**Number range CHAPTER**

**SAMPLING and ASSESSMENT GUIDANCE**

[(From NOAA\'s Emergency Guidance Manual, Version 1.1, July
1994)]{.smallcaps}

**SAMPLING PROTOCOLS AND STRATEGIES**

**INTRODUCTION**

Information regarding the collection of perishable data has been divided
into two sections: chemical analyses and biological resources. Accepted
methodologies for collection of perishable data from the spill source
and water/sediment contamination are presented in the first section,
while quantifying the impacts to wildlife and habitats are detailed in
the next section.

Prior to collecting samples, the NRDA responder must have knowledge of
the proper procedures required for collecting litigation-quality data.
These requirements include:

1\) Developing Sample Collection Strategies

\- Clearly stated objectives

\- Hypotheses

\- Endpoints

\- Sample design

\- Controls

\- Number of samples

2\) Documentation and Reporting Requirements

\- Quality assurance and quality control protocols

\- Sampling equipment and sample containers \"clean" requirements

\- Sample identification and labeling

\- Record keeping

These topics are discussed in detail in the following section. The NRDA
responder should become intimately familiar with these topics as they
define the very structure of the damage assessment response.

**DEVELOPING SAMPLE COLLECTION STRATEGIES**

During a damage assessment response, sample collection strategies and
procedures should be thoroughly defined and documented. Scientific
methodologies that require careful documentation include outlining the
pre-study conditions for analysis. The *a priori* development of formal
objectives, hypotheses, analyses, and endpoints is needed to adequately
outline the course of study.

**Objectives**

Stating a clear objective often seems so obvious that it is not
explicitly mentioned by people considering sampling projects. The early
sampling strategy should include a clear statement of the objectives.
These objectives will help to define how the sample should be collected.
For example, if the objective for collecting a source sample is to have
a reference sample for finger-printing purposes, a smaller sample will
be needed than if the objective is to collect source oil for later
bioassay experiments. During an NRDA response, the objectives are best
defined by asking the following questions:

1\) What questions are to be asked regarding the suspected injuries?

2\) What information and data are needed to evaluate the suspected
injuries?

Often, the first priority of an NRDA response is to survey the extent of
contamination and then to determine the potential for injury to natural
resources and their services. The early NRDA data will ultimately be
used to estimate injuries and loss of services and to focus the later
assessment activities. Thus, it is important that the NRDA responder
develop and follow clearly stated objectives in order to accurately
assess the extent of injury during a spill incident.

**Hypotheses**

A hypothesis is merely a restatement of the objective in statistical
language that can later be subjected to a statistical test, where the
hypothesis will be rejected or not rejected, depending on the data
results. Experiments having several components may have several
different hypotheses.

The development of a formal sampling procedure should also include the
development of a null hypothesis. Daniel (1983) defined a null
hypothesis as follows:

> \"In general, the null hypothesis is set up for the express purpose of
> being discredited. Consequently, the complement of the conclusion that
> the researcher is seeking to reach becomes the statement of the null
> hypothesis. In the testing process, the null hypothesis either is
> rejected or is not rejected. If the null hypothesis is not rejected,
> we will say that the data on which the test is based do not provide
> sufficient evidence to cause rejection. If the testing procedure leads
> to the rejection of the null hypothesis, we will conclude that the
> data at hand are not compatible with the null hypothesis, but are
> supportive of an alternative hypothesis. When we fail to reject a null
> hypothesis, we do not say that it is true, but that it may be true.
> When we accept a null hypothesis, we do not say that it is true, just
> that the evidence supports this hypothesis."

Formulating an explicit hypothesis will also necessitate selecting the
endpoint to be used for determining whether the hypothesis is accepted
or rejected.

**Endpoints**

Endpoints are specific measures of the objectives of the experiment, and
they determine how parameters such as effectiveness, toxicity, or
similarity will be measured. Ideally, endpoints should be a measurable
quantity, such as, \"the concentration of PAH in tissue, measured as dry
weight," or, \"the abundance of selected species counted in intertidal
quadrats." Endpoints may include qualitative data, but the type of data
and the way they will be used should be specified as closely as
possible. As part of establishing endpoints, proper sampling procedures
should be followed and documented to ensure that the samples are
collected and handled appropriately.

Two major problems may result from the failure to select an endpoint for
an experiment:

1\) The data collected may not have been handled appropriately for the
analyses that are needed; and

2\) The type of data or the way they were collected may not be
appropriate to answer the original objective.

Specific selection of endpoints is also very important when several
groups are involved in an experiment, as is frequently the case with
NRDA studies. At the conclusion of the project, it may be found that the
data collected do not answer the question posed. Or, when several
different kinds of data are collected, disagreement may ensue among the
different groups involved over what constitutes the \"real" endpoint,
and thus the \"real" result.

**Sampling Design**

A sample design includes consideration of control (reference) sites, the
number of samples to be collected, the locations where samples will be
collected, the timing of sampling, and sample handling and analysis. For
monitoring or sampling projects other than very simple ones, the best
procedure will be to consult with a statistician while still in the
planning stage. More detailed habitat and resource-specific sampling
design guidelines are developed in later sections.

**Reference Sites.** A crucial part of most experiments is the reference
site. Most objectives will involve detecting differences, or making
comparisons, and thus require a baseline, or reference from which to
measure change. In the absence of some sort of reference, interpreting
the data will be difficult. In theory, reference sites should be
replicates of the tested or experimental site, which are the same in
every way, except for the treatment applied.

In practice, reference sites in field studies are never completely
identical to the test sites. Usually, one must settle for a
\"representative control" that is as similar or as representative as is
feasible. Other options include the use of experimental units placed in
the environment, such as sediment boxes. This technique was used by
Berge (1990) to study colonization of biota to sediments impacted by
oil, compared with unoiled sediments.

One way to establish controls to differentiate or quantify the effects
of the cleanup is by the use of \"set-asides" (areas that are affected
by a spill that are set aside, and left untreated for experimental
purposes). NOAA arranged for such \"set asides" after the *Exxon Valdez*
spill. Having these sites made it possible to conduct the long-term
study of treatment effects in Prince William Sound that is still ongoing
(Houghton *et al*., 1991a; 1992). For damage assessments, however, there
is seldom the need for separating out oil and cleanup injuries; both are
resulting from the spill and both may be included in the injury
determination.

**Number of Samples.** The number of samples to be collected will depend
on:

o The hypothesis being tested;

o Data analysis methods (including the statistical certainty desired);
and

o Practical considerations (area available, access, personnel, cost,
etc.).

A larger number of samples often allows greater power for statistical
analysis, but will be more costly. If the parameter being measured is
highly variable, such as the distribution of oil in sediments, a large
number of samples will be needed to increase the power necessary to
conduct statistical testing. A small number of samples will provide an
indication of the processes occurring, but may not be representative of
the entire study area or adequate to statistically differentiate among
sites. One strategy is to collect a large number of samples in the field
and then analyze only a subset of the samples collected, based on the
early results. However, it is more useful to take fewer carefully
thought out samples than to take numerous samples without proper
planning.

The number of replicate samples to be collected often depends upon the
information to be gained from the samples. During the early sampling
effort, more replicate samples may be needed because of large variations
and patchiness in distribution of the contaminants in the environment.
Sample preservation and holding times must also be considered.
Guidelines on how to define the sampling strategy and determine the
necessary number of samples for chemical and biological sample
collection schemes are included in later sections.

**[QUALITY ASSURANCE AND QUALITY CONTROL PROTOCOLS]{.smallcaps}**

To produce data that will stand up in a court room, it is necessary, at
a minimum, to be able to characterize the quality of the data. If time
permits, it is desirable to specify data quality objectives (DQOs) that
meet the needs of the project, and to establish a quality assurance
strategy for meeting the DQOs (see, for example, Preparing Perfect
Project Plans, EPA, 1989). The following section provides basic
instructions and ideas for meeting only the most fundamental quality
assurance needs during a spill response situation. The responder should
understand these instructions, and apply them to each specific spill and
study. If the basic considerations outlined below cannot be addressed
for any given observation, survey, or study, the NRDA responder should
seriously consider whether the data generated will have any value at all
for a damage assessment.

**Data Quality**

EPA recognizes five measures of data quality:

1\) [Accuracy or bias]{.underline} - the difference between a measured
value and the true or expected value.

2\) [Precision]{.underline} - the degree of mutual agreement among
repeated measurements of the same value or parameter.

3\) [Representativeness]{.underline} - the degree to which data
accurately and precisely represent the characteristic of a population
parameter, variation of a property, a process characteristic, or an
operational condition.

4\) [Completeness]{.underline} - a measure of the amount of data
collected from a measurement process compared to the amount expected to
be obtained under the conditions of measurement.

5\) [Comparability]{.underline} - the confidence with which one data set
can be compared to another.

Under emergency conditions, when time and resources are short, it is
recommended that the responder focus on determining and documenting
accuracy and precision. Listed below are representative types of data
and observations that may be needed in a spill event, with some
recommendations on how to control and document data accuracy and
precision:

**[Counts]{.underline}** - It may be necessary to count carcasses, to
determine densities of live organisms, or to quantify other parameters
through counting.

> Accuracy can be checked in some cases by verifying counts with a more
> reliable methodology. For example, an aerial photograph could be
> closely examined to determine whether aerial counting techniques or
> ground observations were accurate. Another technique for checking
> accuracy might be to mark the objects counted by one person using a
> routine survey technique, and then to check the same area with a
> second more intense counting technique to determine whether all
> objects have been counted and marked. It is recommended that 10
> percent of counts be checked for accuracy.
>
> Precision can be checked by counting the same objects in the same area
> using the same technique. It is recommended that precision be checked
> by a second counter for 10 percent of the counts.

**[Characterizations]{.underline}** - A variety of characterizations may
be encountered during a spill damage assessment. A characterization
occurs when an observer places a situation or observation into a
category. Examples of characterizations include mapping the degree of
oiling on a beach, taxonomic identifications, and naming a
histopathological condition.

> Accuracy may be determined by archiving a standard representation of
> the category and submitting the standard representation for
> independent evaluation. For example, the standard representation could
> be a picture or schematic used for determining level of oiling, or
> voucher specimens for species identifications and histological
> conditions. Each standard or voucher specimen should be evaluated or
> identified by an independent expert to determine whether the
> characterization was correct.
>
> Precision may be evaluated by conducting replicate characterizations
> by the same observer and/or different observers. The degree of
> agreement among replicate characterizations would represent the level
> of precision associated with the data. It is recommended that 10
> percent of the characterizations be checked for precision.

**[Measurements]{.underline}** - A number of parameters might be
measured during a spill damage assessment. Typically, measurements use
equipment to generate a numerical characterization. For example, the
weight or size of an organism, or chemical concentrations might need to
be measured.

> Physical Measurements-
>
> Accuracy may be determined by regular calibration of the measurement
> equipment using standards (e.g., standard weights). All calibration
> measurements should be recorded. The frequency of calibration depends
> on the type of equipment; it is better to do too many calibration
> checks than not enough.
>
> Precision may be determined by repeating the same measurement more
> than once. It is recommended that 10 percent of the measurements be
> replicated to allow evaluation of precision.
>
> Chemical Measurements-
>
> Laboratories with demonstrated capabilities and recent successful
> experience with identical or similar analyses and sample matrices
> should be used. Analytical laboratories should be willing to provide
> their Quality Assurance Plan and related documentation.
>
> Accuracy may be checked using various calibration standards, reference
> materials, laboratory spikes, and blanks. It is recommended that a
> minimum of one reference material and/or other standard be run with
> each batch of analyses. It is generally recommended that one of the
> standards employ the same matrix as that being analyzed for the study.
>
> Precision may be evaluated using replicated analyses of the same
> sample (assuming that it is homogeneous). One sample per analytical
> batch should be split and run as a laboratory duplicate. Field
> duplicates show variations that result from the combined effects of
> both the sampling and analytical procedures. It is recommended that
> 5-10 percent of the samples be submitted as field duplicates.
>
> Both accuracy and precision deteriorate as the method detection limit
> is approached. The responder should know the method of detection limit
> to ensure that the analytical technique is appropriate to the study.

Duplicate counts, calibrations, laboratory replicates, verification by
independent experts, and other types of data that allow an evaluation of
data quality should be included in field notes and data tables so that
an independent evaluation of the data quality may be performed.
Therefore, the responder should take appropriate steps to ensure that
such data are recorded and reported.

**Record keeping and Documentation**

Standard operating procedures for all sampling procedures must be
written in detailed, clear, simple, and easy-to-follow language. The
field team must be knowledgeable and experienced in the prescribed
procedures. Any changes in procedures, including the justification for
the changes, must be recorded in detail in spill-specific logbooks.
These logbooks should be sturdy, bound books with sequentially numbered
pages. Waterproof paper is preferred for field logbooks.

All information pertinent to field activities should be recorded in the
field logbook. The information should be accurate, objective, and
complete. The descriptions should be complete enough to allow anyone
reading the entries to reconstruct the sampling situation. Additional
information should be provided by field data sheets and photographs,
both of which should be referred to in entries in the field logbook.
Sample tags are NOT appropriate forms for recording important field data
unless the information written on each label is transferred to data
sheets or the logbook at the end of each day. There is no guarantee that
the information recorded on the sample tag will be recorded by the
laboratory or made available to the sample collector.

The entries for each day should be closed out with a horizontal line,
dated, and initialed. Errors in entries should be corrected by drawing a
single line through the error, entering the correct information, and
signing and dating the correction. No entry should be erased. Completed
logbooks and field data sheets should be kept under chain-of-custody
procedures until the trustees release them.

As the logbook accompanies the responder in the field and may be lost or
damaged, photocopies of all entries and forms should be made on a daily
basis during the response. The copies should be retained in a secure
place, well removed from the scene (e.g., hotel room) in order to
prevent the loss of information.

**\"Clean" Requirements for Sampling Equipment and Sample Containers**

Because of the trace levels of contaminants in environmental samples, it
is very easy to contaminate a sample unless stringent precautions are
taken. Cross-contamination of samples at spills is of particular concern
because of the wide range in contamination levels_for example, sampling
below oil slicks to collect water samples at parts per billion (ppb)
levels. Furthermore, detailed chemical characterization of the sample
may be needed to identify the source of the contamination, and even
minute amounts of other petroleum products can invalidate the results.

For sample containers:

1\) All sample containers must be either organic-free (solvent-rinsed)
glass or organic-free (solvent-rinsed) aluminum foil.

