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OCR for page 135
135
Remote sensing of dissolved fossil fuel compounds in seawater
appears to be several years in the future and should naturally emerge
from the more general basic research into reroute sensing of chemical
and b iolog ical components of the oceans .
Sampl ing Techniques
The b ias introduced by var ious sampl ing protocols should be r ecogn ized
explicitly. The mechanics of sampling needs close attention in order
to maximize useful data. For example, many grab samples and gravity
cores taken after oil spills did not contain the "flock ~ eye r at the
sediment-water interface, thereby introducing severe doubt as to whether
or not petroleum compounds had reached the benthic ecosystem.
Large volume water samplers designed to avoid contamination during
the sampling process have been developed and should be more extensively
used to obtain useful samples.
In situ, or on deck, water pumping systems capable of obtaining
samples of water for analyses of dissolved and particulate compounds
should be sub ject to further deployments in a variety of oceanic and
coastal regions to further evaluate their usefulness. Initial tests
are quite favorable and suggest that these systems will prove useful to
studies of fossil fuel compounds in the water columns.
PART B
Biological Methods
INTRODUCTION
An impressive amount of research has been done during the past decade
on uptake and effects of petroleum by single species of organisms under
controlled laboratory conditions. In fact, the methods for exposing
organisms are now technically sophisticated in some cases. However,
relatively few experiments have been conducted in the field to validate
laboratory findings. Because of the inadequate comparison of results
of laboratory experiments and postspill f ield investigations, the
specific knowledge needed for predicting the impact of acute petroleum
pollution in the mar ine environment is not yet available. Fortunately,
the study of marine mesocosms, i.e., scaled living models of natural
ecosystems, is a promising new means for developing the needed
comparisons.
Physiological, Behavioral, Population, and Ecosystem Effects
Fundamental to the study of populations, communities, and their
habitats is the identification of species. Because species names are a
key to the biological literature, it is important to know exactly which
animals, plants, and microorganisms are involved in any given study.
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136
In general, physiological data are meaningful only when associated with
reliably identif fed species . Although costly and time consuming,
identif ication is a pr imary ob jective for both f ield and laboratory
studies. Specimen depositories permit verification of identifications
made in f ield surveys, before and after spills, and are vital for
physiological and biochemical analyses during and subsequent to spills .
In fact, depositor ies may provide the only means of establishing
validity of the data gathered at the time of a given event.
Not only is it important to know the species involved in a given
test system or event, the chemistry of the toxicant causing an impact
has to be thoroughly understood. Changes in composition and concentra-
tion during exposure need to be monitored to establish cause-and-effect
relationships.
After establishing the species and toxicant, the choice of methods
for any biological study is determined by the goals of the investiga-
tion, availability of instruments, familiarity of the investigators
with those instruments and methods, and the cost of the overall project.
The biological methods suounar ized in this report may be used in
connection with the following objectives: to measure effects on
physiology and behavior (a) on individual organisms, i.e., primarily
acute (short term lethal or sublethal) effects, and (b) on the life
cycles of organisms, including energy budgets, i.e., primarily chronic
effects on growth, development, and reproduction; to assess population
changes including (c) acute impacts and (d) subsequent changes in
populations; and to obtain information at the ecosystem level (e) for
experimental systems and (f) for natural systems.
Items (a), (b), (c), (d), and (f) are usually investigated in
connection with accidents or chronic discharges. Item (a) , but
particularly (b) and (e), are pr Oedipal approaches for searching out
causal and possible predictive relationships or explanations for the
effects of crude oils or their constituents. Although goal (d) is an
important environmental consideration, a more fundamental concern is to
maintain marine ecosystems (f) in a condition that allows them to be
utilized as society deems appropr late.
Problems of Exposure ~ Type of Oil, Weather ing, and Exposure Medium
Because of changes in composition that beg in as soon as oil is spilled
on the sea (see Chemical Methods section), experiments using unweathered
oils do not indicate those responses expected when the same organisms
are exposed to a-ted oils. ExPer iments designed to assess the impact of
,
oil must take this dispar ity into account.
