Protocols For NRDA Surveys SAMPLING HYDROCARBONS IN ORGANISMS ON
MAN-MADE STRUCTURES

## Introduction

Man-made structures include pilings, rip-rapped shorelines, dikes and
levees, concrete or sheet-pile bulkheads, seawalls, and wharf
structures. These structures are typically hard, present unusual habitat
values, and provide a unique sampling environment. On structures
constructed with concrete, steel, or timber, intertidal "biofouling"
biota is generally relatively sparse. On those constructed of natural
material such as rock (e.g., jetties and breakwaters) with large
interstitial spaces for habitat, the biota can be rich and diverse.

Dock and urban harbor areas are likely to be ground zero for spills and
ultimately may be the focus of cleanup actions. Many structures are also
exposed to contaminants from commercial activities (fuel docks and
harbors), and some are protected from physical deterioration by creosote
and other preservatives.

Structures such as sea walls and pilings that have large vertical
surfaces can be easily accessed by boat and sampled quickly. Also, such
relatively uniform structures are easy to mark for future resampling. In
contrast, riprap slopes are more difficult to sample, but they can
entrap large quantities of oil and other contaminants. Structures such
as jetties can be exposed to high wave energy, compounding sampling
difficulties.

## Sampling Objectives

• To collect tissue samples of living biofouling assemblages on man-made
structures for use in hydrocarbon (HC) analyses.

• Collect samples from structures that exhibit a range of HC
contamination including 1) obviously contaminated, 2) possibly
contaminated, and 3) background or reference.

## Survey Equipment/Guides

• Clipboard, pencils • Camera with macro lens • Field notebook

• Calculator (one per person) • Magic Marker or Sharpie • Plastic label
material

• Chain-of-custody forms • 30 m waterproof measuring tape • 1/16 sq. m
quadrat

• % cover estimator guides • Putty knife or scraper • Forceps

• 1-qt heavy-duty Ziploc bags • Vegetable or toothbrush • 8-oz
pre-cleaned glass jars

• Oven-baked aluminum foil • Ice chest & 10 lbs. of bagged ice •
Decontamination supplies

• Appropriate PPE, incl. disposable rubber gloves

## 

## Sampling Methods

# *General*

Record location, date and time of collection, tide height, and other
appropriate data in your field notebook. Also note landmarks that will
assist in relocating the sampling site, if necessary.

1\. To ensure your notes are complete, provide the following:

> \- **Map:** Sketch a map of the sampled areas, noting distinct
> shoreline features and the sampled structure.
>
> \- **Relative Tidal Zone**: Tidal elevation relative to low, mid,
> high, or supra (above high) tide. Include a descriptor of the
> biological zone (littorine (periwinkle), barnacle, mussel, etc.)
>
> \- **Height of Oiled Assemblages**: Average vertical or slope height
> of the oiled zone.
>
> \- **Length of Oiled Assemblage**: Horizontal distance of the oiled
> zone along the shore parallel to the beach.

-   **Distribution of Oil on Biota**: Average percent cover of oil in
    the zone (nearest 5%).

-   **Fishes Occurring on Pilings**: Report types of fish around
    structures and important activities such as spawning.

-   **Oil Type**: Use SCAT terminology only (fresh oil, mousse,
    tarballs, patties, etc.).

-   **Unusual Weather Conditions:** Rain or snow runoff introducing
    contaminants from street, deck, or pier surface; wave or boat wake
    exposure, proximity to storm water discharge pipes, industrial
    activities adjacent to or on the structure, etc.

-   **Team Member Names and Affiliations**

2\. If possible, permanently mark the sample location by driving a spike
into the structure. Photograph all sampled oiled, reference, and
potentially contaminated assemblages. Take obliquely oriented
photographs of habitat and structure from approximately 30 feet and
nearly normally oriented photos of sampled organisms from 6, 3, and 0.75
feet to provide perspective of exposure. Keep a detailed photo log so
that each photo can be labeled and located as to the oil zone it
represents. Label each roll with a unique code, such as Your
Initials-Abbreviation for Spill-Roll Number (e.g., JM-EV-4).

3\. Once sampling is complete, COC forms for the samples contained in
each ice chest should be placed in a Ziploc bag inside the chest. Seal
ice chest with evidence tape and ship samples and forms to the
analytical lab.

