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Oil in the Sea: Inputs, Fates, and Effects (1985)

Chapter: Part B: Biological Methods

« Previous: Part A: Chemical Methods
Suggested Citation:"Part B: Biological Methods." National Research Council. 1985. Oil in the Sea: Inputs, Fates, and Effects. Washington, DC: The National Academies Press. doi: 10.17226/314.
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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.

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

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.

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

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,

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

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

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,

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

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146 carbons. Thus, although naphthalene has a solubility of about 20 mg/L in seawater (Ross) and Neff, 1978) and No. 2 fuel oil (API reference oil) contains 4,000 mg/L naphthalene, a water-soluble fraction prepared from No. 2 fuel oil contains only 0.84 mg/L naphthalene (Neff and Anderson, 1981~. Salinity of the test medium has little effect on the rate of loss, but temperature has a marked effect (Laughlin and Neff' 19791. For example, half times for naphthalene loss from a 15% water-soluble fraction vary from 3-5 days at 20°C to 2-4 days at 30°C. Loss of naphthalene from the aqueous phase is caused pr imar fly by volatilization. It is clear that hydrocarbons in static tests are lost from the test solution (Vandermealen and Ahern, 1976~. To solve this problem, one can turn to flow-through systems. Water-Soluble Fractions: Flow Through Several attempts have been made to design a continuous flow system for use in petroleum exposure studies. The extreme compositional complex- ity of petroleum and its unusual behavior in water make the design of such a system difficult. A major problem has been, as in static systems, to maintain a constant hydrocarbon concentration and compo- sition in the exposure tanks. Oil slicks, insoluble oil residues, or emulsions may accumulate in parts of the system over time, altering properties of the dispersed or soluble aqueous fraction. Weather ing of oil and buildup of oil-degrading bacteria in the flow-through system may also pose problems. The simplest flow-through systems utilize stock water-soluble fractions, prepared daily or more frequently, which are infused at a constant rate into and mixed with the inflowing seawater. By con- trolling flow rate of dilution water and water-soluble fraction, one can achieve precise exposure concentrations. Prepar ing the water- soluble fraction frequently makes possible the maintenance of relatively constant hydrocarbon concentration and composition. Examples of recent investigations using this approach include those of Moore et al . (1980 and Capuzzo and Lancaster (1981~. An elaborate flow-through solubilizer system was devised by Roubal et al. (1977a) to prepare water-soluble fractions of crude oil for months-long exposures of marine organisms. The system gave an uninter- rupted flow of seawater extracts of Prudhoe Bay crude oil for test periods of up to 5 weeks, with a total hydrocarbon range from 5 to 6 mg/L. The diluted seawater extracts exhibited no Tyndall Effect and were clear. The water-soluble fraction was free of alkanes, which implied the virtual absence of suspended droplets of undissolved oil (Roubal et al., 1977a). Another system, built by Krugel et al. (1978), produced a saturated solution of the water-soluble components of a jet fuel oil (JP8 I, free from droplets of undissolved oil, for flow-through bioassay tests. Water, as droplets, flowed through five consecutive vertical columns containing the oil. One of the advantages of the apparatus was its simplicity: the entire system was gravity fed with no pumps or regulators needed.

