can be mistaken for bivalves, but they are small shrimp-like animals encased in two shells. You can often see their legs protruding from the shell.

  1. Lodge all specimens in an internationally recognised specimen collection (e.g. museum) for curation and public accessibility [Recommended].

  2. If all specimens are unable to be lodged at a museum due to lack of resources or the need for destructive analyses (e.g. biochemical extractions), then a voucher collection should be produced (i.e. at least one animal per OTU). This voucher collection can be held temporarily by the agency undertaking the survey if there are other surveys planned in the region to aid in subsequent identification. Ultimately, this voucher collection should be lodged in an internationally recognised specimen collection (e.g. museum).

Data Release

Produce a technical or post-survey report documenting the purpose of the survey, survey design, sampling locations, sampling equipment specifications, and any challenges or limitations encountered (sample template here). Provide links to this report in all associated metadata [Recommended].

Sedimentology

For samples submitted to GA for sedimentological analysis, sedimentology data will be publicly available in the national Marine Sediments collection (MARS, https://portal.ga.gov.au/persona/marine) following lab analysis and QC checks as part of GA’s internal workflow processes. This database includes sediments from estuaries, coasts, shelf, and the deep-sea.

For samples from which sedimentological analysis were done elsewhere, please submit the data to marine@ga.gov.au, along with required metadata. Data may also be published separately (e.g. Carvalho et al 2022), but submitting to the national Marine Sediments collection ensures the data is discoverable and searchable through the AusSeabed data portal.

Biogeochemistry

Submit all geochemical sample metadata and analysis results to GA including:

  • Reduced sediments (Y/N);
  • Total organic matter content (%); and
  • Chl a (ug g-1 dry sediment).

The easiest way to do this is to add two columns to Table 9.3 for LOI and chl-_a _data and submit this to marine@ga.gov.au.

Biology

All biological data should be publicly released, unless circumstances require otherwise (e.g. confidentiality clause or embargo for commercial work). Even in situations when data cannot be shared, the metadata and deployment information should be made available (Steps 1-2 below). Poor scientific data management and lack of data sharing has been shown to hamper scientific progress (Stocks et al. 2016).

Traditionally, data related to biological specimens have been delivered as presence-only taxonomic identifications, thus reducing the applicability of such databases to monitoring purposes. Data are often managed by individual museum scientists or curators and subsequently harvested by the Atlas of Living Australia (ALA). ALA does not yet include absences or information related to sampling effort, although the Extended Data Model project is working to address this.

OBIS is using the data structure described in the project called OBIS-ENV-DATA that allows the linking of species data to other related information (e.g. environmental data, images, sampling effort) (De Pooter et al. 2017). It now has the capacity to store absence records and sampling effort, and is working to include this information in data downloads.

In the meantime, the steps listed below will ensure appropriate and timely release of both metadata and data:

  1. Create a metadata record describing the data collection. Include information about the collection methods used or cite this field manual and other relevant methods. Provide as much detail as possible on the collection/deployment (either directly in the metadata record itself, or in the form of attached field sheets as .csv, .txt or similar). This should include sampling locations and dates, equipment used, level of sorting applied, etc. All collection/deployment information must be QC-d before inclusion.
  2. Publish metadata record(s) to the Australian Ocean Data Network (AODN) catalogue as soon as possible after metadata has gone through the QC process. This can be done in one of two ways:
    • If metadata from your agency is regularly harvested by the AODN, follow agency-specific protocols for metadata and data release.
    • Otherwise, metadata records can be created and submitted via the AODN Data Submission Tool. Note that this tool requires user registration, but this is free and immediate. As of January 2024, this tool is under maintenance, and metadata submissions should be sent to info@aodn.org.au until it is again active.

    This step provides immediate documentation of the methods and location of the collection of biological material. This stage may also include links to field reports or data sheets.

  3. Produce a technical or post-survey report documenting the purpose of the survey, survey design, sampling locations, sampling equipment specifications, and any challenges or limitations encountered (template available here). Provide links to this report in all associated metadata records [Recommended]
  4. Complete the species identifications and associated abundance for targeted groups identified. This can take quite some time, depending on sample size and available resources. It is not unusual for taxonomic identifications to lag years behind survey completion, but this should not delay publication of initial metadata and deployment information. Care must be taken to ensure consistent nomenclature is used and documented for undescribed or unnamed species (e.g. defined Operational Taxonomic Units, OTUs). Ideally catalogues of OTUs are established such that subsequent surveys may use consistent OTU classification, thereby ensuring comparability of data between surveys.
  5. QC the data. This includes checking for spelling errors, missing data, consistent nomenclature and use of OTUs, and confirmation that outliers are not data entry errors (e.g. 100 individuals really were collected, not just 10). Current taxonomic nomenclature can be checked using the World Register of Marine Species (WoRMS) Taxon Match Tool. This process provides accepted scientific name, the scientific authority and full taxonomic breakdown for each taxon.
  6. Technical specialists should also consider whether the data has to be rationalised. This process is often required to remove potential ‘ecological noise’ which may adversely affect the statistical analysis of the data. Examples include juveniles or non-target taxa (e.g. purely pelagic biota such as chaetognaths or ctenophores; or terrestrial biota such as dipterans in grab samples). Newly-settled juveniles are often ephemeral, with high abundance and post-settlement mortality rates, and are therefore not generally representative of prevailing benthic faunal communities (OSPAR Commission 2004).
  7. Additional analysis of data may be required, including classifying trophic levels of taxa, and classifying or characterising habitat types for each sample (e.g. National Intertidal and Subtidal Benthic classification in Mount et al. 2007; Combined Biotope Classification Scheme in Edmunds and Flynn 2018).

  8. Attach or link the full data spreadsheet (including absences and abundances/biomass) to the metadata record previously created and published to the AODN. This will ensure public discoverability and accessibility of the complete data, including absences.

    To then publish data to OBIS, inform OBIS Australia (OBISAU) using the contact details and information on http://www.obis.org.au.

    OBISAU will download the data from AODN or any other site and apply the following procedures.

  • OBISAU provides a taxa matching service using WoRMS web services and will validate the dataset as best as possible.
  • The data is tested for any temporal or spatial outliers.
  • Any observed parameters (biotic and abiotic) are matched where possible to vocabularies maintained by AODN and BODC.
  • Metadata is authored from any existing metadata or publications.

  • Finally the datasets is published via the OBIS Australia data node

    OBISAU has the option to publish the data at the same time directly to GBIF, and it has developed a service to inform ALA that a new dataset is available to be harvested for inclusion into ALA.