The relative immiscibility of oil and seawater makes the quantita-
tive monitor ing of petroleum in aqueous bioassay solutions cliff icult .
As a result, several methods have been proposed and employed for the
preparation of oil-water bioassay mixtures or for simulating the type
of exposure to oil an organism might encounter in nature (also see
Laboratory Exposure ~Qethods section) .
Behavioral, bioassay, and bioaccumulation studies using organisms
exposed to weathered petroleum in the laboratory are meaningful if they
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137
improve or broaden our understanding of the biological responses of
organisms in their natural habitats. The goal of such research should
be to measure biological effects of a specific compound, or mixture of
compounds of known concentration, on organisms under a prescribed set
of environmental conditions. Often, however, assessment of laboratory
results in terms of field situations is difficult because of the
complexity of the environmental factors involved (G.V. Cox, 19741.
Nevertheless, laboratory studies are important to explore potential
damage caused by various concentrations and exposure times for
pollutants and to assist in designing field studies.
METHODS FOR ASSESSING TOXICITY OF PETROLEUM TO MARINE ORGANISMS
Hi
A full appreciation of petroleum hydrocarbon concentrations that might
actually occur in a given water column, sediment, and/or food found in
different oil-contaminated marine environments is valuable in designing
effects studies. (See Chapter 4.)
In the laboratory, test organisms are best exposed to petroleum
hydrocarbon concentrations similar to those that might realistically be
expected to occur in a contaminated marine environment. A wide range
of exposure concentrations is used whenever possible, including environ-
mentally realistic concentrations and concentrations up to about 10-20
times higher than the latter. Higher concentrations are helpful in
eliciting obvious biological effects and are useful in estimating a
safety factor (difference between lowest concentrations eliciting a
response and expected environmental concentration), when environmentally
realistic concentrations do not elicit a measurable response.
Acute Lethal Toxicity Bioassay
The usual first step in evaluating the toxicity of petroleum and
specific petroleum hydrocarbons for marine organisms is the acute
lethal bioassay (Sprague, 1978~. This is a rapid screening method,
designed to provide an estimate of the relative toxicity of crude or
refined oils or specific hydrocarbons, to different species and life
stages of marine organisms. It is a rough predictor of the maximum
concentration of pollutant material that can be present in the marine
environment for an extended period of time without causing damage to
sensitive organisms and/or ecosystems (Sprague, 1971; Wilson, 1975;
Perkins, 1979~. Chronic bioassays, in which organisms are exposed for
longer periods (most of their life cycle or even for several genera-
tions), and studies of effects of chronic exposure to low concentrations
of pollutant materials on various biochemical, physiological, and
behavioral parameters in the test organisms, are more useful for
deriving maximum acceptable concentrations of pollutant materials. If
results of acute lethal bioassays show that a given pollutant is toxic,
studies of chronic, life-cycle, and sublethal effects are a useful
follow-up, as well as mesocosm studies, as appropriate, to establish
maximum acceptable concentrations.
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138
As methods for acute lethal toxicity bioassay protocols are
improved, eventually they will be standardized. At the present time,
several manuals and reviews are available in which such protocols are
described in sufficient detail to measure the toxicity of petroleum for
mar ine organisms (Amer ican Publ ic Health Association, 1977; American
Society for Testing and Materials, 1980; Environmental Protection
Agency, 1975a,b; Environmental Protection Agency/Corps of Engineers,
1977 ~ .
Flow-through, as opposed to static, petroleum bioassays are pre-
ferred if resources and constraints peculiar to the organisms of choice
allow. Several flow-through systems have been designed for use in
petroleum bioassays (Hyland et al., 1977; Vanderhorst et al., 1977b).
It is imperative in flow-through and static bioassays and in chronic
effects studies that the petroleum hydrocarbons in the aqueous phase in
contact with the test organisms be characterized and measured at
regular intervals.