2\) Lids for glass containers must be lined with either Teflon or
solvent-rinsed aluminum foil.

In most cases, certified-clean sample containers will be available in
sampling kits for use during spill responses. However, in the event that
containers or sampling equipment have to be cleaned in the field, the
following procedures should be followed:

**[First Rinse:]{.smallcaps}** Wash with soap and hot water, rinse with
plenty of warm, distilled water.

**[Second Rinse:]{.smallcaps}** Rinse with methylene chloride or acetone
(solvents)**\*.**

**[Third Rinse:]{.smallcaps}** (if acetone is used) Pentane or
hexane**\*.**

**New Glass Jars or Unused Aluminum Foil:**

> o Do not require first soap and water rinse.
>
> o Require solvent-rinsing.
>
> o Dull side of aluminum foil are solvent-rinsed and stored with clean
> sides folded together.
>
> o Aluminum foil can also be heated at 450_C for over one hour.
>
> o Glass jars are stored tightly capped with either solvent-rinsed
> aluminum foil or Teflon-lined caps.

**Glass Jars:**

> o May be cleaned by heating to 450_C for a minimum of one hour.
>
> o Glass jars are stored tightly capped with either solvent-rinsed
> aluminum foil or Teflon-lined caps.

**Sample Collection Equipment:**

> o Requires solvent-rinsing before collecting each sample.
>
> o Steam-cleaning or a soap wash and hot water rinse is required at the
> end of each day or between sampling locations.

Waste solvents used in the field must be collected (in a waste solvent
jar) and disposed of properly. One alternative is to arrange for sending
the waste solvent to the analytical laboratory with the sample shipment,
for proper disposal.

**\*** *All solvents (methylene chloride, acetone, pentane, and hexane)
used for cleaning **MUST** be of appropriate quality for trace organic
residue analysis and be stored in glass or Teflon containers, **NOT**
plastic. Proper solvent collection and disposal after use is required.*

**Sample Identification and Labeling**

Sample identification and labeling is extremely important, since many
other researchers may be relying upon samples collected during the early
phases for a wide range of purposes. Also, only a subset of the samples
from a site may be analyzed initially, so many samples may be stored for
later study. If multiple teams are collecting samples or establishing
stations, it is extremely important that a numerical coding scheme is
planned ahead of time. Each sample and station must have a unique
identification number.

Recording detailed information, such as the sample location, sampling
equipment used, and other conditions is not essential for sample tags,
but is recommended. Sample containers are discarded at the analytical
laboratories, therefore, the information recorded on sample containers
is not always retrievable. However, for samples that are held over long
periods, sample details on the sample tag may be helpful to identify a
sample or provide a source of missing data.

**Maintenance of Chain-of-Custody**

(see Appendix R)

All collected samples must be maintained under the custody protocol
identified in Appendix R. Each person who accepts custody of a sample is
responsible for maintaining the integrity of that sample. QA/QC
protocols officially define a sample as being in someone\*s custody if:

o the sample is in one\*s actual physical possession or view;

o the sample is retained in a secured place (under lock) with restricted
access, or;

o it is placed in a container and secured with an official seal(s) such
that the sample cannot be reached without breaking the seal.

The field sampler is personally responsible for the care and custody of
the samples collected initially until they are transferred under
chain-of-custody documentation procedures. When samples are transferred
from one individual\*s custody to another\*s, the individuals
relinquishing and receiving the samples need to sign and date the
chain-of-custody form. The original form remains with the samples;
copies may be kept by each custodian, as desired. During shipping, the
original chain-of-custody form is included in the shipment; a copy is
retained by the shipper.

**TYPES OF SAMPLES TO COLLECT**

**Spill Source**

Samples of the known source should be collected in large volumes (one
gallon or more). There is always a great demand for samples of the
source, even years after the spill. It is very important to confirm the
origin and handling of samples of the \"source material" so it\*s
appropriateness for use can be evaluated. A sample of the known spill
source is requested for fingerprinting and weathering analyses so that
future monitoring studies will have the chemical characterization and
documentation for identifying the spilled oil over time. Usually the
USCG or spiller\*s representative can provide a source sample. But, be
aware that a source sample could have been collected from floating
slicks, recovery vessels, storage tanks, etc., and may have been
contaminated or changed by weathering. Temporary storage facilities
usually have not been cleaned prior to use, so they most likely are
contaminated with other materials. Get written documentation of the
sample collection and storage history copies of chain-of-custody forms
or signed statements from the collector and custodian. The source sample
can be evidence vital to the claim, and it is imperative to have
complete and valid documentation of the validity of the sample. Source
samples should be properly preserved and turned over to a laboratory as
soon as possible. Table 1 lists sample volume and storage requirements
for different types of samples.

**Environmental Samples for Fingerprinting**

Samples for fingerprinting are collected to answer the question \"Is
this the same material that was spilled?", thus they do not have to be
quantified, or related to a measured amount of sample. When collecting
oil samples for fingerprinting, thick surface slicks are scooped up away
from any other petroleum sources, and quickly transferred into the
pre-cleaned glass containers and labeled. Thin sheens can be collected
with a special sheen sampler composed of Teflon strips on a line which
is pulled through the sheens, then stored in a glass jar. Samples for
fingerprinting can also include tarballs, oiled sediments, oiled animal
parts (feathers preferably), and oiled vegetation. Approximately 100 ml
of the oil is needed for accurate fingerprinting analyses. All samples
should be kept cold or preferably frozen, even in transit, until
delivered to the analytical lab.

**Oil Analysis Quantity Storage Other Conditions**

Fingerprinting \~ 1 gallon Pre-cleaned glass Kept cold or frozen until

source samples containers delivered to the lab.

Weathering \~ 1 liter Pre-cleaned glass Kept cold or frozen until

source samples containers delivered to the lab.

Water Samples \~ 1 gallon Super-clean glass Kept cold and dark for

containers less than 8 hours. Preser-

vation of sample may be

done_contact analytical

lab for preservation

requirements.

Sediment 100 gm Pre-cleaned glass jars Extreme care should be

Samples, Finger- taken to prevent sample

printing contamination. Kept cold

or frozen until delivered

to the lab.

Sediment 100 gm Pre-cleaned glass jars Composite subsamples,

Samples, Total 10-15 samples from site.

Petroleum Kept cold or frozen until

Hydrocarbons delivered to the lab.

Sediment 50-100 gm Ziploc or whirl-pak No special conditions

Samples, Grain bags required.

Size Analysis

**Water Samples**

Water samples are very difficult to collect, especially where surface
oil slicks are present. When sampling in areas covered by a surface
slick, extreme care is needed to \"knock" the surface slick aside so
that the sampling device can be lowered into the water without becoming
contaminated. The depth at which the sample is taken should be recorded
and should be consistent among samples. If there is concern that the
pollutant may be stratified, samples should be taken at several depth
intervals. This is especially important in areas with salinity or
temperature gradients.

At oil spills, water samples should be collected directly in the sample
container, since oil droplets readily adhere to the inside of samplers.
Ideally, water samples should be stored no longer than eight hours,
although, if they are kept cold and dark, water samples may be stored
for as long as five days. Background contamination samples should be
collected well away from any oiled areas, being careful not to sample
along the vessel path or near any boat or aircraft engines, to reduce
the chance of contamination. As petroleum products and other organic
chemicals will readily adhere to glass, several preservatives (methylene
chloride, freon 116) may be added to the sample to begin the extraction
process. Contact the analytical laboratory to confirm that this
preservation technique can be accommodated by their analytical
capabilities.

**Sediment Samples**

Sediment samples are collected differently for different analyses. The
responder must understand why the sample is required and the analysis to
be conducted. In particular, the responder should determine whether
quantitative or qualitative information is necessary. Routine scrape
samples collected for fingerprinting, for example, may provide the
responder with information on the type and degree of weathering of the
oil, but generally will not provide an estimate of the concentration of
the contaminant in sample matrix. In order to obtain estimates of
concentration, the sampling objectives need to be carefully defined, and
the volume of the sample need to be documented.

There are three sediment samples typically required for an NRDA,
including: samples for fingerprinting; samples for determining the total
petroleum hydrocarbon (TPH) contamination; and grain-size analysis to
assist in the determination of the likely extent of injury. For all
sediment sample needs, approximately 100-200 grams (approximately one
cup) of oiled sediment samples of surface and/or subsurface oil
contamination are usually collected. In gravel areas, surface samples
may be individual clasts placed into the sample jars for analysis,
usually for weathering characterization. In non-gravel areas, the
surface samples usually come from the top 2 cm of sediment (generally
the layer of sediment most likely to be exposed) unless a specific
sampling need warrents deeper samples. Subsurface samples from the
intertidal zone are collected from discrete, well-defined intervals,
usually from the sides of trenches dug into the beach. Core samples can
also be used, where the core sample is divided into discrete layers and
subsampled by layer to determine concentrations by depth. Detailed
record-keepign on the depth and volume of the sample is necessary if the
goal of the sampling is to determine contaminant concentration.

Subtidal sediments are usually collected from a larger box corer. The
sampling interval should be specified in the sampling plan. All sediment
samples should be photographed prior to collection from the trench or
sediment core. All samples should be numbered uniquely; it is important
to have pre-assigned numbering series when multiple teams are in the
field collecting samples. Samples can be collected with clean metal
utensils or wooden tongue depressors and placed in pre-cleaned glass
jars with Teflon-lined caps, taking care to prevent contamination with
other sources of petroleum. For sediment samples used for fingerprinting
of TPH analysis, all samples should be kept frozen, even during transit,
until delivered to the analytical lab. The samples collected for
grain-size analysis require no special handling/storage conditions.

**Shipping Guidelines for Samples and Other Chemicals**

There are two kinds of shipping issues at spills: 1) shipment of
pollutant samples (and exposed water, sediment, and biota) to the
laboratory; and 2) sampling chemicals (e.g., cleaning solvents and
preservatives) to and from the field. Sediment, oil, and biological
samples can be transported by most carriers. Some are reluctant to
transport samples with dry ice, so blue ice or freezer packs may be
needed. For most conditions, the amount of oil/chemical in environmental
samples is below those classified as hazardous under shipping
regulations. However, when shipping a sample of the product, the shipper
should be checked for any volume restrictions.

There are many restrictions on the shipment of chemicals used in
sampling to/from a spill. The United Parcel Service (UPS), Federal
Express, and the U.S. Postal Service Express Mail.

To maintain sample integrity during shipping, the following
recommendations should be followed:

1\) Small (0.5 liter or less) glass jars should be shipped in their
original boxes or individually wrapped in bubble-wrap.

2\) Large (\> 1 liter) glass jars containing liquids need to be shipped
in Styrofoam holders to prevent breakage.

3\) There are several types of disposable, inexpensive shipping
containers on the market which meet shipping requirements. These should
be part of a standard response kit.

4\) Always contact the recipient with the shipment details.

5\) Send the original chain-of-custody forms with samples.

6\) Keep a copy of the packing list and chain-of-custody forms for your
files.

**EARLY SAMPLING METHODS FOR ASSESSMENT**

**OF INJURY TO NATURAL RESOURCES**

This section provides the NRDA responder with guidelines for conducting
early assessments on natural resources, although governmental or
contracted experts will likely be called upon to design and conduct
these surveys. The NRDA responder is not expected to be an \"expert" in
the identification and analysis of all natural resources and services
that may be impacted during a spill. However, the NRDA responder may be
required to coordinate assessment efforts and should be knowledgeable of
methods used and information gained during these studies, to assure that
the studies are conducted in a manner which will meet the overall NRDA
objectives.

This section outlines the type of information needed and early survey
methodologies for a variety of natural resources that may be impacted as
a result of a spill. Some alteration of the methodologies will likely be
necessary for each specific spill. It should be noted that NOAA is
encouraging the establishment of approved protocols for sampling and
survey methods, as part of other pre-planning activities for NRDA. If
such protocols are available, they should be followed instead of those
presented here. It is important that the actual methods used be
documented.

Guidelines for twelve natural resources are presented here. Studies of
past spills have indicated that these resources are susceptible to
spills, and may therefore be of concern during future spills. The reader
should keep in mind that these guidelines are not comprehensive and are
designed only to be a starting point for the NRDA. If injuries are
suspected to these categories of natural resources, the responder should
consult with experts on the specific resources. The resources addressed
in this manual include:

+-----------------------------------+-----------------------------------+
| > 1\. Non-vegetated Shorelines    | > 7\. Fish                        |
+-----------------------------------+-----------------------------------+
| > 2\. Marshes                     | > 8\. Shellfish                   |
+-----------------------------------+-----------------------------------+
| > 3\. Mangroves                   | > 9\. Turtles and Other Reptiles  |
+-----------------------------------+-----------------------------------+
| > 4\. Seagrasses                  | > 10\. Birds                      |
+-----------------------------------+-----------------------------------+
| > 5\. Corals                      | > 11\. Marine Mammals             |
+-----------------------------------+-----------------------------------+
| > 6\. Intertidal Communities      | > 12\. Terrestrial Mammals        |
+-----------------------------------+-----------------------------------+

For each resource, the known impacts for each resource based on
historical spills are summarized, and potential methods for documenting
impacts or injuries are outlined.

**ASSESSMENT OF INJURY TO NON-VEGETATED SHORELINES**

**Beaches**

A beach is an accumulation of unconsolidated sediment that is
transported and molded into characteristic forms by wave-generated water
motion. Beaches will form virtually anywhere sediment is available and
there is a site for sediment accumulation. Inherent to beaches is
change, over time scales ranging from seconds to years. Knowing the
patterns of change allows prediction of the behavior of oil spills and
the persistence of oiled sediments.

Coastal sediments are classified according to the dominant size of the
individual clasts into three general categories: mud (silt and clay),
sand, and gravel (from granule to boulder). The variation between the
different beach classifications is related to the grain size of the
sediments composing the beach. Wentworth (1922) developed the following
classification system:

**[Size Description]{.underline} [Size Range]{.underline}**

clay 0.0002-0.004 mm

silt 0.004-0.0625 mm

fine-grained sand 0.0625-0.25 mm

coarse-grained sand 0.25 - 2 mm

granule 2 - 4 mm

pebble 4 - 64 mm

cobble 64 - 256 mm

boulder \> 256 mm

There is a general relationship between the sediment size and the slope
of the beachface_the coarser the grain size, the steeper the beachface.
The beach slope is also controlled by wave activity in that eroding
beaches tend to flatten and accreting beach steepen. Knowledge of the
sediment transport patterns for beaches (sand in particular) is
important for understanding how oil behaves on them.