147 Giam et al. (1980) developed a dosing method for exposing marine organ isms to low levels of poor ly water-soluble compounds in a recirculating system using a column containing sand coated with a contaminant. Another type of solubilizer, described by Nunes and Ben~rille (1979a,b) and Benville et al . (1981), consisted of an oil reservoir (Figure 3-6a), oil pumps, modif fed solubilizer glass bottle (Figure 3-6b), and oil waste reservoir. The water-soluble components of crude oil were dissolved without serious loss of more volatile compounds and without the formation of emulsions or oil droplets, and the apparatus could be assembled as a recirculating system (Figure 3-6c). Benville and Korn (1974) and Maynard and Weber (1981) described simple devices for metering low-molecular-weight hydrocarbons into seawater for use in either static or flow-through experiments. The devices produced water-soluble fractions with constant concentrations of low-molecular-weight aromatic hydrocarbons and reduced emulsion formation. Oil- in-Water Disper signs Oil-in-water dispersions were used in the early 1970s, particularly for short term static exposures and acute toxicity bioassays. These provide higher concentrations of oil in water because the Volubility of oil can be exceeded. Interpretation of data from this early research was often difficult because of uncontrollable variations in exposure conditions or lack of reproducible results (Varanasi and Malins, 1977~. Methods for preparing oil-in-water dispersions included mixing oil with seawater and shak ing, stirr ing, blending, ultrasonically emulsifying, passing through baffle plates, and turbulent mixing in a water jet. The stability of oil-in-seawater dispersions is dependent upon a number of factors, including the size of the droplets and the presence of air or vapor space above the dispersions. For instance, very fine (<1 um) oil droplets may stay in suspension for long periods, although some loss occurs from volatilization. Different hydrocarbon classes and molecular weight ranges are selectively partitioned between aqueous and oil phases (Boylan and Tripp, 1971~. For example, the low-molecular-weight aromatics (e.g., benzenes and naphthalenes) are more highly accommodated in the aqueous phase than higher weight aromatics. Consequently, in static exposures, organisms are initially exposed to relatively high concentrations of aromatic hydrocarbons in the water, but because of their volatile nature, these aromatics are soon lost through evaporation. Emulsified oil-in-water preparations can be prepared by shaking oil in seawater on a mechanical shaker (J.W. Anderson et al. , 1974; Winters et al., 1977), by homogenizing without ultrasonification (C.K. Wong et al., 1981), or with ultrasonificatzon (Wells and Sprague, 1976; Gruenfeld and Freder lck, 1977 ~ . A continuous flow-through dosing apparatus for prepar ing water-acconunodated fractions of oil was descr ibed by Hyland et al . (1977 ~ . No. 2 fuel oil was metered into a horseshoe-shaped oil separation chamber in which a constant input of

148 SEAWAT tar 'fluid meter i ng pump AL \ INFLUEN] ~ '2..~. 1 A. Continuous flow system ER :3L SOLUBI LIZER SEAWATER INFLUENT _ n v stainless steel diffuser Dlate Hoffman // clamp /D vent '' tube ll~=il ~ vent Kuhn {2in. glass ears , holes ( 1/16 inch) _E F; LUE NT /~/~ ~ ~ / / SE AWATE R / / EFFLUENT // WITH WSF // _ ~1 ,~ Lo ) ~ S-shaped glass tube to 24/40 joint B. Solubilizer bottle ,' SEAWATER CRUDE OIL fluid metering pump C. Recirculating system using the solubilizer oil reservoir ~7 FIGURE 3-6 Solubil izer systems for exposing organisms to water-soluble fractions of crude oil: (a) continuous flow system (Nunes and Benville, 1979a), (b) solubilizer bottle (Whipple et al., 1978; Benville et al., 1981), (c) recirculating system (Nunes and Benville, 1979b) .