The LC50 (median lethal concentration) is currently the term most
often used to report results of acute mar ine bioassays. The LC50 and
its error may be estimated by simple graphical methods (American Public
Health Association, 1977; Lichtfield and Wilcoxon, 19491, more precise
prob~t (Finney, 1971) , logit (Ashton, 1972; Hamilton et al., 1977), and
nonparametric (Stephen, 1977) methods and by methods that make use of
computer capabilities, e.g., BED 03S Fortran program (Dixon, 19701.
H.J.W. Anderson et al. (1980) recommended use of the product of
LCso and exposure time as a toxicity index, to compare toxicity of
d ifferent oils or sensitivity of different species. This method is
stil 1 under study.
If log time is plotted versus log LC50, a straight line can be
produced which can then be extrapolated to predict mortal ity for
exposure intervals likely to be encountered dur ing an oil spill.
Chronic and Sublethal Ef facts Studies
To study chronic and sublethal effects, employment of full 1 if e-cycle
assays are desirable but not always practical. In a life-cycle bio-
assay, test organisms are exposed over a complete life cycle, or a
major portion of it, to sublethal concentrations of a given pollutant.
Biological parameters usually measured include mortality, growth rate,
time to maturation, fecundity, offspring survival, and physiological or
genetic adaptation. The most sensitive stages in the life cycle of an
organism are detected and effects of the pollutant on sensitive and
ecologically important parameters such as growth and reproduction are
determined tHansen et al., 1978; Nimmo et al., 1977; Reish, 1980~.
Life-cycle bioassays using petroleum have been performed with poly-
chaete worms (Car r and Reish, 1977; Rossi and Anderson, 1978) and
crustaceans (Laughlin et al., 1978; W.Y. Lee, 1978~.
Besides the mentioned biological parameters, others have been
measured in an effort to define sublethal concentrations of petroleum
causing deleterious responses to marine organisms (J.W. Anderson,
1977a,b; Malins, 1977; Neff and Anderson, 1981~. The behavior of
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139
mar ine animals has been shown to be highly sensitive to petroleum-
induced perturbation. Methods are cited therein for monitor ing
behavior, chemosensing, locomotory, and feeding responses, among others .
Change in respiration (oxygen consumption) has been used frequently
as a Or iter ion of sublethal response of mar ine organisms to oil pollu-
tion. Results have been highly variable because a great many endogenous
and exogenous factors, other than pollution, influence respiratory
r ate . A fruitful approach is to combine respiration rate with other
biological parameters (food consumption, growth, and excretions to
construct an energy budget for the animal (Bayne et al ., 1976 , 1979 ;
Widdows, 1978 ~ . Several indices of stress can be der ived from the
energy budget, including scope for growth {energy available for growth
and reproduction) and growth eff iciency.
The ratio of oxygen consumed to nitrogen excreted (O: N ratio) can
also be used as an index of pollutant stress, although there can be
considerable variability arising from other environmental factors.
Nevertheless, it provides an estimate of the proportion of metabolic
energy derived from catabolism of proteins and amino acids. Bioener-
getics methods, or variations of them, of which the O:N ratio is an
example, have been used in several recent investigations of the effects
of sublethal concentrations of petroleum on marine animals (Capuzzo and
Lancaster, 1981; Edwards, 1978; Gilfillan and Vandermenlen, 1978; Johns
and Pechenik, 1980; Stekoll et al., 1980~.
Biochemical enzyme assays, pr imar fly of blood serum, are a powerful
diagnostic tool in human clinical medicine. Many of these enzyme
assays have been appl fed to t issue samples of mar ine an imals in an
effort to detect changes in enzyme activity attributable to pollutant
exposure, but such efforts have met with only limited success. This is
a growing f ield and of fer s great promise .
The activity of the microsomal cytochrome P450 mixed function
oxygenase (MFO) system of fish and, possibly, marine polychaete worms
is increased (induced) by exposure of the animal to petroleum and
selected aromatic hydrocarbons {Neff, 1979; Stegeman, 1981; Varanas i
and Malins, 1977~. Because it is induced by exposure to petroleum, the
hepatic MFO in fish has been recommended as an index of petroleum
contamination in the marine environment (J.F. Payne, 1976; Walton et
al., 1978; J.F. Payne and Fancey, 19823. Several other pollutants,
including heavy metals and chlorinated hydrocarbons, as well as natural
environmental and biological factors, may influence MFO activity. It
must be used with caution as a specific index of petroleum pollution,
because of the ef feats of other pollutants.