**Summary of Known Impacts from Oil Spills**

From the perspective of oil behavior on beaches, there are two basic
factors:

1\) The depth of oil penetration into the sediments, and

2\) The potential for burial of oiled layers by clean sediments.

The behavior of oil on beach type (fine- and coarse-grained sand
beaches, gravel beaches, and mixed sand and gravel beaches) is
summarized below.

**[Exposed Beaches, Fine-Grained Sand]{.smallcaps}**

o During small spills, oil will concentrate in a band along the
high-tide line.

o Under heavy accumulations, oil can cover the entire intertidal area,
although the oil will be lifted off the lower part of the beach with the
rising tide.

o Oil penetration into fine-grained sand will be less than 10 cm.

o Burial of oiled layers by clean sand within the first few weeks after
the spill will be limited usually to less than 30 cm along the upper
beachface.

o Deeper burial is possible if the oil is deposited at the beginning of
an accretionary period.

o Much of the oil will be removed during the next storm.

o Biological impacts include temporary declines in infaunal populations,
which can also affect feeding shorebirds.

o The usually hard, compact sediments will support pedestrian and
vehicular traffic.

> **[Exposed Beaches, Coarse-Grained Sand]{.smallcaps}**

o During small spills, oil will concentrate in a band along the
high-tide line.

o Under heavy accumulations, oil can cover the entire intertidal zone,
although the oil will be lifted off the lower part of the beach with the
rising tide.

o Large amounts of oil can accumulate in the berm runnel where it is
unable to drain off the beach at low tide.

o Oil penetration into coarse-grained sand can reach 25 cm.

o Burial of oiled layers by clean sand within the first few weeks after
the spill can be rapid, and up to 60 cm or more.

o Burial over 1 m is possible if the oil is deposited at the beginning
of an accretionary period.

o Persistence of deeply buried oil could be long, depending upon the
season of year and beach cycle.

o Biological impacts include temporary declines in infaunal populations,
which can also affect feeding shorebirds.

o The sediment can be very soft, making vehicular access difficult.

> **[Sheltered Beaches, Both Fine- and Coarse-Grained
> Sand]{.smallcaps}**

o Oil penetration will be less where the sediments are finer and more
poorly sorted.

o Depending on the degree of exposure to any waves, oil persistence can
increase to months or years.

o A moderately rich biological community can be supported.

o Burial by clean sand is still significant but less than exposed
beaches.

o Asphalt pavements can form under heavy accumulations; pavements will
change the nature and stability of the substrate and thus its biological
utilization.

> **[Exposed Beaches, Mixed Sand and Gravel]{.smallcaps}**

o During small spills, oil will be deposited along and above the
high-tide swash.

o Large spills will spread across the entire intertidal area.

o Oil penetration into the beach sediments may be up to 50 cm.

o Burial of oil may be deep at and above the high-tide berm, where oil
tends to persist.

o Oil can be stranded on low-tide terraces composed of gravel,
particularly if the oil is weathered or emulsified.

> **[Sheltered Beaches, Mixed Sand and Gravel]{.smallcaps}**

o Pavements are likely to form wherever heavy accumulations of oil can
fill the voids between the sediments.

o Once formed, these pavements are very stable and can persist for
years.

o Any oil stranded above the high-tide line will be highly persistent.

**Methods for Assessment of Impact to Beaches**

Hayes *et al*. (1973) developed a systematic sampling program for
rapidly classifying large sections of coastline in Alaska. This method,
called the [zonal method]{.underline}, has been modified for application
in the assessment of the extent and persistence of stranded oil at oil
spills. There are six steps for documenting the impact of a spill to
beaches:

> 1\) Collect and study available literature, aerial photographs, maps,
> and charts.
>
> 2\) Conduct overflights to map the extent and degree of shoreline
> contamination.
>
> 3\) Select specific areas for detailed study, based on the aerial
> survey, local maps, and oil distribution. The sampling interval
> depends on the variety of coastal types, as well as logistical and
> financial considerations. Areas selected are representative of the
> local geomorphology, habitat type, and oil distribution.
>
> 4\) Set up two types of stations for groundtruthing the aerial survey
> observations: (a) visual inspections stations and (b) zonal stations.
> These station types are outlined below. Figure 6 is a checklist of
> equipment to be used for shoreline surveys.
>
> 5\) Categorize the coast into geomorphological types onto base maps
> and superimpose the oil distribution. Oil distribution may be
> categorized as follows\*:
>
> Very light - \<5 percent coverage (of the intertidal zone)
>
> Light coverage - 25 percent coverage
>
> Moderate coverage - 25-65 percent coverage
>
> Heavy coverage - \>65 percent coverage
>
> 6\) Follow-up surveys and overflights are necessary to determine oil
> behavior and persistence, cleanup effectiveness, and geomorphic
> changes.
>
> \* These boundaries and terms (i.e., very light, light, moderate,
> heavy) are frequently in dispute at a spill. The measures need to be
> standardized for the spill conditions.

o Cameras (SLR, 35 mm, and video cameras)

o Two profile rods (1.5 m long, marked at 2 cm intervals)

> o Day pack, which contains:
>
> o plastic sediment sample bags o survey tapes (30 m, in cm)
>
> o photo scales o film
>
> o hand level o Rite-in-the-Rain field
>
> o notebook
>
> o compass o orange surveyor\*s marking
>
> tape and orange spray paint
>
> o hand lens o a tool for scraping sides of
>
> trenches (e.g., plasterer\*s
>
> trowel)
>
> o Plastic, box-type clip board, which contains:
>
> o profile sheets o sketch sheets
>
> o gravel grain-size comparator o percentage estimate chart

o sand grain-size comparator o plastic one-foot rule (in cm)

> o extra lead pencils with erasers o magic markers
>
> o sample-labeling tape
>
> o Four-foot stakes to mark survey sites, two per site_2 x 2 wood stake
> for front stake and metal fence post for back stake
>
> o shovels and sledge hammers

**FIGURE 6.** Equipment checklist for shoreline surveys.

**Visual inspection stations.** These sites are surveyed rapidly to
determine shoreline type, oil coverage, and the thickness and depth of
buried oil layers as observed in small trenches dug across the
beachface. Photographs are taken to document oil distribution,
biological impact, and coastal morphology. Observations are recorded in
a field notebook and verbally on tape using the standard shoreline
terminology/codes. Sketches are made to record and locate measurements.
These surveys are repeated at appropriate intervals to monitor the
short-term exposure and behavior of oil on shorelines. If detailed
measurements are made of the length, width, depth, and coverage of the
oil, these surveys can be used to calculate the amount of oil stranded
on the shoreline as part of a mass balance. These surveys are also used
to groundtruth the oil distribution as determined from overflights. Even
quick field sketches of the oiling conditions during the survey may
provide valuable information needed for other components of the damage
assessment.

**Zonal stations.** The oil distribution, sediment type, biota, and
geomorphology at zonal stations are studied in much greater detail than
at the visual inspection stations. The following tasks are performed at
each station:

**a) [Topographic Profiles]{.underline}.** A shore-normal topographic
profile is run from the back beach to the low water line, using a
horizon-leveling technique. Two permanent stakes are established, a back
stake (BS) located well back of the normal storm wave erosion zone, and
a front stake (FS) just behind the spring high-tide swash line. The
profile is run within two hours of low tide in most areas. In macrotidal
areas (tidal range = \>4 m), the upper intertidal zone is exposed for a
greater amount of time, and profiles can be run within three hours of
low tide.

> Standard shoreline oil terminology/codes should be used. Recorded at
> each interval (usually a maximum of 3 m), is:

o The horizontal distance of the interval.

o The change in elevation (in cm) over the interval.

> o The percent oil coverage (estimated for a distance of about 3 m on
> either side of the transect).
>
> o The oil thickness

o Comments noted on the profile sheet should include sediment type,
geomorphic variations (berm crest, cusp horn, etc.), oil appearance, and
biological information (species and abundance). Use of grain-size
comparators improves estimation of sediment types.

o The high-tide swash line and water level should be indicated.

o Stake heights are recorded in case erosion or deposition occurs
between repetitive profiles.

**[Sediment Samples]{.underline}.** These are taken at selected sites
along the profile. Sampling sites are usually marked before running the
profile, so they can be appropriately located on the profile sheet. If
needed, an unoiled sample is taken for textural or grain-size analysis;
otherwise, the oil will have to be chemically removed later. If the
objective of the sampling is to ascertain the degree of subsurface
contamination, the surface oil should be scraped away.

> If the sediment is coarse gravel, a photograph (with scale) is taken
> of the site and the grain size is estimated in the field. Sediment
> size may also be measured from the projected image after the
> photograph is developed.

**c) [Biological Communities]{.underline}.** Visible effects on
biological communities are observed and recorded. The location and
distribution of oiled (and clean) sessile organisms are photographed and
described in order to determine temporal changes in the population. In
each case, a 15-cm or 30-cm photo scale is placed in the picture to
determine the surface area photographed. In most cases, more detailed
biological surveys augment and expand upon the findings of the
reconnaissance surveys.

**d) [Trenches]{.underline}.** Trenches are dug along the profile line
to describe the distribution of buried oil. The thickness, depth of
burial, and type of oil are described for each trench. Photographs are
taken of all the trenches, both close-up and overview. On some sand
beaches, it is useful to dig a trench across the entire beachface. Data
concerning the extent of buried oil are necessary to accurately
determine the quantity of oil incorporated within the intertidal zone.
Samples may be taken of the oiled layers within the trenches to
determine quantity and composition of the oil.

> **e) [Documentation]{.underline}.** A hand-drawn sketch is made of the
> zone surrounding the profile line to force careful observations of all
> aspects of the site. On each sketch, the following should be noted:
>
> 1\) Beach morphology 7) Dominant epifauna and flora
>
> 2\) Profile line 8) Clean-up operations if in progress
>
> 3\) Sample locations (or effects if completed)
>
> 4\) Oil distribution 9) All samples taken
>
> 5\) Trench locations 10) All photographs taken
>
> 6\) Depth of buried oil

Prior to conducting shoreline surveys, all team members should conduct
an inter-calibration exercise at an oiled site, to assure that the terms
and estimates are being uniformly applied. The team should practice
estimating until all members independently make similar estimates
(within about 10 percentage units). Also, descriptors for oil type
should be agreed upon, using field examples. If new terms or codes are
needed for a spill, they should be reviewed by the team and added to the
coding sheet for distribution to all members. This field training step
should be repeated as new members join teams. Also, staff should
occasionally switch teams to maintain uniformity.

Detailed records should be made during all surveys on wildlife
observations. These records include data on number of individuals
present by species or species group, behavior, readily apparent impacts,
etc.

**ASSESSMENT OF INJURY TO MARSHES**

**Summary of Known Impacts from Oil Spills**

Most information regarding the impact of oil on marshes comes from study
of spills affecting vegetation under tidal influence in coastal
estuaries. Most of the studies have been of marshes dominated by
*Spartina alterniflora*. Based on the available data, there are
significant differences among species assemblages.

When oil comes in contact with a marsh in a tidal setting, it generally
behaves in the following manner:

o Oil adheres readily to the vegetation; in fact, marsh vegetation is a
very effective oil sorbent.

o The band of coating will vary widely, depending upon the tidal stage
at the time that the oil slicks are in the vegetation. There can be
multiple bands.

o Large slicks will persist through multiple tidal cycles and coat the
entire stem from the high-tide line to the base.

o Fresh crudes and heavy oils will tend to \"slide" down the stem over
time in warmer weather and pool on the sediments at the base of the
plant.

o Weathered oils do not \"slide" as much; the oil stays on the
vegetation.

o If the vegetation is thick, heavy oil contamination can be restricted
to the outer fringe, with penetration and lighter oiling up to a 10 m
width.

o Lighter oils (light refined, fresh crudes) can penetrate deeply, to
the limit of tidal influence.

o Medium to heavy oils do not readily adhere to or penetrate the wet,
muddy sediments, but they can pool on the surface and in burrows.

o Light oils can penetrate the top few centimeters of sediment and
deeply into burrows and cracks (up to 100 cm).

Although every spill is a unique combination of events, there are
several factors which affect the behavior and impact of the oil on the
marsh ecosystem, as discussed below.

**Impacts by Oil Type.** It has been shown that light refined products
have the greatest acute toxicity to marsh vegetation, when compared to
other types of oil. In contrast, observations of spills of crude oils
and heavy refined products show mostly short-term impacts, and recovery
within 1-3 years (Baker, 1971; Baca *et al.*, 1983; Bender *et al.*,
1980; Michel, 1989). There are even studies which show that an increase
in standing crop of the marsh grass occurs following some spills
(Hershner and Moore, 1977). Crude oils contain nitrogen, and when
nitrogen is limiting, there can be growth simulation under some
conditions. However, the net impact is always negative.

**Extent of Vegetation Contamination.** The extent of vegetation
contamination is another very important factor. Many plants can survive
partial oiling; few survive when all or most of the above-ground
vegetation is coated.

**Degree of Sediment Contamination.** The degree of contamination of
sediments is another consideration which can prolong impacts to marsh
ecosystems for many years, compared with the early loss of oiled
vegetation. Slower recolonization rates are frequently related to
hydrocarbon levels in the sediments, though it should be noted that the
composition of the oil is as important as the total petroleum content.
That is, fresher oil and refined products have higher percentages of the
more toxic fractions in oil, whereas heavy oils have lower initial and
long-term toxicities.

**Exposure.** The physical setting of the oiled marsh, relative to
exposure to waves and currents, is one of the most important factors
controlling the persistence of oiled vegetation and overall rate of
recovery. Exposure can work to speed recovery, but, in some cases, it
can also work to increase erosion after plant roots die and before new
growth can occur.

**Seasonal Effects.** The timing of an oil spill, relative to the
plant\*s growing season, can affect the nature and duration of the spill
impact. In general, oiling during the dormant winter season has the
lowest impact, whereas oiling of vegetation during the growing season
will likely have longer effects. Mechanisms responsible for the slower
recoveries from a spill during the growing season have not been
adequately studied, but probably are related to plant stress at a time
when the plant\*s resources are being fully expended.