149 oil and unfiltered seawater was turbulently mixed and then allowed to separate over a series of baffles . The water-accommodated fraction was removed at the end of the chamber at mid-depth and distributed to the experiment in exposure tanks. Clement et al. (1980) combined features of the designs of both Hyland et al. (1977) and Roubal et al. (1977a) to simulate chronic exposure to dispersions, using an apparatus capable of simultaneously delivering dispersions with nominal crude oil concen- trations in water of 30, 300, and 3,000 ng/L. Only a relatively small fraction of the total oil added to seawater becomes dispersed as fine droplets in the water column during prepara- tion of an oil-in-water dispersion. For example, vigorous mixing of 100,000 pL/L of South Louisiana crude oil (API reference oil) with seawater produces a dispersion containing only 81.2 uL/L total _ , _ ~ _ _ _ _, _ _ , _, hydrocarbons (J.W. Anderson et al., 1974~. The hydrocarbon composition of the dispersion can be expected to resemble that of the parent oil, at least initially, as most of the hydrocarbons are still present in the form of dispersed oil droplets. Oil-in-water dispersions are usually unstable under static bioassay conditions (vice infra1. The concentration of total hydrocarbons in the water dispersions of South Louisiana crude oil decreases almost 90% within 24 hours during gentle aeration (J.W. Anderson et al., 1974~. Similar results have been reported by other investigators. Concentra- tions of aliphatic hydrocarbons in dispersions decrease more rapidly than concentrations of aromatic hydrocarbons because of a much lower aqueous Volubility of the former and as a result of their presence in oil droplets r ising to the surface. Petroleum dispersions in seawater that remain stable over periods of several months have been produced in an apparatus described by Vanderhorst et al. (1977b) (Figures 3-7a, b, and c). The oil and seawater are mixed as they are introduced through a funnel into the first compartment of the contactor. The mixture passes through a small hole in the side of the compartment into a second larger compartment, where the undispersed floating oil is separated by a baffle and discarded. The dispersion is directed to a second tank (Figure 3-7d), where baffles allow for further removal of undispersea ana suspenaea oil. Finally, the solution is directed to a metering tank (Figure 3-7e) where the flow and dilution factors for the exposure tanks are controlled by the size of the tubing and by gravity. Composition and concentration of individual and total hydrocarbons in dispersions produced by this system- have been measured (Bean and Blaylock, 1977; Bean et al., 1980; J.W. Anderson et al., 1980~. Monoaroma tic hydro- carbon concentrations var fed somewhat over time, but maintenance of relatively constant hydrocarbon concentrations in the water column over several days was quite good. Chemically dispersed oil can be prepared in this apparatus by adding the appropr late chemical at the initial mixing funnel (J.W. Anderson et al., 19817. Water-accommodated extracts prepared by hand or by mechanical shaking exhibit a similar range of var lability in preparation as that observed for water-soluble extracts. However, shaking or turbulent stirring yields an extract which includes oil in particulate form (Gordon et al ., 1973 ~ . Thus , the composition and concentration may

50 - L [A D Hi; b Glass Tube! Glass Bottle. 7 Elevation Dispersion Surface C' / Baffles ~< Fiberglass Tank Am,- ~9,< from Contactor Floating Oil Discard a ~ Seawater 1 No. 2 Fuel Water Level ~GIass Funnel Partition \1: Water Level -Water Level ~ / Baffle f,~,,_~,, Fiberglass Tank ~ ~ ,4 Ad. ~ ~ Dispersion Discharge ~ :~ Floating Oil Discard to Separation Tank Fiberglass Tank Dispersion Supply Dripper Arm for :\~ Flow Adjustment A \ al:' Dispersion Discharge to Metering A_ ~ Dispersion Col lection - , :: Am' e ~' Floating Oil Discard FIGURE 3-7 Fuel oil dispersion and bioassay apparatus: (a) arrangement of components, (b) fuel oil metering, (c) contactor, (d) separation tank, and (e) dispersion metering tank. SOURCE: Vanderhorst et al. (1977b).

151 differ considerably as a result of inconsistent equilibr ium times. AS specific fractions of oil (aromatics) appeared to elicit pronounced cellular and physiological responses from phytoplank ton populations, Winters et al. (1977), Pulich et al. (1974), Karydis (1979), and Batterton et al. (1978), among others, obtained and tested specific fractions of a variety of hydrocarbons by either distilling and collecting the required fractions or driving off specific fractions, such as volatile aromatics by heating or bubbling with air. Subsequent handling of the oil-water preparation, such as preparing a dilution ser ies, may also result in change in composition and concentration. There are other factors to be considered, aside from the phys ical, such as avoiding contamination of preparations, ach ieved by use of axenic (bacter ia-free) cultures . Prouse et al . (1976) found a significant change in the C17:pristane ratio within 30 minutes of adding algae to the media--suggesting bacteria degradation of the n-alkane, relative to the isoprenoid. Such variation in oil-water preparation methods and subsequent handling, reported above, make interpretations of the presence or absence of effects difficult. AS pointed out by Vandermuelen and Ahern (1976), Prouse et al. (1976), O'Brien and Dixon (1976), and Corner (1978), among others, the specific details of the methodology used in each experiment must be known--specifically, the method of extract preparation, whether sterilization or filtration was used, and if the actual concentration and composition of both the extracts and the mixtures used for experiments were determined analytically. Filtering, heating, or autoclaving whole oils or their extracts in an attempt to obtain sterile preparations for use with axenic cultures further modify composition and concentration (Vandermuelen and Ahern, 1976), although Prouse et al. (1976) reported that autoclaving whole oils in bulk did not appreciably alter the aromatic content or relative concentrations of n-alkanes. Surface Slicks: Laboratory In the case of surface slick exposure studies and bioassays the oil is usually poured onto the seawater surface in the exposure chamber, and the oil components are then allowed to leach into the underlying seawater without mixing. The seawater underneath the slick may not necessarily be changed over the duration of the exposure (Clark and Finley, 1974a; Wells and Sprague, 1976~. It may be exchanged in a flow-through system where the seawater contaminated by the slick is slowly exchanged or diluted with uncontaminated seawater (Bott et al., 1976; Taylor and Karinen, 1977; Shaw et al., 1977; Payne et al., 1978~. Another type of flow-through system allows fresh seawater to fall through the surface slick and contaminated seawater to be drained from the bottom of the exposure tank (Eisler, 1975; Rinkevich and Loya, 1979 ~ . Direct additions of crude oil as a surface slick were used by Shiels et al. (1973) with natural phytoplankton populations. Lacaze (1974) added crude oils with and without emulsifiers to outdoor