Acute or chronic exposure to petroleum may cause a variety of
tissue lesions, increased incidence of parasitism, or increased
susceptibil ity to bacter ial or viral disease. These can be detected
and evaluated by examination using the light or electron microscope
tHodgins et al., 1977; Sinderman, 1979~. Some success has been
achieved using the light and electron microscope for histopathology and
h istochemistry to detect sublethal damage in laboratory and f ield
populations of mar ine f ish (Blanton and Robinson , 1973; Hawkes , 1977;
DiMichele and Taylor, 1978; Payne et al., 1978; Hawkes et al., 1980;
Eurell and Haensly, 1981; Haensly et al., 1982) . These methods could,
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140
indeed, be useful for diagnosing characteristics of damage in marine
invertebrates and fish caused by pollutants, provided they can be
related directly to the pollutant.
Field Studies
There is a growing interest in adapting physiological, biochemical, and
h istopathological methods, such as those descr ibed above, for diagnosing
the state of health of f ield populations of mar ine animals in the vicin-
ity of oil spills or chronic oil inputs to the mar ine environment. Such
methods, if validated and adapted for field use, could be useful for
monitoring petroleum contamination of the marine environment. For
example, two large interdisciplinary programs currently involve devel-
oping and evaluating field monitoring methods. These include the
Pollutant Responses in Marine Animals (PRIMA) Program suppor ted by the
National Science Foundation and NOAA'S Ocean Pulse Program, now the
Northeast Monitoring Program. Suites of biochemical, physiological,
and histopathological tests provide a diagnostic profile of the health
of the test animal. Such characterization may prove more useful than
any single test for assessing pollutant stress in populations of marine
animals (McIntyre and Pearce, 1980~. There are problems in such an
approach, however, For example, plaice (Pleuronectes platessa) from
two estuaries heavily contaminated with oil from the Amoco Cadiz crude
oil spill were examined for histopathology, and a wide variety of
biochemical changes over a 2-year period were recorded. A progression
of biochemical changes and pathological lesions was observed, which
indicated an initial deal ine in the health of the f ish, followed by
improvement in these indices 27 months after the spill (Haensly et al.,
1982~. However, it is not clear that the effects were, in fact, due to
the oil alone. Thus, one must be confident that a cause-and-effect
relationship has been established.
Selection of Test Organisms
Several criteria are important in selecting test organisms for labor-
atory toxicity and accumulation/release studies. Because marine
organisms vary widely in their sensitivity to oil and ability to
metabolize and excrete petroleum hydrocarbons (Neff et al. , 1976;
Craddock, 1977; Varanasi and Malins, 1977; Neff and Anderson, 1981),
several species, representing different major taxonomic groups provide
a more useful system. Most frequently used are microalgae, polychaete
worms, bivalve mollusks, crustaceans, and fish.
Ideally, test species should meet several criteria. However, the
criteria for selection of a test species will depend on the question
being asked. At the minimum, the test species ought to be available in
large numbers, occur over an extended geographic range, represent
important members of the ecosystem, and come from, or represent, marine
habitats likely to be severely impacted by oil spills. Species used
for hydrocarbon accumulation/release studies ought to include taxa
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141
possessing different types of hydrocarbon metabolizing ability or
response.
Several lists of mar ine species have been descr ibed in the 1 itera-
ture which fulfill some or all of these criteria, and several of these
species have been recommended as standard bioassay/biological effects
test organisms, including the microalga Skeletonema costatum, the
copepod Acartia tonsa, the opossum shrimp Mysidopsis bahia and the
cyprinodont fishes Fundulus heteroclitus and Cyprinodon variegates
(Becker et al., 1973; Environmental Protection Agency, 1975a,b; American
Public Health Association, 1977; Environmental Protection Agency/Corps
of Engineers, 1977; Reish, 1980 ~ . In many cases, however, it is prefer-
able to use species indigenous to, or representative of, habitats of
particular concern, such as coral reefs, f ishing banks or continental
shelves, estuar ies, and arctic reg imes .