**Methods for Assessment of Impacts to Marshes**

The early surveys of marshes or wetlands should focus on documenting the
areal extent of contamination and distribution of the various types of
impacted wetlands using both aerial and ground-survey methods. Aerial
surveys should be conducted within a 2-hour window around low tide,
using 7.5 minute topographic quadrangles or aerial photographs as the
base maps. Observations should include estimates of the width and degree
of contamination along each shoreline segment, penetration into
channels, wetland zonations, and preliminary selection of stations for
groundtruthing. Extensive photo-documentation is highly suggested during
aerial surveys.

Ground surveys provide the most detail for documenting impacts. Ground
stations should be established at regular intervals appropriate to the
length of shoreline impacted (every 0.5 or 1.0 kilometer) at locations
representative of the zones mapped during the aerial surveys. If
possible, permanent markers should be located at each station. A
topographic profile should be measured at each station, also within a
2-hour window around low tide. The profile intervals should be short
enough to adequately represent the topography of the station and changes
in the distribution of plants and contamination, but no longer than 3
meters apart. For each profile a detailed sample profile sheet needs to
be completed. The information required for each profile interval
includes:

o Horizontal distance.

o Change in elevation.

o Percent oil coverage in the interval.

o Oil thickness on the sediment surface.

o Height and thickness of oil on the vegetation.

o Vegetation types.

o Biological resources present.

o Extent of biological resource oiling/contamination.

o Comments on the effects of clean-up activities.

o Location of sediment samples collected.

o Photographs taken (roll and exposure numbers).

The survey results provide the basis for estimating the acreage of
wetlands impacted and the degree of these impacts, in a
semi-quantitative manner. If classes of vegetation contamination are
used, i.e., light, moderate, and heavy, these classes must be carefully
defined (e.g., light contamination is less than 15 percent oil coverage
on the vegetation). Field charts to guide estimates of percent cover
reduce variations among field personnel. The degree of contamination of
vegetation and sediments can be used to predict injury, estimate the
duration of recovery, and thus predict damages, through lost use, or to
justify restoration. The profile stations can be used to monitor
shoreline erosion as a result of vegetation die-off or clean-up
activities. In these cases, sequential profiles are run to determine the
amount of erosion over time. For assessments of shoreline erosion, the
profile must be \"closed" so that measured changes can be shown to be a
result of erosion and not measurement errors. Closure of a profile
entails running the profile back up the transect to the original
starting point, and calculation of the measurement error in the
elevation readings. On most shoreline types, the error should not exceed
10 cm in elevation if the intent is to quantify sediment loss.

Detailed photography is very important to further document oil impacts
and changes over time. Photographs should be taken of the various oiling
classes, so that visual records are kept.

Further identification of the impact of oiling to marshes can be
quantified by conducting square quadrat measurements of number, height,
and biomass of the vegetation. Multiple quadrats are established at
known intervals along a profile within the vegetated areas. Using the
methods of Morris and Haskins (1990), the net aboveground effect on
production is conducted by censusing the number and height of all stems
within permanent quadrats. Estimates of individual stem biomass can be
accomplished by harvesting the vegetation from selected quadrats within
the marsh.

Following the initial survey, repeated surveys should be conducted at an
appropriate interval to monitor for oil persistence and vegetative
response to oiling.

**ASSESSMENT OF INJURY TO MANGROVES**

Mangroves often comprise the dominant estuarine and nearshore coastal
communities in tropical and some subtropical regions. These environments
are very important to the ecological balance of many of the marine
ecosystems in the tropics. Like the salt marshes of the temperate zones,
mangroves serve as nursery habitats and have a high diversity and
density of animal and plant species.

The two most common species of mangroves found in the United States are
the red mangrove (*Rhizophora mangle*) and the black mangrove
(*Avicennia* *germinans*). Red mangroves have gracefully curving prop
roots which extend a meter or more above ground and are covered with
small pores, called lenticels, through which oxygen diffuses when
exposed to the air at low tide. Because mangroves grow in anaerobic
soils, they obtain most of the oxygen needed from the atmosphere. Black
mangroves uptake oxygen through small roots, called pnematophores, which
extend 10-30 centimeters straight up above the soil from an underground
root system. Where both species exist, red mangroves occur in the low-
to mid-intertidal zones, whereas black mangroves are more common in the
upper intertidal and supratidal zones. Black mangroves are less tolerant
of high salinities, so they grow better where there is less exposure to
salt-water inundation and some fresh groundwater influence. The white
mangrove (*Laguncularia* *racemosa*) occurs primarily seaward of the
black mangrove.

Where mangrove forests occur, water depths are very shallow and most of
the forest is exposed during low tide on a regular basis. They are
frequently fronted by shallow seagrass beds or reef flats.

**Summary of Known Impacts from Oil Spills**

The nature and degree of oil spill impacts to mangroves are related to
three variables: the type of oil, the life stage, and the topographic
elevation on which the mangroves are colonizing. In general, the impacts
vary with the degree of contamination. Mangroves can suffer impacts when
their roots are coated or smothered with oil, preventing gas exchange.
The mangrove may be directly exposed or poisoned via absorption of the
toxic soluble fractions through the pnematophores and prop roots.
Mangroves may also be affected by chronic exposure to oil-contaminated
sediments. Impacted mangroves may exhibit one or more of the following
symptoms:

o Partial to complete defoliation.

o Low survival of propagules.

o Leaf deformities.

o Reduced leaf size and flower production.

o Increased insect infestation.

o Death of the individual plant.

The type of oil greatly affects the nature and degree of impacts to
mangrove ecosystems. Light, refined petroleum hydrocarbons, with large
amounts of the water-soluble aromatic compounds (e.g., jet fuels,
gasoline), are more acutely toxic to mangroves at lower concentrations
than are crude oils and heavy refined products. Based on both laboratory
and field studies of oil spills in mangroves, the following guidelines
have been developed.

**[Light Oils (Gasoline, Jet Fuel, No. 2 Fuel Oil)]{.smallcaps}**

o Fresh spills will have acute, toxic impacts to both trees and
intertidal biota.

o Sunny, windy weather will speed evaporation which will lessen
water-column and intertidal impacts.

o These light products will penetrate deeply into the forests, stopping
only at the high-tide line. Highest concentrations will occur at the
high-tide line and in detrital material.

o Persistence of oil in sediments should not be great, unless there has
been physical mixing into the substrate by wave or clean-up efforts.

o No. 2 fuel oil will have the greatest persistence; it can persist and
remain toxic for many years if it penetrates burrows and prop root
cavities.

**[Crude Oil/Heavy Refined Products]{.smallcaps}**

o Oil will coat intertidal zone, with heaviest concentrations either at
the outer fringe or the high-tide line.

o Penetration into the forest will be limited by the amount of oil,
forest density, and oil viscosity; weathered, emulsified oils penetrate
less.

o Oil can pool onto sediment surfaces and accumulate in detrital
material such as seagrass wrack.

o Toxicity is due to coating and sediment contamination; there is little
difference in toxicity among crude oils and the heavier refined
products.

o Because of low physical energy in mangrove swamps, oil persistence is
great and sheens can be generated for months to years.

Life stage is also important when determining the extent of impacts.
Seedling and propagules may die when they are coated with oil, even when
the oil has weathered for a few days before stranding. It should be
noted that where defoliation and death of adult trees occurs following a
spill, seedling density increases significantly. Because of the newly
opened canopy, more light reaches the seedlings and they have higher
sprouting rates. However, where the sediments remain contaminated,
long-term seedling survival is lower.

Topographic elevations in the forest further control the location and
extent of impact from floating slicks. There are essentially four types
of physical impacts likely to occur during oil spills. These include:

1\) Inner Fringe Impacts

2\) Outer Fringe Impacts

3\) Inner Basin Impact

4\) Riverine Impact

Detailed descriptions of the four types of expected impacts to mangroves
follows.

**Inner Fringe Impacts.** These impacts can occur when there is a berm
behind mangroves growing in subtidal or low intertidal areas. In
general:

o Oil passes through the open network of prop roots and collects at the
front of the berm. This interior berm is either inherited topography or
built by the accumulation of sediment and debris carried into the forest
by storm waves.

o The berm serves to prevent the transport of oil deeper into the
forest, so where present, the berms are the *de facto* interior limit of
oil penetration.

o Prop roots of the outer mangroves are oiled, but only as a thin band.

o Defoliation and mortality of both seedling and adult trees at the
inner stand of mangroves occurs.

o Large concentrations of the spilled oil may collect in sediments and
wrack piled on top of the berm. Large amounts of debris may be
indicative of accumulation zones with slow removal rates.

o Heavy debris also acts as a natural sorbent for oil, with the
potential as a long-term leaching source.

o Natural rates of flushing of oil from these inner berms are highly
variable.

**Outer Fringe Impacts.** These impacts can occur along relatively steep
inter-tidal zones where red mangroves commonly occur in a narrow band.
With this type of impact the following generalities may apply.

o There can be heavy oiling of the sediments and any accumulated debris.

o The energy processes affecting the shoreline will affect the natural
removal of stranded oil.

> \- If a wave-built sand beach or steep rocky shore exists, the
> potential for removal of stranded oil by natural processes increases.
> A rocky substrate may indicate wave exposure, but is not as diagnostic
> as a depositional beach.
>
> \- In sheltered bays where mangroves have been established on a rocky
> substrate, the oil is less likely to contaminate and persist in the
> rocky substrate.

**Inner Basin Impacts.** These impacts occur where there is a low
overwash berm in front of a shallow depression or interior basin. In
general:

o Oil gets washed over the low berm and is trapped in the basin.

o Although the oil can spread over a large area, the oil is less likely
to be concentrated in a narrow band, and may result in partial
defoliation.

o Persistence of the oil can be long term, depending on the degree of
natural flushing in the basin and oil type.

> \- In more exposed areas, oil can be removed from the system within
> several months, particularly for refined products and light crudes.
>
> \- Heavy oils are always more persistent because of their higher
> viscosities.
>
> \- In sheltered areas, oil is likely to persist for years.

**Riverine Impacts.** This type of impact is very similar to outer
fringe impact, however, it occurs only on river point bars, and it can
be more extensive than outer fringe impacts. Of course, if the spill
occurs during unusually high water levels, the oil can be carried over
the bars and river levees, into the interior basin forests. Riverine
environments are, however, relatively high energy, being exposed to both
riverine and tidal currents which are effective in natural removal of
the oil.

Impacts to the associated fauna and flora can also be very severe. Red
mangrove prop roots support a dense community of attached algae,
mussels, oysters, and barnacles, which are frequently directly killed
under moderate to heavy oil coating. Crabs are particularly hard hit,
whereas gastropods appear to be able to shift to less contaminated
areas, if present (Getter *et al.*, 1981; Jernalov *et al.*, 1976).

**Methods for Assessment of Impact to Mangroves**

These methods are designed to assess biological impacts by testing the
null hypothesis that \"no significant biological changes resulted from
the spill event". Assessment of impacts are determined by comparing
measurements between impacted and non-impacted sites, assuming that the
presence of the contaminant is the only original difference between the
stations. Thus, it is very important that non-impacted sites be as
similar as possible to the impacted sites, in terms of substrate type,
exposure to waves and tidal flow, and topography of the intertidal zone.
An important part of the NRDA team\*s responsibility is to identify
areas which are good candidates for establishment of permanent profiles
and plots for documentation of impacts. Strategies should include
classes of degree of oiling and treatment for the different forest types
and physical settings. The physical setting may control the rate of
recovery, particularly in terms of detoxification of contaminated
substrate and recolonization by seedlings.

There are two approaches that are commonly used to survey mangrove
forests: the profile method and the station plot method. For the profile
method, each station consists of a topographic profile of the intertidal
zone, along which the following parameters are measured at selected
intervals:

1\) [Adult tree oiling]{.underline}. The extent of oiling on adult trees
is estimated, using intervals such as: heavy (81-100 percent oil
coverage); moderate (21-80 percent coverage); light (1-20 percent oil
coverage); and no oil observed.

2\) [Height of oiling of the trunk and prop roots]{.underline}. If there
are multiple bands of oiling, the coverage and height of each are
recorded.

3\) [Adult tree mortality]{.underline}. The trees without leaves,
expressed as a percent of the total area (either a band of a defined
width or a circle of a defined circumference), measured with an angle
gauge, are recorded. Mortality is seldom observed early in the spill.
Mortality for each species is also measured.

4\) [Adult tree defoliation]{.underline}. The change in percent of
ambient light reaching the forest floor due to defoliation is determined
by averaging three light measurements at each interval. Percent open
canopy can also be measured using a spherical densitometer, a hand-held
device that uses a convex mirror over which a grid pattern is etched.
The amount of open space in the tree canopy observed on the mirror
survey is then converted to percent open canopy.

5\) [Basal area measurements]{.underline}. To determine the stand
development in terms of tree size and density, adult trees are measured
as to their height and diameter at breast height (DBH) at 1.3 m.

6\) [Seedling density and condition]{.underline}. At five randomly
selected locations, seedling density and condition (live, dead,
stressed) are recorded using 0.5 x 0.5 m^2^ quadrats.

7\) [Black mangrove root mortality]{.underline}. At five randomly
selected locations, ten subsamples of black mangrove roots are collected
from 0.5 x 0.5 m^2^ plots and examined for living cambium.

8\) [Observations of mangrove fauna]{.underline}. Observations of crabs,
snails, and other organisms are recorded, noting the presence of
contamination, behavior, distribution relative to the oil, and percent
mortality estimates.

9\) [Other indicators of stress]{.underline}. Observations on bark
fissuring, epithelial scarring, lenticel expansion, leaf deformities and
chlorosis, and propagule stunting or bending are noted at each interval.
These are all indicators of stress, which may eventually result in
mortality.

The station plot method includes these same measurements, however, they
are measured at 10 x 10 m^2^ plots established at selected locations
representative of areas with similar degrees of contamination, position
in the intertidal zone, species composition, etc. At these plots, all
the trees and seedlings are measured and marked, so that repeated
surveys can be made of the same population.

Sediment samples are collected to document the source and degree of the
contamination. Composite samples, collected from 5-10 locations within
the plot, will best represent the degree of substrate contamination.
Sediment samples should be taken with clean aluminum cores, which are
then sectioned and placed in clean glass jars, with either Teflon- or
aluminum-lined lids.

More detailed methodologies are provided in Getter *et al*. (1981),
Getter and Ballou (1985), and RPI (1987b).

**ASSESSMENT OF INJURY TO SEAGRASS ECOSYSTEMS**

Seagrasses and their associated animal communities are often a concern
during spills because of the susceptibility of these resources to
oiling, the general decline of seagrasses (Thayer et al. 1975) and the
important functions that seagrasses provide in nearshore and intertidal
marine environments (Marshall, 1991).