152 mesocosms containing natural populations. Laceze and Villedon de Naide ~ 1976 ~ made direct additions of fresh crude oil, but also aged or weathered the oil in the 1 ight and afar k in open and closed f tasks as a means of assessing effects on growth arising from photooxidation and/or losses of volatile fractions . Again, in water slick exposure exper i- ments, quantif i cation of the hydrocarbons enter ing the water phase is essential . Oil in Food Var ious investigators have incorporated petroleum, ref ined products, or fractions thereof into food for the organisms; exposure is via consumption of contaminated food (Corner et al., 1973; Hardy et al., 1974; Roubal et al., 1977b; Whittle et al., 1977; Varanasi et al., 1979~. Another means is to introduce oil through the food web via an intermediate step, such as feeding oysters containing radiolabeled hydrocarbons to crabs (R.F. Lee et al., 1977), or dimethylnaphthalene- contaminated detr itus to benthic deposit-feeding organisms (Roes) jadi et al ., 1978 ~ . In another example of food web transfer (Mel ins and Roubal , 1982 ), a tr itiated 2 , 6-dimethylnaphthalene , accumulated by Fucus sp. algae from seawater without conversion to metabol ites, was fed to sea urchins. Waldon et al. (1978) have used oiled food in MFO induction studies in fish. Oiled Sediment Studies Starting in the early 1970s, laboratory research was initiated to study the uptake of hydrocarbons from oiled sediments that serve both as a habitat and as a food-containing segment of the food web. Various devices have been used for expos ing bottom-dwell ing organisms to oiled sediments, ranging from simple beakers (Prouse and Gordon, 1976; Wells and Sprague , 1976 ; Gordon et al ., 1978 ~ and aquar ia (Taylor and Kar inen , 1977 ; Howgate et al ., 1977 ), polyvinylchlor ide trays (J .W. Anderson et al., 1977), and aluminum pans suspended in aquaria (Straw et al., 1977), to systems containing aquaria within aquaria or water tables, often with provisions for tidal simulation (Mc'Cain et al., 1978; J.W. Anderson et al., 1978; Roesijadi and Anderson, 1979~. Studies have been conducted in which externally oiled sediments were placed in the test tanks (Taylor and Rar inen, 1977; J.W. Anderson et al ., 1977 , 1979; Rossi, 1977; Howgate et al ., 1977; BUSdosh et al., 1978; McCain et al., 1978; Gordon et al., 1978; Roesi jadi and Anderson, 1979; Varanasi and Gmur, 1981a,b) or where clean sediments were con- taminated by oil slicks deposited by simulated falling tides (Prouse and Gordon, 1976; Taylor and Karinen, 1977; Shaw et al., 1977) . McCain et al. {1978) described a typical laboratory system for exposing flatfish to oiled sediments for extended periods of time. Crude oil was mixed into wet sediment as a sediment-oil-seawater slurry in a commercial cement mixer . The sediment, initially containing 2 ,000 mg/L oil was placed on the bottom of an aquarium situated inside a