In the design of f ield studies, the choice of suitable exper imental
species may be limited by what is available locally at a field site.
Many of the Or iter ia used to select laboratory sub jects can be applied
here also. In most cases, particular species are especially suitable
for use in answering a particular environmental question; for example,
bivalve mollusks are good subjects for studying petroleum contamination
of biota because, in general, they accumulate hydrocarbons readily.
Preparation of Oil-Water Solutions
Test organisms can be exposed to petroleum in the laboratory in the form
of water-soluble fractions, oil-in-water dispersions, surface slicks,
oil-contaminated food, or oil-contaminated sediments. No single method
of exposure to petroleum is applicable for all marine organisms.
Exper iments using microorganisms require different approaches from
uptake studies with mar ine macroorganisms. The latter, in turn, need
different methods applied than those used with birds or marine mammals.
The following discussion describes how petroleum and its components
have been presented to a variety of marine organisms, recognizing, of
course, that specif ic me~hods often are needed for different organisms
or when different exper imental approaches are applied.
Preparations of petroleum solutions should represent situations that
can occur in the environment as a result of an accidental discharge of
petroleum or from chronic inputs. Many methods used in preparing
petroleum solutions for laboratory exposures can also be used for flow-
through systems, particularly when larger organisms held in aquaria or
tanks are to be exposed. Similarly, birds and mar ine manunals require
different approaches for exposure studies; the former has been reviewed
by Holmes and Cronshaw (1977) and the latter by Geraci and Smith (19777.
See Chapter 5.
Water-Soluble Fractions: Static
When oil is mixed with seawater, the oil can form macroparticles (dro~
let dispersions), microparticles (collodial dispersions and oil-in-water
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142
emulsions), and single-phase, homogeneous mixtures Water-soluble frac-
tions) of hydrocarbons. There are no definitive demarcations between
these states of dissolution, although arbitrarily, decisions have been
made, such as using filters having 0.45- and 1.2-um pore size to
differentiate between particulate oil (retained on the filter) from
subparticulate and soluble oil (passing through the filter) (Gordon et
al. 1973; Wells and Sprague, 19761. However, reaggregation may occur
after filtering. Recent developments resulting in improved chemical
analyses have permitted a more critical distinction between states of
dissolution.
Published accounts of laboratory exposure studies conducted through
the mid-1970s frequently described a test solution as a Water-soluble
fraction" (WSF). Unfortunately, many of the reports contain no
descr iption of the exposure medium, whereas in others, an attempt was
made to define the water-soluble fraction by reporting chemical
analyses of only the major hydrocarbon compounds, providing limited
data on oil particle sizes, and results only of visual examinations of
the clarity of the fractions. Such information is inadequate because
oil particles of 100 um diameter or less are not readily discernible
to the human eye (Nelson-Smith, 1973), and oil droplets smaller than
1-2 um in diameter remain suspended in seawater for hours or days
(Parker et al. 19713--much longer than the settling period used
routinely in preparing water-soluble fractions. In addition to the
problems cited above, it is difficult to determine whether water-
soluble fractions used in the tests reported in the early literature
were truly single-phase solutions, dispersions of fine droplets of oil
in water, or a combination of these, described as "accommodated" by
Gordon et al. (1973) and R.C. Clark and MacLeod (19771. Unfortunately,
an additional difficulty is that most dispersions of oil and seawater
are unstable over time.
A water-soluble fraction is an artificial mixture and cannot be
used to simulate precisely the conditions of hydrocarbon composition
and concentration in a water column when oil is spilled in the marine
environment. Equilibration conditions in nature may be quite different
from those used to produce water-soluble fractions in the laboratory.