Eelgrass (*Zostera spp*.) is the most common seagrass taxon in temperate
regions of North America (Thom, 1990), ranging from Alaska to Mexico on
the Pacific coast, and extending from Greenland to North Carolina on the
Atlantic coast (Thayer et al. 1975). The dominant species in southern
waters include turtle-grass (*Thalassia testudinum*), manatee-grass
(*Syringodium filiforme)* and shoal-grass (*Halodule wrightii*).
Seagrass beds occur both intertidally and subtidally. Subtidal beds are
generally less sensitive to oil spills, since they usually do not come
into direct contact with the oil, while intertidal plants are at greater
risk of oiling (HMRAD, 1992). Some of the services provided by
seagrasses include:

o Sediment stabilization.

> o Detritus production which provides a major basis of food chains,
> although the bulk of the biomass is in the sediments (in the
> rhizomes);
>
> o Substrate for a highly productive epiphytic community, with a total
> biomass which often approaches or exceeds that of the plants
> themselves;
>
> o A directly used food source for a few organisms, namely green
> turtles and manatees, which graze on seagrasses;
>
> o Habitat which is utilized by fish and shellfish as nursery areas;
> and
>
> o Key role in nutrient cycling, including nitrogen, phosphorus, and
> sulfur.

**Summary of Known Impacts from Oil Spills**

Oil effects on tropical, subtropical, and temperate seagrass ecosystems
were discussed in Zieman *et al*. (1984) and are summarized below:

> o Greatest impacts occur on seagrasses that are intertidal, where the
> oil comes in direct contact with exposed blades;
>
> o Oil readily adheres to exposed blades, particularly when the oil is
> heavy or weathered;
>
> o Unless the sediments are also oiled, oiled blades are quickly
> defoliated and the plants have the capacity to grow new leaves (the
> leaves grow from a relatively protected meristem). Recovery can occur
> within 6-12 months;
>
> o Plant mortality has been observed at spills when the sediments were
> contaminated by oil, although such incidents have been rare.;
>
> o The most sensitive components of the seagrass ecosystem are the
> epiphytic community and juvenile organisms using the grass beds as a
> nursery. These species and life stages can be highly sensitive to both
> the water-soluble and insoluble fractions of oil; and
>
> o The plants can uptake hydrocarbons from the water column and
> sediments, potentially lowering their tolerances to other stresses.
>
> o Mortality to seagrasses can lead to loss of sediments, prolonging or
> preventing the re-establishment of the seagrass meadows (Marshall,
> 1991).
>
> o Oil retained in adjacent mangrove beds and shorelines can result in
> chronic exposure to seagrass beds, leading to loss of fringing edges
> of seagrass meadows (Marshall, 1991).
>
> o In certain instances, the main impacts to seagrass beds may be
> physical injury related to response activities. In particular, shallow
> seagrass beds may be disturbed by outboard motors, and other equipment
> (HMRAD, 1992). Shoreline washing may also move oil into subtidal
> areas, potentially impacted seagrass beds (Houghton et al. 1991b).

**Methods for Assessing Impact to Seagrass Systems**

The key parameters to measure oil effects on seagrass systems are listed
in Table 2. These measures mostly concentrate on documenting exposure
and injury to the seagrass bed itself because it is the basis for a
highly productive ecosystem. The objective is to quantify the injury in
terms of reductions in the key measures of seagrass productivity and
function. The areal extent and duration of both exposure and injury
should be determined as well. These measures can be translated into lost
services and support later restoration planning. Although seagrass
restoration is difficult, viable restoration strategies for seagrass
systems are being improved (Fonseca, 1992).

**Exposure.** During the preassessment phase of the NRDA response, the
field teams should focus on documenting the degree and extent of the
exposure of oil to seagrass beds. The pathways of exposure, including
direct smothering by submerged oil, water column exposure from passing
slicks, and impacts resulting from response operations should be
documented. This exposure and pathway information may be critical to
establishing later, more detailed studies of the seagrass beds.

**Baseline information.** Historical information and the immediate
post-spill status of the seagrass beds should be collected in order to
assist in identifying any changes in the areal extent of the seagrasses.
Historical aerial surveys and maps may indicate the pre-spill extent of
the seagrasses. Evidence of recent declines of seagrass beds may be
observable from the presence of roots and rhizomes. After the 1986 Bahia
Las Minas oil spill in Panama, Cubit and Conner (1993) noted that in
areas of heavy sediment contamination, the leaves of the seagrasses died
and sloughed off within a month, but the roots and rhizomes took over a
year to decompose.

> **Biomass.** Measurements of biomass can have extremely high
> variability, thus many replicates (at least 5-8) per site are needed
> to support statistical analysis. Although the standing crop of leaves
> is significant, the majority of the biomass is in the rhizomes and
> roots. To measure both above- and below-ground biomass requires coring
> and separating out the various plant components for weighing.
> Above-ground biomass can be measured by repeated clipping of the
> leaves (Kenworthy *et al*., 1993).
>
> \_ Exposure
>
> \_ Baseline
>
> \_ Biomass
>
> \_ Species abundance and density
>
> \_ Growth rates
>
> \_ Morphological measures
>
> \_ Physiological measures

**Species Abundance and Density.** Studies of seagrasses in Florida show
that seagrasses follow standard successional sequences (Zieman, 1982)
into beds dominated by single plant species. *Thalassia* is the climax
species but frequently the succession steps are \"reset" by
perturbations or environmental conditions such that the climax is not
reached. Thus, relative species abundance is generally not useful in
detecting oil effects. Instead, they are used to characterize the
seagrass habitat in general. Relative abundance and density most
frequently are measured using standard quadrat counting methods at
randomly located sites.

**Growth Rates.** Sublethal effects of oil exposure can result in
reduced productivity and growth rates. There are various methods to
measure productivity in terms of specific growth rates: 1) Short-term
growth of leaves can be measured by perforation with a needle at the
base of shoots in quadrats and measuring growth over a time period
usually of days to weeks (Thom, 1990). Eventually the leaves can be
harvested to measure growth in terms of leaf area and dry weight. 2)
Long-term growth can be measured by tagging rhizomes at the base of the
most recent shoot, then returning months later to collect the tagged
segments and measure any new growth (Houghton *et al*., 1992).

**Morphological Measures.** Leaf area index, the ratio of leaf area to
substrate surface area, provides an estimate of secondary surface area
available for epibiota, habitat complexity, and photosynthetic
potential. Short-shoot and leaf-pair densities may be a better indicator
of biomass where there are large seasonal fluctuations in standard
biomass measurements (Kenworthy, 1992).

**Physiological Measures.** Acute, sub-lethal effects of oil on
seagrasses can be measured by changes in the photosynthesis and
respiration rates of exposed plants. Durako *et al*. (1993) used
photosynthesis versus irradiance responses of leaf tissues exposed to
oil to assess oil toxicity to seagrasses. Such laboratory experiments
may be needed to link the injury to exposure of the water-accommodated
fraction for the specific oil type and seagrass species.

**Application to Injury Quantification**

All of these methods are directly applicable for injury quantification.
Special cases that may require additional studies include spills where
impacts are likely from chronic oiling or where a significant amount of
the spilled oil was transported into the seagrass bed.

Where there are inadequate pre-spill data, injury can be measured
through comparison of oiled versus unoiled, reference sites. Care should
be taken in site selection, to document that oiled and reference sites
have similar physical settings (in terms of currents and wave energy)
and substrate. In some cases, it may be necessary to demonstrate
similarity of oiled and reference sites by continuing the evaluation of
injury over time until natural recovery has progressed and the
parameters converge. The high degree of natural variability in these
parameters for seagrasses is an extremely important consideration in the
sample design. The results of the assessment should be able to generate
data on: 1) severity of injury, in terms of a percent reduction in
function and services; 2) total acreage of injured seagrasses; and 3)
duration of the injury. If there are two or more degrees of exposure,
such as heavily and lightly oiled seagrasses, then these measures of
injury can be determined for each exposure category.

**ASSESSMENT OF INJURY TO TROPICAL REEF SYSTEMS**

Coral reefs, like mangroves, are one of the dominant intertidal and
subtidal communities in tropical and some subtropical environments. Like
the salt marshes of the temperate zones, coral reefs serve as nursery
habitats and have a high diversity and density of animal and plant
species.

Because coral reef systems are mostly subtidal in nature, they are
seldom directly impacted by floating pollutants. Since coral reefs are
complex ecosystems, assessment of impacts from spills should be
conducted only by experts in the field. If impacts to reefs are
suspected, then the trustees should be notified and special teams
brought in to assess potential impacts. Conditions which might result in
reef impacts include:

o Reefs were in direct contact with the oil, because they are intertidal
or the spill occurred during unusually low water levels.

o There is likelihood that oil stranded on adjacent shorelines will be a
chronic source of sheens.

o Large amounts of oil on the adjacent shoreline were eroded and
deposited on the nearshore subtidal habitats.

o Light products were spilled or transported directly in shallow reef
waters.

Note that significant mechanical injury to the reefs can also occur from
vessel groundings, salvage efforts, and debris from wrecked vessels.
Again, assessment of these impacts are best conducted by experts in the
field.

**ASSESSMENT OF INJURY TO ROCKY SHORES**

Rocky coasts encompass many types and sizes of substrates. Included in
this general class are:

o Vertical bedrock cliffs, such as along fjords.

o Wide, rocky ledges which may be strewn with boulders.

o Rocky, rubble slopes which are formed by passive accumulation of sand
to boulder-sized talus on bedrock slopes.

There can be wide variations in the width of the intertidal zone and the
degree of \"permeability" of the rocky substrate. Since oil seldom
strands on exposed rocky shorelines, emphasis is placed on sheltered
intertidal rocky habitats. Because of the low wave energy, there is
little sorting of sediments, and there can be a jumbled mix of grain
sizes, from boulders to clay overlying the bedrock. This poorly sorted
mixture usually does not allow deep penetration of oil into the
subsurface. However, determination of the \"surface" on a shoreline with
boulder- and cobble-sized rubble layer overlying an irregular bedrock
platform with patches of muddy sand and granules is complex. Detailed
information regarding the effect of oil on the three types of sheltered
rocky coasts is summarized below.

**Summary of Known Impacts from Oil Spills**

**[Vertical Rocky Shores]{.smallcaps}**

o Oil will adhere readily to the rough rocky surface, particularly along
the high tide line, forming a distinct oil band.

o The lower half of the rock face usually stays wet enough to prevent
oil from adhering and remaining.

o Heavy oils and weathered oils can coat the upper zone with little
impacts to the rich biological communities of the lower zone.

o Fresh oil and light refined products have high acute toxicities which
can affect attached organisms after even short exposures.

**[Rocky Ledges]{.smallcaps}**

o Oil can adhere readily to the rough rocky surface, particularly along
the high tide line, forming distinct oil bands.

o If a beach is present, the oil will penetrate the sediments, with
long-term persistence very likely.

o Fractures in the bedrock surface are sites of oil pooling and
persistence.

o Even for wide ledges, the lower intertidal zone usually stays wet
enough to prevent oil from adhering to the rock surface.

o Heavy oils and weathered oils can persist on the lower intertidal zone
by penetrating surficial sediments.

o Fresh oil and light refined products have high acute toxicities which
can affect attached organisms after even short exposures.

**[Rubble Slopes]{.smallcaps}**

o Oil can adhere readily to the rough rocky surface, particularly along
the high tide line, forming distinct oil bands.

o Oil will penetrate into the crevices formed by the surface rubble and
pool at the contact of the rubble and the surface.

o Oil can form pools and eventually pavements under heavy oiling.

> o Oil can penetrate deeply into loosely packed rubble, causing
> long-term contamination of the subsurface sediments.

**Methods for Assessment of Impacts to Sheltered Rocky Coasts**

Sheltered rocky coasts can be surveyed for documentation of the extent
and degree of oil contamination using the same profiling methodology for
beaches. Although this survey method may be difficult, a permanent
profile should be established to document the long-term persistence and
impacts of the spilled oil.

Documenting the extent of oil penetration into the rocky coastlines may
be difficult. In areas where loose rock or gravel are present, shallow
trenches may be dug. In areas where there are extensive cracks or
fissures in the bedrock, extensive photo-documentation is required to
document the degree and persistence of oil.

**ASSESSMENT OF INJURY TO INTERTIDAL COMMUNITIES**

Intertidal communities are oftentimes severely impacted when floating
contaminants, such as oil, strand on the shoreline. Infauna are
categorized as animals that live buried in sediments, while epifauna and
epiflora are animals and plants that live on the sediment surface or
attached to rocks. Information regarding infauna and epifauna that are
considered shellfish (crabs, mollusks, and shrimp) is presented in the
section on Shellfish.

**Summary of Likely Impacts from Oil Spills**

**Plants**

The main plants in the intertidal zone are the attached macroalgae. In
general, intertidal plants:

o May be subject to smothering by oil.

o Can be quite resilient and survive even heavy oiling.

o May incur short-term effects on reproduction due to oiling; duration
of impacts lasted only for a period of less than one month after the
*World* *Prodigy* spill in Narragansett Bay in 1989 (Thursby *et al*.,
1990).

During the Santa Barbara blowout in 1969, surf grass (*Phyllospadix*)
growing in the intertidal zone was observed to be heavily oiled at some
sites; many of these plants turned brown and died (Foster *et al*.,
1971). NOAA studies of intertidal communities impacted by the *Exxon*
*Valdez* spill in Alaska found that attached macroalgae, specifically
*Fucus*, survived oiling at numerous sites but were heavily impacted by
hot water washing of shorelines to remove oil (Houghton *et al*.,
1991b).

**Infauna**

Many of the infaunal species (e.g., polychaetes, oligochaetes, and
meiofauna) appear to be moderately sensitive to oiling. Polychaetes,
including lugworms (*Arenicola* sp.), have been attributed to playing an
important role in the biodegradation of residual oil in sediments
whereas several oligochaete species are known as opportunistic species
that are commonly found in polluted areas. The extent of impacts to
infaunal species will be dependent upon the type and volume of oil they
are exposed to. In general:

o Some invertebrates survive in heavily oiled sediments, including
polychaetes, oligochaetes, and copepods.

o Polychaetes may facilitate biodegradation processes.

o Intertidal species are most likely to experience acute toxicity,
especially those in the upper half of the intertidal zone.