153 second aquar ium. Uncontaminated seawater was percolated through the oiled sediment. The concentration of oil dropped to 700 mg/L after an overnight flushing in running seawater before the addition of test fish. After 4 months under running seawater, the concentration in the sediment stabilized to cat 350 mg/L {total extractable petroleum- derived hydrocarbons), at which time the fish were introduced. In another approach to preparing contaminated sediments, J.W. Anderson et al. (1979) first emulsified the oil in seawater using a blender at "high. speed before adding the suspension to the sediment in a cement mixer. Solid intertidal substrata (e.g., rock, natural floating materials, artificial substrates) can be removed, complete with the attached organisms and placed in a laboratory environment in order to expose sessile microorganisms to petroleum pollutants. The size of the exposure chamber limits the amount of substrate used. Further, care must be exercised in transporting the attached organisms to minimize phys iolog ical stress . BACTERIA, YEASTS, AND FI=MENTOUS FUNGI Methods for Estimating Microbial Numbers and Biomass The microbial population size, i.e., numbers or biomass of microbes, must be measured so that changes can be normal ized for total microbial numbers or biomass to quantify changes in microbial populations resulting from interaction with oil. This is particularly important for natural populations. Microbial interactions with oil may increase or decrease the number of petroleum-transforming microorganisms (PTM). In general, lethal events are not measured when aerobic heterotrophic microorganisms interact with oil, especially after the oil has aged and the lighter solvent fractions have evaporated. The immediate response of a micro- bial population to addition of oil is an enrichment of PTM, both in relative and absolute terms. In fact, such an increase in number of PTM has been used to practical advantage for locating oils (Atlas, 1981). Epifluorescent direct counts are useful for estimating total numbers of bacteria and fungi (Zimmerman and Meyer-Reil , 1974 ; Daley and Hobble, 1975; Por ter and Feig, 1980~. Results of direct counts provide a reference to which PTM counts can be compared. Epifluorescent tech- n agues may also be used to assess growth of bacter ia on a specific carbon source, e.g., petroleum, in the presence of nalidixic acid (Kogure et al., 1979~. The resulting elongated cells indicate the number of cells capable of growth on the substrate, in this case, petroleum. However, precise interactions of fluorescein dyes and nalidixic acid with petroleum are unknown. Jones (1981) successfully employed measurement of adenosine triphosphate (ATP) to estimate the biomass of pure cultures associated with petroleum, and Griffiths et al. (1981) did similar work with mixed cultures. The effects of petroleum associated with losses of ATP from

154 stress alone have not yet been quantif led, creating a potential source of error in this measurement. The most probable number (MPN) technique and the techniques 1 isted below provide indirect estimates of PTM after samples of water or sediment are inoculated into media containing petroleum as the sole carbon and energy source. The MPN technique appears to have been first applied to the estimation of PTM by Gunkel and Trekel (1967~. J.D. Walker and Col~ell (1976a) modified this technique by using antibiotic- supplemented media to provide separate estimates of counts for bacter ia and fungi growing on petroleum. They also compared results obtained using the MB?N method with those of several other methods listed below. The 14C-hydrocarbon method for estimating PTM has been used to estimate numbers as well as activity of PTM (Caparello and LaRock, 1975; Atlas, 1979 ) . A silica gel-oil (SGO) medium was developed independently by Seki {1973) and J.D. Walker and Colwell (1975b) as a method of estimating Pl?M growing on petroleum as the sole carbon and energy source. The advantages and disadvantages of agar plate procedures have been dis- cussed, and the silica gel medium was developed to eliminate problems encountered with growth of non-PTM on oil-agar. Oil-agar was first described by Barush et al., (1967~ but was modified and employed by Atlas and Bartha (1972) and J.D. Walker and Colwell (1973, 1975a) to estimate PTM in estuar ine samples. use of oil-agar as a medium in estimating PTM is not encouraged. If a solid medium is desired, SGO is r ecommended . However, problems of s ineres is and l iquefaction may occur if the medium is not prepared fresh before use. Recently, a medium was developed that permits detection of microorganisms using a given hydr~ carbon as the sole carbon and energy source, but the medium is 1 imited to those hydrocarbons r e .g ., phenanthrene, around which a hydrolytic zone is produced upon growth of microorganisms (Shiaris and Cooney, 1982 ) . As cited above, the number obtained and the biomass calculated from the numbers of cells enumerated must be interpreted, with the under- standing that the entire population is not cultured, and the organisms in culture are not capable of using oil as a sole carbon and energy source. Enumeration and biomass estimates of PTM are valuable as a means of compar ing differences among localities and times, particularly if dif- ferences observed are of several orders of magnitude. It is especially impor tent to note that, as indicator s of microbial response, the number of PTM normalized to the total population of aerobic heterotrophic microbes is more meaningful than the number of PTM alone. Methods for Estimating Metabol ic Effects of Oil on Microorganisms Not a great deal is known about the more subtle microbe-oil inter- actions. Some information exists on the effects of oil on the chemoreceptor system of bacteria and on the effects of oil on the biochemistry of sediment bacteria.