The water-soluble fraction represents a compromise, a means of gener-
ating a highly reproducible and relatively stable oil-in-water mixture
and is, therefore, very useful when comparing the relative toxicity of
different crude and refined petroleums for marine organisms.
Laboratory studies have employed low-molecular-weight aromatic
hydrocarbons because of their relatively high, short term toxicity for
marine organisms; however, in the case of oil spills the partitioning
of the simultaneously volatile and soluble low-molecular-weight hydro-
carbons is a dynamic process, dependent upon a set of parameters unique
to each spill {e.g., water and atmospheric mixing energies, temperature,
salinity, presence of natural and human-contributed polar materials in
the seawater, and type of petroleum. See Chapter 4 for details.
Table 3-4 provides a summary of data for four American Petroleum
Institute (APT) reference oils and Prudhoe Bay crude oil employed In
many studies in recent years. The compositional analysis of the whole
reference oils is compared to that of the water-soluble fraction,
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143
prepared by mixing one part of oil with nine parts of seawater for 20
hours. Analyses of water from the Prudhoe Bay crude oil exposures are
quite different, since a flowing exposure system was used in extracting
the latter. The e-paraffin compounds, which range in carbon chain
length from 12 to 24, represent a large amount of the total measured
components in the oils, even though their contribution to the water
extracts is relatively small.
Measurements of monoaromatic compounds present in the oils and their
extracts are not always quantitative, and in fact, low-bo~ling compo-
nents frequently are not measured in the oil. However, the contribution
of monoaromatics to the total concentrations of hydrocarbons in water-
soluble fractions is significant if they occur in fresh oil; this is
particularly true for crude oils that have not undergone any refining
process. From an examination of the concentrations present in the
water extract, the contribution of compounds higher in molecular weight
than the alkylnaphthalenes is very small and may not be significant in
producing acute toxicity. One could conclude that either the mono-
aromatics or the diaromatics are the major contributors to the acute
toxicity associated with these extracts.
Table 3-5 was prepared to summarize the data in Table 3-4 as
percent of the classes of compounds, relative to the total amounts of
hydrocarbon actually measured. The percent composition of individual
hydrocarbons routinely measured in the oil by environmental chemists is
relatively low (5-15%) in compar ison with the numbers of compounds
present (Mel ins, 1980 ~ .
Water-soluble fractions have been prepared by stirring varying
ratios of petroleum compounds and experimental media for varying per lads
of time and allowing these to stand so as to err ive at a stabilized
water-soluble fraction. Stirr ing times range from several hours to
days, e.g., 12 hour stirring (Kauss and Hutchinson, 1975) and 72 hour
stirring (}Mahoney and Haskin, 19801. Following an equilibrium period
of several minutes (e.g., Winters et al., 1977; Pulich et al., 1974) to
several hours (e.g., Kauss and Hutchinson, 1974 ~ for separation of the
aromatic and aqueous phases, the aqueous phase may be f iltered through
materials which range from glass wool to 0.45-pm Mill~pore(R) filters.
Subsequent dilution with filtered or unfiltered seawater or media
provides a range of concentrations. Soto et al. (197Sa,b) determined
that the type of stirring affects the composition and, therefore, the
biological effects of a petroleum extract, whereas Wells and Sprague
(1976) determined that the type of stirring affected the concentration
of the extractable organics measured by W and fluorescence (i.e.,
aromatics) and, hence, influenced the toxicity of the preparations.
If mixing conditions are carefully standardized, highly reproducible
results can be obtained in preparing water-soluble fractions (Linden et
al., 1980~. However, chemical and physical characteristics of the
petroleum will affect the actual composition and concentrations of
hydrocarbons in the water-soluble fraction preparations (J.W. Anderson
et al ., 1974 ; Neff and Anderson, 1981) . Inasmuch as the oil-water
partition coefficients of hydrocarbons favor retention in the oil phase,
and evaporation and solubilization are competing processes, the aqueous
phase of a water-soluble fraction never becomes saturated with hydro
OCR for page 144
144
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Representative terms from entire chapter:
oil spill