**Epifauna**

Epifauna, which include crabs, snails, barnacles and mussels, are all
susceptible to oiling. Effects may include behavioral changes, reduced
feeding rates, bioaccumulation, and direct mortality. These effects vary
with species type and are related to the degree of oiling, oil type, as
well as the duration of exposure. In general:

o Mussels, and other attached bivalves often survive direct oiling, but
may also bioaccumulate hydrocarbons.

o Many crustaceans, including barnacles and crabs, are sensitive to
acute and chronic effects of oiling.

o With heavy accumulations, smothering may result in high mortalities.

**Methods for Assessment of Impacts to Intertidal Communities**

To accurately document the extent of impact oiling has had on intertidal
communities, a monitoring study is conducted where both control and
oiled sites are examined simultaneously. Methods include establishing
permanent survey stations or objective selection of plot locations. The
type of sampling strategy to be employed will be determined by the
sampling design developed prior to this step.

**Permanent Stations**

For permanent survey stations:

1\) Permanent profile line(s) are established during low tide so that
the intertidal zone is exposed. Extreme care must be taken so that
impacted and non-impacted sites are comparable in substrate, slope,
degree of exposure to waves, and orientation relative to sun exposures.

2\) Permanent stakes are driven into the sediment (rock) at known
intervals along the transect. Detailed information regarding the angle
of the profile, positioning of the permanent stakes, and any other
pertinent details is necessary for revisitation.

3\) At each stake, quadrats are established, usually with a 50 x 50 cm
(0.25 m^2^) plastic frame. Quadrat sizes range from the 10
centimeter-square to 2 meter-square based on properties of the resources
affected (evenness of distribution, clump size, etc.).

4\) Core samples are taken for infauna.

The primary goal is to document the intertidal communities response to
oiling over time by establishing enough permanent plots within the study
area to statistically quantify the changes in abundance, density, and
diversity. It may be important to classify stations according to the
degree of contamination, exposure to wave and tidal energy, substrate,
and type of clean-up conducted at the site. Another alternative is to
utilize previously established stations (Mussel Watch, State or
University long-term monitoring sites, etc.) located in the area of
impacts. These sites can provide historical data on population
compositions and natural variations. However, the same sampling
methodologies should be used so that comparative analyses (pre- versus
post-spill) are possible.

**Objective Plot Selection Sites**

There are three sampling strategies employed when objectively selecting
plots for documenting exposure. These include:

1\) [Systematic strategy]{.underline}-locating plots known distances
along transect lines or systematically sampling a grid system.

2\) [Random strategy]{.underline}-using a random number table to
generate plot locations.

3\) [Stratified random strategy]{.underline}-sites are located
systematically or with respect to predetermined divisions of the area
into different environmental categories, but replicate stations at each
site are randomized.

**Quantitative Sampling**

For both permanent stations and objective sampling strategy sites, there
are several parameters that can be recorded for each quadrat/sampling
site. These parameters include:

+------------------------------------+---------------------------------+
| > 1\) Species composition.         | > 5\) Degree of oiling          |
| >                                  | >                               |
| > 2\) Percent live cover or        | > 6\) Clean-up index (when      |
| > density, total or by species.    | > applicable) for permanent     |
| >                                  | > plots.                        |
| > 3\) Percent dead cover or        | >                               |
| > density, total or by species.    | > 7\) Species behavior and/or   |
| >                                  | > condition.                    |
| > 4\) Percent bare ground.         |                                 |
+------------------------------------+---------------------------------+

Quadrat sizes should be chosen to best represent the resident
populations and allow the accurate estimation of percent live or dead
cover. Additional information to be recorded includes the number of
active clam siphons, mussels, crab burrows, etc., as well as the animals
behavior and/or condition. Each quadrat/study site should be
photographed extensively; whole site/quadrat shots as well as more
detailed location shots should be taken.

As discussed in the QA/QC section, the field teams should be very
careful to document and use consistent and precise terminology. Avoid
using ambiguous descriptions such as several, or abundant, if more
precise observations can be obtained (i.e., approximately 20 organisms,
or 80% cover.)

**ASSESSMENT OF INJURY TO FISH**

**Summary of Likely Impacts from Oil Spills**

The probability of adverse impacts to marine fishes from spilled oil is
influenced by the inherent sensitivity of each species. Sensitivity and
susceptibility are functions of life-history, stage and, in turn,
habitat preference, behavior, diet, and other factors. Each stage has
characteristics that directly control the likelihood and degree of
impact during an oil spill.

The sensitivity of fish eggs is high, but reduced somewhat in comparison
to larval fish due to the presence of protective membranes that may
reduce exposure. Susceptibility of eggs is highly variable. Benthic eggs
released in deep water are unlikely to be exposed to oil during a spill.
Benthic eggs released in shallow waters are vulnerable to exposure to
light oils having a significant water-soluble fraction, but usually the
impacts are very localized. Benthic eggs spawned on intertidal or very
shallow subtidal substrates are highly vulnerable to direct mortality
from contact with floating slicks and from contamination of nearshore,
subtidal sediments.

The larval stages of most marine fish are planktonic; their large-scale
movements are controlled by water currents because they have limited
swimming capabilities. Planktonic larvae use an attached yolk sac during
the first few days or weeks, then feed on phytoplankton and zooplankton,
which are concentrated in the upper water column. However, dilution of
oil under a slick is rapid, and there is a low probability of acute
toxic effects on significant percentages of a larval population during
an oil spill. However, large spills of light types of oil or those which
occur in restricted areas with low flushing rates may produce
significant mortalities.

Later life stages of marine fish (juvenile and adult) are considered to
be the least sensitive to oil spill impacts. They are very mobile and
better able to detect and avoid spills. Acute toxicity is likely to
occur only when light, refined products are spilled in lakes, small
streams, and rivers. However, there may be concern for long-term,
sublethal impacts when sediments in nursery areas have been
contaminated.

**Field Methods for Fisheries Assessments**

The easiest type of fisheries assessment is the collection of dead fish
(fish-kill investigations) along all or segments of an impacted
estuarine waterbody. The newly revised American Fisheries Society (AFS)
publication on fish-kill investigations details the methods to be used.
Most of the time, however, this approach will greatly under-estimate the
total injuries from the spill because it only counts the number of dead
adult fish.

Fishery management agency personnel will usually take the lead in the
field assessments of impacts to fish. However, the trustees should brief
the fishery scientists on the NRDA process to ensure that the field
biologists understand the data and evidence requirements. The trustees
may need to provide appropriate sampling containers are chain-of-custody
forms. The trustees or NRDA team members can also assist by providing
information on possible sampling locations, degree of oiling of fish
habitats, coordination of sampling and sharing of results of sediment
and water analyses, and predicting the duration of the exposure.

Fish trawls may be needed to collect samples for chemical analysis of
tissues and organs to document exposure to the oil. Fish metabolize
petroleum hydrocarbons, and therefore high levels of these compounds are
seldom found in fish tissue. Other indicators of exposure are the
presence of hydrocarbon metabolites in the bile and enzymes in the
liver.

In some instances where an expedited or model-based assessment is used,
a qualitative assessment of fish/biota may be useful in justifying later
assumptions (i.e., PRP can challenge whether the resource is there to be
exposed if field verification is not conducted).

**Modeling Techniques for Impacts to Fish**

Assessment of impacts to eggs and larvae is extremely difficult because
of their patchy spatial distribution over time. It may be preferable to
develop models which use bioassay data (either from the literature or
using local communities and the spilled material), exposure calculated
from physical properties of the material and hydrological conditions,
and species density to calculate mortality rates and sublethal impacts.
The Type A model uses this approach to calculate the mortality of both
adult and larval fish resulting from exposure to toxic fractions of the
oil during the spill. The bioassay can be for the water-soluble fraction
or exposure via contaminated sediments and food.

The Type A model or other water-quality models can be used to estimate
the oil distribution in the water column at selected depths over time.
When combined with toxicity and population data for selected species and
life stages, an estimate of the potential injury can be made. Care
should be taken so that seasonal variations in distribution and
abundance are considered, as well as the actual spill conditions.

**ASSESSMENT OF INJURY TO SHELLFISH**

**Summary of Likely Impacts from Oil Spills**

The primary shellfish of concern in coastal U.S. waters are shrimp,
mollusks, and crabs, because of their importance to both commercial and
recreational users. Shrimp are highly sensitive to the toxic effects of
oil in the water column; they usually have the lowest LC~50~ of all
shellfish. The eggs of shrimp are the most sensitive life-stage, but not
always the one at greatest risk, depending upon the species. For
example, brown shrimp eggs are present in deep water where there is
little possibility of oil exposure. The larval shrimp also remain mostly
offshore in pelagic habitats, moving into estuaries as postlarvae. Thus,
juvenile and adult brown shrimp are most susceptible because of their
estuarine and shallow coastal distribution. White shrimp, on the other
hand, can spawn in shallow water and near the surface, so all life
stages could be vulnerable. The probability of impacts to shrimp from
spilled oil is influenced by the inherent sensitivity and susceptibility
of the species and is a function of their life-history, stage, habitat
preference, behavior, diet, etc. Each stage has characteristics that
directly control the likelihood and degree of impact during an oil
spill.

Impacts to crab species are also dependent upon their life stage,
habitat preferences, behavior, etc. Female blue crabs are most
susceptible to the toxic effects of oil when the female is carrying
eggs. Depending on the species, the spawning period may occur year
round, often with a peak spawning period during one portion of the year.
Crabs can be exposed to heavy oil spills which sink or are transported
along the bottom in brackish water environments. There is also a
possibility of crab species scavenging dead, oiled wildlife that are
transported into their preferred habitats. The effects of the
consumption of petroleum hydrocarbons has not been fully documented in
crab species.

Mollusks (primarily clams, mussels, and oysters) often exhibit a wide
diversity of impacts when exposed to spilled oil. The extent of impact
is most often controlled by life stage and habitat preference. Most
molluscan larvae are planktonic and are found in estuaries and nearshore
waters with the notable exception of the deep water species. Thus, they
are susceptible to acute toxic effects during the early period of a
spill. Many of the dominant molluscan species_oysters, clams, and
mussels_because of their intertidal, sessile distribution, are very
likely to be exposed to contaminants spilled in nearshore areas. Seldom
are mollusks killed outright by oil, except when smothered by heavy
slicks. Usually, the biggest concerns are uptake and sublethal effects,
such as decreased or abnormal growth, increased mucous production, and
decreased respiration rates. Uptake of petroleum hydrocarbons takes
place at all levels of water concentrations and can occur for weeks to
years following a spill, causing concern about the safety of shellfish
consumption. Contaminated shellfish can also provide a pathway for
exposure of other resources which feed heavily on intertidal shellfish,
such as oystercatchers, harlequin ducks, and sea otters. Once the source
of exposure is removed, depuration can occur within a few days to weeks.
Therefore, shellfish are an excellent indicator of the bioavailability
of oil in the environment.

**Field Methods for Population/Behavioral Changes**

For most spills, it will be extremely difficult to quantify the impact
to mobile species such as shrimp using field surveys of distribution and
abundance, pre- and post-spill, or oiled versus unoiled areas, because
of the high degree of spatial variability. An alternative approach would
be to use an exposure model, as described previously for finfish, to
calculate the mortality based on the toxicity of the material,
water-column levels of the pollutant, and existing distribution and
abundance data for larvae, juveniles, and adults. The area affected by
the spill can be confirmed using slick distribution maps, reports of
fouled nets by shrimpers, and analysis of water samples.

Monitoring studies to determine lethal and sublethal impacts could
consist of comparative studies of oiled and unoiled mollusk beds and
bivalve transplant studies, making sure that the sites are similar with
respect to salinity, temperature, dissolved oxygen levels, and degree of
exposure to tidal flushing. The study plan should include measurement of
enough samples so that differences can be quantified statistically.
Measured parameters could include mortality rates, growth rate over
time, frequency of abnormalities and fungal infections, and
physiological parameters, such as fecal production, condition index,
gonadal index, and gill tissue respiration (Scott *et al*., 1984). These
studies may be appropriate for spills where large areas of mollusk beds
were impacted but not directly killed.

Bivalves, such as mussels and oysters, can be used as indicators of
exposure and bioeffects. They provide integrated information about the
bioavailability and effects of oil which cannot be defined by chemical
analysis of discrete water samples. This capability is particularly
important in monitoring of oil spills where exposure is often ephemeral
and highly variable. The main advantage in using transplanted mussels
over monitoring resident populations is the experimental control over
sampling sites, sampling intervals, and test animals. Bivalves can be
strategically transplanted along physical and chemical gradients and in
assessment areas where they might normally settle to provide a surrogate
monitoring population. The exposure period is controlled by measuring
the caged animals at selected intervals. The statistical power of the
data is increased by monitoring individuals before, during, and after
the exposure to oil.

Measuring bioaccumulation and growth in transplanted bivalves is a
potentially powerful tool in the NRDA process for injury determination,
establishing pathways, and restoration monitoring. Transplanted mussels
have been used to monitor the *Amoco Cadiz* and *Exxon Valdez* oil
spills by measuring the accumulation and depuration of oil in mussel
tissues before, during, and after the spills. These chemical
measurements can be used to establish a pathway from the discharge to
the injured resource. Biological measurements of bivalve weights and
lengths can be used to estimate injuries to biological resources.
Juveniles should be used for growth measurements since they grow faster
and adults for bioaccumulation since they provide more tissue for
chemical analysis. Minimizing the size range reduces variability in the
test.

The following guidelines should be considered when using transplanted
bivalves in natural resource damage assessment:

1\) Maximize number of test animals (\$50);

2\) Minimize size range (.2-4 mm);

3\) Use juveniles (\<30 mm) for growth;

4\) Use adults (\>50 mm) for bioaccumulation; and

5\) Minimum beginning and end-of-test measurements should include:
number of test animals; tissue concentrations of oil; and bivalve
weights and lengths.

Monitoring studies to determine the uptake and subsequent depuration of
the spilled material should also consist of oiled and unoiled site
comparisons, to account for the often widespread background levels of
organic contaminants in coastal waters. Comparisons can be made with
data from long-term monitoring sites such as Mussel Watch or National
Status and Trends, which record PAH levels in shellfish and bottom
sediments when collecting samples in oiled areas. Extreme care should be
taken not to contaminate the tissue with oil from the shell or
sediments. Figure 12 outlines protocols for sampling bivalve mollusks.

Histological samples should be collected only by experienced or trained
staff. The general method for sample collection is as follows:

> 1\) New, clean, sharp scalpels are used for tissue collection for each
> animal.