# *Tissue Sample Collection*

Several types of animal tissue samples may be collected from man-made
structures at each site. Typically, the most appropriate organisms on
man-made structures are molluscs (i.e., bivalves or snails). Potential
organisms include species of mussels and other large bivalves, limpets,
periwinkles (littorines), and other snails. Organisms such as barnacles,
generally small and difficult to collect without breakage, are not good
candidates for tissue sampling. Large specimens are most useful because
they require less effort to separate tissue from the shells. Below a
certain size, collection of animals for tissue samples is not feasible.
Suggested minimal sizes are: mussels = 25 mm length; oysters = 20 mm
diameter; limpets = 20 mm length; periwinkles = 15 mm diameter; drills =
30 mm length. Preference hierarchy for sampled organism is as follows,
from most preferred to least preferred:
mussels\>\>oysters\>\>limpets\>\>periwinkles\>\>drills. Avoid: 1)
collecting animals with broken shells as this exposes tissues to
surficial contaminants; and 2) contaminating the surface of the tissue
(foot or mantle) on snails or bivalves.

At each area selected for sampling: 1) Identify 3 sampling sites within
each tidal zone. Each sampling site should be 2 or more meters in width
and less than a meter in elevation. 2) Using forceps or scraper, collect
three 20-gm (\~1 oz.) samples of [TISSUE]{.underline} (not whole weight)
from each sampling site in each zone. Each sample should consist of
several organisms. For most organisms, at least 5 specimens are needed
to obtain the target weight. Samples of limpets and periwinkles may
require 20-30 individuals. 3) Clean the exterior of the shell with a
brush and paper towel to remove gross HC contamination and sediment. For
mussels, remove the byssal mass (beard), which may also be contaminated.
4) Place cleaned and drained specimens on a sheet of aluminum foil large
enough to completely wrap the sample twice and fold edges in a manner
that excludes fluids from entering or draining from the package. Write
sample ID, location, and sampling data on the package with a Magic
Marker. Fill out a similar label and insert the label and sample package
in a similarly labeled glass jar or Ziploc bag.

# *Substrate Considerations and Collection*

The NOAA National Status and Trends Program sampling protocols
specifically exclude bivalve collection from buoys and preserved wooden
structures due to potential PAH contamination from creosote and other
preservatives (NOAA 1993). In instances where no other choices for
collection of biofouling organisms are available, samples of creosote
seeps on pilings should be collected for chemical comparisons from well
above the splash zone. If that is not possible, samples of wood
splinters (again well above the splash zone or obvious oil
contamination) should be removed from the pilings for chemical analyses.
As little as 1 or 2 drops of creosote oil (\< 1 g) will suffice for neat
creosote oil characterization, while several splinters (constituting
20-30 g) of piling substrate (wood) should be collected if possible.
Distribution of alkylated PAH homologues from creosote is dominated by
C~0~\>C~1~\>C~2~\>C~3~ substituted components, as opposed to
C~0~\<C~1~\<C~2~\<C~3~ in most crude oils (Bence and Burns 1995 and
Stout et al. 1998), so detailed chemical analyses should be able to
differentiate between PAH contamination from the spill and background
PAH associated with the substrate. Likewise, if asphalt is present in
riprap, a 2-3cm^2^ piece should be included for background analyses.
Prepare, label, and pack samples as described above.

# *Sample Preservation and Storage*

Samples should be shipped immediately via overnight courier. Include a
generous quantity of ice or frozen blue-ice packs in the cooler. If
samples cannot be shipped until the day after they are collected, they
should be stored in custody-sealed coolers in a cold room or freezer at
a local university or laboratory (a local supermarket or meat locker may
be used in emergencies). If samples will be shipped more than 36 hours
after collection, they should be frozen as soon as possible after
collection. Do not use dry ice because courier requirements inevitably
delay sample shipments up to several days.

## Key References

Bence, A.E. and W.A. Burns. 1995. Fingerprinting Hydrocarbons in the
Biological Resources of the Exxon Valdez Spill Area. Exxon Valdez Oil
Spill: Fate and Effects in Alaskan Waters, ASTM STP 1219, Peter G.
Wells, James N. Butler, and Jane S. Hughes, Eds., American Society for
Testing and Materials, Philadelphia p. 84-140.

NOAA. 1993. Sampling and Analytical Methods of the National Status and
Trends Program. 1984-1992. Volume 1 -- Overview and Summary of Methods.
NOAA Technical Memorandum NOS ORCA 71.

NOAA. 2000. Shoreline Assessment Manual, Third Edition. HAZMAT Report
No. 2000-1, Hazardous Materials Response and Assessment Division, NOAA,
Seattle, WA. 57 pp. + appendices.

Stout, S.A., A.D. Uhler, and K.J. McCarthy. \"PAH Can Provide a Unique
Forensic Fingerprint for Hydrocarbons Products,\" Environmental
Forensics, Battelle Memorial Institute, October 1998.