155 Methods employed to date for measuring metabolic changes in bacteria associated with oil are essentially modifications of standard methods employed in microbial ecology. Similarly, var iation in population species composition can be determined using taxonomic methods and analyses. Microbial processes commonly used in oil spill assessment include the changes in SO4 reduction, sulphide oxidation, N2 fixation, nitr~fication, denitrification, methane production, activity of enzymes "hydrolytic, chitinase, chitosanase, cellulose, and amylase), and ribo- nucleic acid (RNA) synthesis. Lipid content has also been examined. Physiological activity of microorganisms associated with oil can be estimated us ing ~eomicrobiolog ical methods or one of the following: (1) release of 1 CO2 from labeled glutamate; (2) heterotrophic potential, with changes in vmax measures; (3) change in specific activity of selected key enzymefs); (4) relative rate of change in RNA or DNA synthesis; (5) changes in adenylate pools, and/or change in ATP cell; and (6) alteration of a measurable function such as N2 fixation or N2O production. Methods for Obtaining Indirect and Direct Measurements of Oil Degradation Measurement of oxygen uptake, carbon dioxide evolution, and 14C- hydrocarbon degradation can be used to estimate degradation of oil indirectly. These methods do not require extraction of oil. Measure- ment of oxygen uptake is a s imple method that provides a r apid estimate of microbial activity for samples containing large numbers of micro- organisms and can be carried out by Winkler titration, or with a suit- able respirometer, or oxygen electrode (Bridle and Bos, 1971; Gibbs, 1972) . Measurement of CO2 evolution is also a simple method providing a rapid estimate of the activity of samples containing large numbers of microorganisms. CO2 can be quantified by titration of BaCO3 or by infrared gas analysis (Atlas and Bar tha, 19721. Use of oxygen uptake and CO2 evolution for estimating short term activity (i.e., minutes or hours) creates the problem of determining effects of oil, or oil degradation products, on endogenous respiration. Use of ~ 4C hydro- carbons eliminates this problem, because the fate of products resulting from the metabolism of the labeled substrate can be measured (e.g., evolution of )4C02) by incorporation of 14C into microbial cells and metabolic products and persistence of unreacted 14C hydrocarbon (J.D. Walker and Colwell, 1976b). Each method of determining activity is very much dependent upon the experimental conditions employed, including type of oil and the physical state of the oil being utilized and/or degraded. A variety of chemical methods can be used to monitor microbial degradation of oil. These include high pressure liquid chromatography, infrared spectrophotometry, ultraviolet and fluorescence spectropho- tometry, gas and/or column chromatography, and mass spectrometry. These are described elsewhere in this methods chapter.