2\) Forceps should be cleaned prior to each tissue collection.

3\) Sample size should be about the size of a small walnut.

4\) All samples from the same animal can go into a single Ziploc bag.

6\) Proper QA/QC procedures should be followed for sample labeling.
Internal container labels are required.

7\) Samples should be fixed for eight hours with a buffered ten percent
formalin solution. This solution can be prepared by mixing nine parts
sea water and one part 37 percent formaldehyde.

8\) After eight hours, the formalin solution should be completely
replaced.

9\) Formalin should be replaced every 24 hours for several days.

Samples should be collected on a regular basis, such as monthly, until
levels drop to background levels. During the *Apex* barge spill in
Galveston in 1990, hydrocarbon levels in shellfish decreased to
background levels within five months (Brooks *et al*., 1991). In
comparison, at the *Exxon Valdez* oil spill, elevated levels of PAHs
have been found in mussels for over 3.5 years, because of the long-term
persistence of subsurface oil in adjacent gravel beaches (Houghton *et
al*., 1992). Such sampling is usually required to monitor for the
potential for human exposure from consumption of contaminated oysters,
but is also a means of monitoring for the bioavailability of oil in the
ecosystem. It is very important to document all field protocols used
during an injury assessment, including any site/case-specific changes
made to the standard procedures throughout the long-term monitoring
effort.

**o** Identify oiled and unoiled sites for comparison.

**o** Clean areas should be sampled first to minimize the potential for
cross contamination.

**o** Tissue samples (**minimum** of three grams; 30+ grams is better)
from each area should be composited into triplicate samples:

> \- two for each duplicate analysis;
>
> \- one for archiving for later use if needed.

**o** Extreme care should be taken when collecting samples in oiled
areas not to contaminate the tissue with oil from the shell or
sediments. This step can be conducted by the contract laboratory. In
this case, the whole organism, shell and all, is sent to the laboratory
for analysis.

**o** Use required QA/QC protocols when sampling.

**[Additional Documentation Requirements]{.smallcaps}**

**o** Describe in detail oiling conditions of the site.

**o** Extensive photography of the sampling sites, including:

> \- overall views to document the location and sediment type;
>
> \- close-up shots of clusters prior to sampling.

**o** Salinity.

**o** Temperature, air and water.

**o** Dissolved oxygen levels.

**o** Degree of exposure to tidal flushing.

**[Sample Preservation and Analysis]{.smallcaps}**

**o** Samples are kept cold until frozen and are shipped frozen to the
laboratory for analysis.

**o** Only GC or GC/MS analyses should be conducted, so that the source
of the contamination can be confirmed.

**ASSESSMENT OF INJURY TO SEA TURTLES AND OTHER REPTILES**

The direct and indirect effects of an oil on sea turtles are not very
well documented. Even less information is available regarding the
effects of oil on other coastal/marine reptiles. Although there have
been many oil spills in areas populated by turtles, it is still unclear
how oil physiologically affects turtles in open water. Because the range
of adult turtles is wide, it is unusual to have large numbers of turtles
directly impacted by an oil spill. Spill-related turtle impacts are
mostly anecdotal and poorly documented as to cause of death. In the
United States, the other reptiles likely to be affected by a marine or
brackish water spill are the American alligator and crocodiles. In some
of the U.S. territories, marine iguanas are also present.

**Summary of Known Impacts from Oil Spills**

There are several studies which have discussed oil and sea turtles. Some
of these reports are only observations of oiled or dead turtles as a
result of an oil spill (Rützler and Sterrer, 1970; Delikat, 1980; Hooper
1981; Gitschlag, 1991). Other studies have directly examined the
physiological and behavioral effect of oil on various sea turtle life
stages (Fritts and McGehee, 1982; Vargo *et al*., 1986). Research has
also been performed on the impact of pelagic tar on sea turtles (Witham,
1978, 1983; Vargo *et al*., 1986; Van Vleet and Pauly, 1987; Gramentz,
1988). Pelagic tar primarily comes from the discharge of tanker ballast
water, although weathered oil from spills does make up a small portion
of the tar.

RPI (1991) has outlined the results of these studies, specifically the
laboratory and field oiling ones, which indicate that crude oil has
detrimental affects on sea turtle eggs, hatchlings, and juveniles. The
major conclusions from these studies are summarized below:

o The number of unhatched eggs in a nest are much higher when fresh
crude oil is on the surface of the sand during the last half or quarter
of incubation.

o Hatchling morphology is affected by the amount of oil and time of
oiling. The hatchling weight is lower and the size is smaller when the
eggs are exposed to a light dosage of oil mixed in the sand, or when the
oil is applied to the surface of the sand half way through the
incubation process.

o Crude oil aromatics may displace or inhibit the use of available
oxygen in the nest atmosphere when coating the sand above a nest.

o Removal of oiled sediment overlying the nest is ineffective; the nest
is lost and the sediment structure has been altered.

o Weathered crude oils are less toxic to turtle eggs than fresh crude
oil.

o Crude oil causes reddening and sloughing off of the skin of juveniles,
which can reduce the viability and increase the chance of infection.

o The oil causes an immune system response as indicated by an increase
in the number of white blood cells.

o The oil also interferes with the functioning of the salt gland,
thereby upsetting the water balance and internal ion regulation.

o In the natural environment, if an affected turtle survives the oiling,
disease and predators may kill the already weakened animal.

o Turtles feed on objects floating at the water surface, therefore they
are susceptible to tar balls. Oil ingested by the turtles does not pass
rapidly through the digestive tract, but is instead retained for several
days. During this time toxic components of the oil can be passed on to
other organs or tissues.

o Tar may coat the turtle\*s flippers. As they try to clean their
flippers, they additionally contaminate their mouths resulting in the
further inhibition of feeding which may ultimately lead to starvation.

**Methods for Assessment of Impact to Sea Turtles and Other Reptiles**

In the event of a spill in sea turtle or other reptile habitats, there
are several actions that can be undertaken to assist any future damage
assessment actions. First, the visible extent of contamination of
turtles, their nesting beaches, and other reptiles of concern should be
documented. Second, accurate records identifying the number and species
(when possible) of animals that are within contaminated areas or along
the projected path of contamination will be helpful when determining the
extent of impact from an individual spill. In the event of a spill
impacting nesting beaches during the nesting season, the primary
response is to provide accurate documentation on the extent and duration
of contamination on suspected nesting beaches.

There have been several instances where sea turtle nests have hatched
while there was extensive contamination on the lower beach face.
Historically, these new hatchlings have been manually transported to
areas outside of the contamination or further offshore outside of the
oil\*s influence. Such activities could be initiated and undertaken by
resource managers from the trustee state or federal agency.

> **Proviso:** Permits may be needed, either through the Fish and
> Wildlife Service or the National Marine Fisheries Service. Any
> assessment activity should be cleared with these agencies first.

**ASSESSMENT OF INJURY TO BIRDS**

**Summary of Known Impacts From Oil Spills**

Seabirds, wading birds, waterfowl, and shorebirds are extremely
vulnerable to spilled oil. Many of exposed birds die after direct
contact, due to the inability to fly and forage after plumage becomes
oil soaked and waterlogged. Thermo-regulatory demands increase when the
insulative and waterproofing effects of feathers are reduced, and the
inability to feed leads quickly to starvation. Lightly exposed birds may
survive, but birds exposed immediately before or during the breeding
season will be highly stressed and will probably cease breeding after
oil exposure.

During cleanup, disturbance to individual birds, to breeding colonies,
and to roosting areas may increase damage to birds. Disruption of
breeding can result in significant losses. Aircraft and boat traffic
associated with cleanup activities will disturb waterbird colonies and
raptor nesting. The magnitude of disruption should be factored into any
damage assessment, as the clean-up disturbance is oil-related and would
not have occurred if the oil had not been spilled.

Sublethally oiled birds may experience stress and breeding disruption.
Birds which are exposed to as little as 0.1 milliliter of crude oil may
abandon the breeding season, with loss of production for the year (Fry
*et al*., 1986; 1987). Those birds remaining in the colony may have
reduced egg or chick survival through several mechanisms: 1) direct
exposure of eggs to oil, which is toxic at very low amounts (Hoffman,
1990); 2) reduced incubation attentiveness resulting in lowered
hatch-ability of eggs (Fry *et al*., 1987); 3) reduced survival of
chicks because of reduced feeding rates by oil-exposed adults
(Trivelpiece *et al*., 1984); and 4) reduced growth of chicks fed oiled
foods by adults (Peakall *et al*., 1982).

Quantification of breeding loss may be very difficult to document, but
exposure documentation will aid in recovery of damages from the spiller,
because these effects are well documented in the literature.

**Assessment of Bird Populations at Risk**

Because oiled birds are vulnerable to predation, they often will hide to
avoid being discovered by predators, including man. Some oiled carcasses
will sink, be scavenged, or will drift offshore and never be found.
Because the number of bird carcasses recovered after an oil spill is
often only 5-25 percent of the actual number oiled, it is very important
to make as good an estimate as possible for the number of birds at risk
of exposure from the beginning of a spill. For this reason, several
methods have been devised to assess the numbers and species of birds at
risk from spilled oil.

During an emergency it becomes very difficult to plan and conduct a
complete and timely survey to inventory all the birds at risk within the
anticipated path of the spilled oil. It is therefore useful to have
baseline information on numbers of birds and species present by season,
so that a survey of critical areas of high bird concentrations can be
made in advance of the spill to confirm the bird numbers and current
timing of migration, breeding, colony use, etc. It is highly desirable
for any bird resource inventory to have data on the following factors.

**Breeding Colony Data:**

1\) Location of known colonies of vulnerable wading birds, waterfowl,
seabirds, and shorebirds.

2\) The species composition and numbers of birds using each of the
colonies, and estimates of the annual variation in breeding populations.

3\) The feeding areas used by birds from each colony, and locations
where high numbers of birds will be found during the day.

4\) The variability of locations of each colony from year to year. Some
colonial waders may change the location of their colony each year, while
others will use the same trees and nests for many years. Terns and gulls
may use barrier islands with changing beaches and dune patterns,
resulting in different annual locations. Colonies of cormorants, gulls,
and terns may use abandoned man-made structures such as piers,
platforms, levees, and breakwaters which may vary in importance from
year to year.

The numbers of birds breeding in any given year may depend on many
variables, such as food availability, seasonal storm conditions, water
temperature, rainfall patterns, and local water conditions. Since any of
these may change in a given year, a prior knowledge of the annual
variation in timing of breeding is particularly useful in anticipating
bird species at risk at the beginning of a spill.

State resource agency inventories compiled and summarized for different
years are particularly useful inventory tools. Additionally,
non-government surveys may also be incorporated, such as Audubon
Christmas bird counts and breeding bird surveys for specific localities.
U.S. Fish and Wildlife Service surveys of migratory waterfowl will be
useful for species information, migration timing, numbers of birds, and
locations of important migratory populations. The recovery plans for any
threatened or endangered species will provide detailed information on
habitats, locations of known populations, and numbers of birds at risk.

**Non-breeding Season Data:**

1\) Locations and numbers of overwintering birds.

2\) Migrating species and important stopover sites.

3\) Timing of migration by vulnerable species.

Lower latitudes provide valuable areas for winter use by migratory birds
inhabiting marshes and coastal waters. Most high-use areas are
traditional, with birds returning every year to overwinter. The
locations and anticipated numbers of birds using each area should be
included in the bird inventory.

Annual waterfowl migration patterns result in peak numbers of birds
concentrating in some areas in fall and spring. The anticipated timing
of such high concentrations of birds should also be incorporated into
the bird inventory.

The species composition of flocks migrating through or overwintering in
an area should be determined, as specific habits of many species make
them particularly vulnerable to spilled oil. Oil vulnerability is
discussed in a section below. Each species group will choose particular
habitats for roosting and feeding. For example, bay and sea ducks
(canvasbacks, scaup, ruddy ducks, etc.) stay in coastal bays and
offshore from coastal beaches for feeding; dabbling ducks (mallards,
gadwall, teal, pintails, etc.) remain in marshes, ponds, and bayous,
rarely venturing into salt water. Herons, egrets, rails, ibises, storks,
and spoonbills may use traditional marsh and coastal areas which are
vulnerable to an oil spill.

The timing of migration may vary within a few weeks each year, based on
global weather variations and water conditions. Having current knowledge
of use patterns by migrating birds will greatly enhance the ability to
respond to a spill and quickly identify critical areas which will need
to be checked for birds at risk of exposure.

**Oil Vulnerability Indices**

Several summaries have been published which quantify the vulnerability
of specific species to spilled oil. King and Sanger (1979) have
developed an \"Oil Vulnerability Index" (OVI), based upon feeding and
roosting habits, habitat use, population concentrations and numbers, and
other factors, which ranks each species on a scale from 1-100. Wahl *et
al*. (1981) have constructed a similar inventory with a geographical
component centered on Puget Sound, Washington. Each of these indices
have been modified to provide a more general summary of the
vulnerability of each species independent of geographical area (RPI,
1988).

The most vulnerable species are birds which both feed and roost in
coastal waters. Loons, grebes, and sea ducks spend 24 hours per day on
water, and they have no way to avoid locally spilled oil. Pelicans,
cormorants, and anhingas are almost as vulnerable because of their
feeding habits (diving for food in nearshore waters), although these
birds roost out of water on trees or coastal structures. Freshwater
ducks and most waders (herons, ibis, spoonbills, egrets, etc.) also
spend a smaller proportion of the day on water, and usually in protected
areas less likely to be impacted by an offshore spill. Spills in
marshes, however, will greatly increase the risk to these species.
Shorebirds, such as sanderlings, snowy plovers, and oystercatchers, are
at high risk on oiled beaches, where they become exposed through direct
contact and consumption of contaminated food. Gulls, terns, and skimmers
are at high risk of being oiled on beaches or on open water, and these
species are often seen with patches of oil on their plumage. While
perhaps not being directly killed by small amounts of oil, the stress of
being oiled, and the toxicity of the oil when ingested during preening,
will probably stop many lightly oiled birds from breeding after exposure
to even small amounts of oil.

**Methods of Assessment of Impacts to Birds**

A detailed methodology for assessing the numbers and species composition
of dead birds that come ashore as a result of an offshore oil spill is
included in Appendix 8. This methodology was developed as part of the
Central California Oil Spill Contingency Plan for the Gulf of the
Farallones National Marine Sanctuary. Their complete plan includes
detailed strategies for obtaining reliable estimates of the total number
of dead, oiled birds by conducting surveys along a prescribed set of
coastal segments.

This methodology was developed from the experience of the Point Reyes
Bird Observatory in conducting damage assessments for impacts to marine
birds during the *Puerto Rican* and *Apex Houston* oil spills in coastal
California. The objective is to quantify the number of birds, live and
dead, impacted by the spill. Live, oiled birds are tracked through
records at rehabilitation centers. Estimates of dead, oiled birds must
be derived from counts of the dead birds washed ashore and estimates of
those that did not wash ashore.

There are currently two approaches in use to estimate the total dead
birds based on the number of carcasses recovered:

1\) A correction factor based on field experiments which show that only
5-25 percent of the birds that die at sea wash in or beach themselves on
shore (Jones, 1980; Page *et al*., 1982; PRBO, 1985). The correction
factor would be based on the spill conditions, which would increase or
decrease the likelihood of beaching of carcasses.

2\) A modeling system which simulates the oil slick trajectories and
marine bird distribution to predict the number of birds killed. The
model uses the beached bird census for calibration and verification. The
model was used to estimate total mortality from the *Puerto Rican*
(James Dobbin Associates, 1986), *Apex Houston* (Page and Carter, 1986),
and the *Exxon Valdez* (ECI, 1991) oil spills.

A team of trained observers should be identified to walk beaches
specifically to count oiled carcasses and document live oiled birds.
Photographic or video documentation is useful for providing evidence of
species and numbers of lightly and moderately exposed birds not able to
be captured. Prompt processing of film and labeling prints or slides is
essential. A \"databack" attached to the camera for identifying
individual frames is very useful, but photographs of hand-labeled cards
identifying each new location help identify and document each frame.

The beached bird census is designed to generate a best estimate for the
number of dead and live oiled birds along a beach segment on a specific
day. Tides, winds, and especially storms will bury carcasses in sand, or
rewash carcasses into the water to be deposited on a different beach or
lost through movement offshore or sinking. Many carcasses will be missed
during a single search, even on beaches which are relatively free from
debris. Several studies have documented the inability to find carcasses
even on clean beaches (Hope-Jones *et al*., 1970; Page and Carter,
1986). Because it is usually not possible to census the entire
shoreline, representative segments are used for more detailed surveys.
Data from these selected segments are then used to estimate the total
number of birds killed. The method includes:

o Dividing the coastline into regions by the general direction the
shoreline faces.

o Further subdivide the regions into beach segments by:

\- Beach type

\- Accessibility

o Classifying each beach segment according to the relative degree to
which it is predicted to accumulate and retain carcasses.

o Surveying each selected segment daily, recording all dead birds by:

\- Species

\- Age and sex, if possible

\- Cause of death

\- Carcass condition

o Marking each carcass in a distinctive manner, or removing it from the
beach.

o Recording length of censused segment.

Scavenging of carcasses may occur rapidly, and many birds may be lost
from beaches if gulls, otters, raccoons, opossums, foxes, or even dogs
and cats frequent the beaches. Loss rates can be extremely variable, and
are difficult to estimate without radiotelemetry of carcasses.

If logistics permit, fixed-wing aircraft or helicopter surveys of bird
populations in the vicinity of a spill are the best method to enumerate
most species at risk. Concentrations of ducks or other open water birds
are easily identified, as are breeding colonies of herons and egrets.
Gulls, terns, skimmers, and pelicans are also highly visible. Aerial
surveys are not very useful for marsh birds and other secretive birds
which cannot be seen from the air. Aerial surveys provide an excellent
method for tracking oil in remote or difficult areas, and provide data
for future beach search locations. The NRDA team should be prepared to
record observations of the numbers of birds in the vicinity of the spill
by species or species groups, early in the spill. Sometimes, these early
observations are the only data for estimating the number of birds
potentially exposed.

Specific bird observation forms can provide a framework for recording
observations, and provide entry blanks for data frequently overlooked by
field crews under emergency conditions.

Sublethally oiled birds, especially shorebirds and marsh birds, will
usually try to avoid humans, keeping out of sight to avoid capture.
Birds which are vulnerable to raptors or gulls, such as sandpipers and
other small shorebirds, are particularly difficult to document. Several
techniques have been devised to estimate the number of oiled birds from
limited recovery data (Hope-Jones *et al*., 1970; Bibby and Lloyd, 1977;
Piatt *et al*., 1990), but each spill has unique characteristics
limiting generalized formulas for estimating losses.

In addition to the immediate losses due to bird mortality during the
spill, the impacts will be continued into the future in terms of:

o Loss of reproductive capacity of the dead birds. Using the natural
reproductive rate, future annual losses can be calculated.

o Loss of reproductive capacity of oiled and released birds. Research
has shown that oiled birds have a very low successful nesting rate for
the nesting year that the spill occurred. Therefore, the number of oiled
birds observed in the field or released from rehabilitation centers
should be multiplied by the expected successful breeding rate to
determine the lost reproductive capacity.

o Losses from oiled nesting colonies. Contaminated eggs and the future
losses to the adult population should be considered.

o Losses associated with decreases in prey availability.

The objective of these studies should be to quantify impacts to each
affected species, in terms of the number of animals killed, sublethal
impacts, and recovery rates. The majority of these studies cannot be
carried out by anyone but trained experts. However, the NRDA responder
can assist these trained experts by documenting any known impacts and
keeping track of animal groups that are within or near impacted areas
during the early phases of a spill, as well as encourage systematic
surveys and documentation and provide forms to other observers involved
in bird rescue and surveys.

**ASSESSMENT OF INJURY TO MARINE MAMMALS**

**Summary of Known Impacts from Oil Spills**

Marine mammals spend most or all of their lives in the ocean and have a
number of behavioral, anatomical, and physiological adaptations that
enable them to live in the ocean. One of the most important adaptations
in relation to oil spill response is the various mechanisms used to
control body temperature. The ability to maintain normal body
temperatures when immersed in relatively cool water is the greatest
single factor affecting the survival of individual mammals. Whales and
dolphins are believed to be relatively insensitive to spilled oil,
because they rely entirely on blubber and skin for thermoinsulation, and
the insulative properties of blubber and skin are not affected by
exposure to oil, however, the furred mammals such as sea otters, seals,
and sea lions are severely impacted when exposed to oil.

To review the known impacts of oil spill on marine mammals, the
information must be separated into the three marine mammal groups.
Although there are some general similarities relative to oiling, the
impacts vary between mammal groups. These groups are:

o Cetaceans (whales, dolphins, and porpoises)

o Pinnipeds (seals, sea lions, and walruses)

o Sea otters

There are three anticipated interactions that may occur for marine
mammals when exposed to oil. These include:

1\) direct surface fouling;

2\) ingestion; and

3\) inhalation of the toxic vapors.

The pertinence of these three methods of exposure or pathways is
presented for the three marine mammal groups below.

**Cetaceans.** In general, whales, dolphins, and porpoises are
considered to have the ability to detect and avoid oil and other
petroleum hydrocarbons. The extent and effects of ingestion of oil by
dolphins are unknown. There is evidence of oil accumulation by toothed
and baleen whales, but there are no data on any lethal or sublethal
effects of either ingestion or accumulation in tissues. There are also
few data on the effects of direct contact of oil on sensitive tissues.
Direct long-term exposure of gasoline on dolphin skin produces mild,
transient damage; effects on eye or nasal membranes are unknown. During
the early stages of large spills, there could be general anesthetic
effects of volatile hydrocarbons on breathing in marine mammals (Geraci,
1990). After the *Exxon Valdez* oil spill in Prince William Sound,
thirteen members of a very stable pod of killer whales disappeared. It
has been postulated that the whales could have been killed by exposure
to the volatile hydrocarbons during the early days of the 10.8 million
gallon spill (Dahlheim and Matkin, 1993).

Observations during spills have documented few direct impacts of oil
spills on dolphins. It is thought that dolphins tend to avoid large
slicks; captive dolphins are known to actively avoid floating oil.
During the June 1990 *Mega Borg* oil spill, surveys of the distribution
and behavior of dolphins during the spill were conducted by NOAA
scientists (Mullin *et al*., 1992; Smultea and Würsig, 1992). They found
that dolphins did not appear to detect oil slicks, though they were able
to detect and avoid areas covered with emulsified oil. There were no
observed marine mammal mortalities. For more information on the impacts
of oil spills on marine mammals, refer to Geraci and St. Aubin (1990)
and RPI (1987a).

**Pinnipeds.** All walrus, seal, and sea lion species are considered to
have the ability to detect and avoid oil and other petroleum
hydrocarbons. To date, no studies have been conducted to determine their
detection abilities. However, in the wild, there have been many
incidents where seals, sea lions, and fur seals have entered into oiled
areas, not seeming to notice the oil slicks. Numerous deaths have been
related to direct and indirect exposure of seals and sea lions to
petroleum hydrocarbons (St. Aubin, 1990).

Most pinniped species rely on thick layers of blubber to protect them
from the cold water they live in and to retain their core temperatures.
Surface oiling, as with cetaceans, does not present a thermoregulatory
problem. Direct surface contact for most pinnipeds is suspected to
result in local irritation of a temporary nature. However, several
furred pinniped species, such as the northern fur seals, are susceptible
to surface oiling. With the reduced insulative properties of their fur,
hypothermia often results (St. Aubin, 1990).

Little information is available regarding the effects or impacts from
pinnipeds inhaling the volatile, toxic fractions of petroleum
hydrocarbons. As with cetaceans, there could be general anesthetic
effects of volatile hydrocarbons on breathing in pinnipeds during the
early stages of large spills. The consumption of petroleum hydrocarbons
has been implicated in numerous seal and sea lion deaths. Experimental
results have revealed a wide variety of effects that may result from oil
ingestion by different species. Symptoms related to oil ingestion range
from organ diseases to permanent damage or death (St. Aubin, 1990).

**Sea Otters.** Sea otters are the smallest marine mammals and also the
most susceptible to the impacts from an oil spill. Numerous deaths have
been related to direct and indirect exposure of sea otters to petroleum
hydrocarbons. Sea otters have no subcutaneous blubber layers and depend
entirely on their fur pelage for insulation. If their coats become even
partially oiled, they usually succumb to hypothermia (Geraci and
Williams, 1990).

Inhalation of hydrocarbon vapors results in a wide variety of effects
ranging from mild irritation of mucous membranes to permanent damage or
even death. Ingestion of petroleum hydrocarbons also results in organ
diseases to permanent damage and death. There are many behaviors and
habits which predispose sea otters to exposure (Geraci and Williams,
1990).

**Field Methods for Population/Behavioral Changes**

Field methods for assessing injuries to marine mammals as a result of an
oil spill will be a function of the size and type of spill, the spill
trajectory, and the seasonal distribution of the animals. Expert marine
mammalogists will certainly be involved and use existing studies as a
basis for conducting impact assessments. A general outline of the types
of studies to be conducted follows:

o Aerial surveys of marine mammal populations and distribution patterns
during and following the spill, for comparison with pre-spill survey
data. These surveys are conducted by trained observers who fly along
line transects at set altitudes and time periods so that the viewing
zone is a known area. Observations on the number of individuals, groups,
behavior adjacent to slicks, etc., are recorded either on sighting forms
or directly into portable computers.

o Quantification of number of dead animals, by shoreline and boat
surveys and recoveries by clean-up crews.

o Sampling of dead animals using established protocols.

o Necropsy of dead animals and collection of tissues for histopathology
and toxicology, with control tissues of animals from clean areas if
possible.

o Relation of distribution patterns and mortality data with slick
trajectories, shoreline contamination maps, and other observations on
oil distribution.

o Observations of sublethal and behavioral responses to oiling.

o Long-term surveys to examine longer-term impacts, through study of
movements, reproduction, survival, and dispersal.

o Assessment of potential impacts of the spill on prey items.

The NRDA responder can assist in the marine mammal counting surveys if
training in species recognition has been provided. Field guides and
field marks for local species are important tools. The collections of
samples from impacted species should be left to the expert mammalogists
and veterinarians. Therefore, where marine mammals are potentially at
risk, preplanning and advance contact with experts should be undertaken.

**Proviso:** Permits may be needed, either through the Fish and Wildlife
Service or the National Marine Fisheries Service. Any assessment
activity should be cleared with these agencies first.

**ASSESSMENT OF INJURY TO TERRESTRIAL MAMMALS**

**Summary of Known Impacts from Oil Spills**

Very little is known about the impacts of oil spills on terrestrial
mammals such as river otters, beavers, muskrats, minks, nutrias, and
raccoons. The most likely impact would be contamination of fur and
ingestion of oil during preening. Studies are being conducted on
farm-bred minks as part of the *Exxon Valdez* damage assessment, and the
results of this study will greatly extend our knowledge of lethal and
sublethal impacts from oil ingestion on small mammals. Though the *Exxon
Valdez* damage assessment has been settled, many of the study results
have not yet been made public.

**Field Surveys for Population/Behavioral Changes**

Inland and coastal spills can have impacts to a wide variety of
terrestrial and aquatic animals by direct oiling and loss of
habitat/prey, particularly those which feed on aquatic organisms and
vegetation or use aquatic habitats for shelter. Field surveys are
important in documenting that exposure and mortality has occurred to
each species of concern. It may be necessary to search for dead animals
for necropsy and collection of tissues for chemical and histological
analysis. Field methods for damage assessment would be very similar to
those used for ongoing studies of each species. There are two
approaches:

1\) Surveys of the affected areas to count the number of animals killed
(body count) by the spill. Transects or quadrats would be systematically
surveyed to count/collect dead or oiled animals. The total number of
animals killed would be extrapolated from the sampled data, using actual
mortality rates for the known survey area modified with correction
factors to account for differences between the surveyed area and the
entire spill impact zone.

2\) Impact versus control (or pre-spill) surveys to determine impacts
based on changes in population distribution.

The appropriate method should be selected based on the behavioral
characteristics of each species and availability of historical
population distribution data. High natural variability may be factored
out by using experimental data or observations from other incidents to
assume type and frequency of impact. For example, using normal species
reproductive rates and density for a certain type of habitat, and the
assumption of a 50 percent loss in reproductive success based on
bioassay data, the number of young lost can be estimated if the
reproductive population and area of impact are known. This approach is
oftentimes preferred, rather than having to determine the actual
reproductive rate in the impacted area versus control or historical
data. Measurement of significant differences between impacted and
control, particularly for larger animals with low densities and long
lifetimes, is extremely difficult.

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