156 PLANKTON Spatial and temporal distr ibution of plankton in the environment is constantly changing. Thus, demonstration of oil pollution effects is difficult in the field. Sampling design must be rigorously defined (Venrick, 1978a,b; Wiebe et al., 1973) if results are to be unequivocal. In every case, the concentration of the oil and its components should be determined. Furthermore, a defined control community is essential. In many field situations, rigorous experimental design may be impossible logistically, or prohibitive in cost. Nevertheless, for valid eco- logical predictions , laboratory experiments should be linked with field investigations (Wilson et al., 19741. General references providing useful methods for the study of phytoplankton include the publications of Sournia (1978) and for zooplankton, Edmondson and Winberg (1971) and Steedman {1975~. Plankton studies of an acute spill are desirable but will usually require extensive resources for adequate sampling and analysis. Both phytoplank ton and zooplankton are notoriously patchy in distribution, with variations often approaching an order of magnitude in normal, unstressed situations. A few samples taken in oiled and control areas are unlikely to yield statistically valid information concerning r elative standing crops. Continuous records of hor izontal distr ibution are highly desirable for preliminary surveys. However, there is some question as to whether instruments ordinarily used for this purpose (towed fluorometers for phytoplankton, Hardy-Longhurst samplers or electronic particle counters for zooplankton} will operate effectively in an oiled situation. This problem needs study. If continuous records cannot be obtained, intensive spot sampling of species composition and physiological properties may yield useful information on the effects of an oil spill. After the initial survey, resources should be focused at the popula- tion level, as well as on assessing changes in species composition of the community. Derived indices, such as diversity, should not be used alone as estimators of community health, in the absence of supporting data. More attention paid to effects at the cellular level and on modes of action of petroleum hydrocarbons on planktonic organisms will improve interpretation and prediction of environmental changes Wells, 1982). Many previous laboratory studies utilizing physiological responses of phytoplankton and zooplankton to assess the impact of oil have been undertaken under varying environmental conditions. Thus attention must be focused on maintaining known environmental conditions, such that the organisms being studied have a known history. A discussion of methods follows, but some of the methods cited may not be expedient in a spill, and judgment must be used. The ranges of size within each of the algal and animal plankton and nekton are such that one type of sampling device will not catch repre- sentative species of any of the three groups. Thus, f ield samples from a single sampling device cause an immediate bias. Moreover, the ranges of size and density of bacter ia, algae, and animals will overlap, not only with each other but also with that of particulate matter (i.e.,

organic and inorganic detritus, or tripton). Hence, only the smallest free-living bacteria, the larger zooplankton, and the nekton fall into size classes distinct enough to be estimated on the basis of mechanical separation. In contrast, samples of phytoplankton will always be heavily contaminated by bacteria, zooplankton, and nonliving organic matter. Nevertheless, the quantity of algae can be crudely estimated by chlorophyll measures, and their activity can be measured by their photosynthesis. Microscope analyses permit the separation and enumera- tion of algae, zooplankton, and tripton; allow estimates of biomass; and provide the means for identifying the species under study. Phytoplankton Field Methods Sampling procedures should be quantitative. Therefore, nets should not be used to collect all size classes of phytoplankton. Large species can be collected quantitatively with nets, provided clogging does not occur (Tangen, 19783. In working under oil slicks, nets and open water bottles generally employed for quantitative sampling can be subject to contamination when lowered through the slick. A method that was tried to avoid the surface slick problem was oblique towing (Grose and Mattson, 1977~. Sampling gear that can be opened below the surface should be employed if samples are to be analyzed for accumulation of petroleum products in plankton. Most routine laboratory analytical methods are amenable to field situations, given ideal working conditions (i.e., sufficient electrical power, space, and stability of the platform). However, serious con- sideration should be given to methods in which initial sample processing involves a minimum of equipment outlay and produces samples amenable to storage, with final analyses done in the laboratory. Historically, field work has included taking measurements of community composition from cell counts (Ignatiades and Mimicos, 1977; Wilhm and Dorris, 1966, 1968; R.F. Lee and Takahashi, 1977; Federle et al., 1979; G.A. Vargo et al., 1981), pigment concentration, and photosynthesis, principally by 4C uptake. Community composition and biomass determinations are critical for evaluating long term effects in phytoplankton communities, because changes in populations will result in changes throughout the food web (R.F. Lee and Takahashi, 1977; Federle et al., 1979; Elmgren et al., 1980a,b). Chlorophyll measurements are useful, but species counts can be used for estimating biomass. However, the lack of reliability of species counts should be recognized. Methods and precautions for sampling procedures, preservation and counting techniques, biomass conversions, and lists of taxonomic literature can be found in the Phytoplankton Manual prepared by Sournia (1978~. Measurement of photosynthesis, using either the oxygen evolution method or 14C uptake, has been the most widely used method for assessing community response to petroleum hydrocarbons {Gordon and Prouse, 1973; Shiels et al., 1973; Bender et al., 1979; Hsiao et al.,

158 1978~. Samples can be incubated in on-deck, seawater-cooled boxes retained in the sunlight and equipped with neutral density light screens, or attached to anchored and buoyed lines (Johansson, 1980~. Maximum photosynthetic rates (PmaX) normalized to chlorophyll will provide the most meaningful data. The index is independent of irradi- ance variability to some extent and permits rather unambiguous compari- son between stations and samples. Alternatively, measurement of photo- synthesis below light saturation, i.e., the initial slope, normalized to chlorophyll and light, is also recommended. Productivity rates, expressed as incorporation rate per volume and time (me Cm~3 hr~l), are less useful than normal ized rates. Additional information con- cerning responses by different size categories of phytoplankton within the community can be gained by size fractionation (Malone and Chervin, 1979; O'Reilly and Thomas, 1980; Lannergren, 1978~. Field comparisons of productivity rates between control and impacted regions, whether based on oxygen or 4C methods, must take into con- sideration potential bias arising from microbial activity and contain- ment effects, a result of differential growth rates, bottle size, and duration of incubation (Venrick et al., 1977; Gieskes et al., 1979~. More recently, Carpenter and Lively (1980) demonstrated that the type of glass bottle used for incubation, its W transmission character- istics, and trace metal contamination from 14C stock solutions and other equipment can seriously affect 14C uptake rates, particularly in oligotrophic waters. Cage cultures (dialysis encapsulation and Nucleopore. filter or nitex mesh cages), for either laboratory or field studies, offer another method potentially useful for evaluating responses of cultured or natural populations. Jensen et al. (1976) and Eide et al. (1979) employed cage cultures to monitor heavy metal pollution, while O'Connors et al. (1978) evaluated effects of PCB on unialgal and natural popula- tions. Dialysis encapsulation (Jensen et al., 1972) and other types of cage cultures {Owens et al., 1977) may ; provide a unique method for studying in situ effects of petroleum hydrocarbons. Laboratory Methods Statistical comparison of growth rates for control and treated popula- tions is more reliably done using cultures in the exponential growth phase. Regressions of exponential growth, compared by suitable statis- tics, e.g., covariance analysis, have been used by Hsiao (1978) and Prouse et al. (1976~. Problems associated with extrapolating results obtained from laboratory cultures to the field (Braarud, 1961) have increased since it is now known that clonal cultures of the same species vary in physiological response to environmental and chemical perturbants (Eppley et al. , 1969; Hargraves and Guillard, 1974; Fisher, 1977; Mahoney and Haskin, 1980; Murphy and Belastock, 1980~. Therefore, results obtained using cultures of one genotype may not necessarily be representative of a species response.

159 Photosynthesis and Respiration Photosynthesis has been the most common physiological index used to measure response of phytoplankton popula- tions to petroleum hydrocarbons. - ~ ~ 1 A_ . . .. . . sots one oxygen evolution and the '~C uptake methods have been used extensively. Oxygen evolution can be measured by the standard Winkler titration (Strickland and Parsons, 1972), as detailed by Shiels et al. (1973), or by using a Clark-type electrode (Pulich et al., 1974; Armstrong and Calder, 1978; Rusk, 1978; Batterton et al., 1978) . Effects of heavy metals on oxygen evolution should be considered. Potentiometer end point determinations for Winkler titrations can increase reproducibility (Vargo and Force, 1981~. The 14C method offers greater sensitivity, but concerns raised with respect to trace metal Problems in the f ield (vice suPra) also apply to laboratory use and also for O2 evolution. Clark-type electrodes and Gilson respirometry have been used to measure respiration rate in the presence of petroleum (Kusk, 1978; Karydis, 1919} . Axenic cultures are required, and species with demonstrated heterotrophic metabolism are best selected for studies of oil effects on dark respiration. Electron-transport-estimated respiratory activity (Christensen and Packard, 1979) has not yet been used to assess response to oil. Preexposure periods, with and without oil present (Shiels et al., 1973 ; Trudel, 1978 ; Hsiao et al ., 1978), vary from short term, i.e., 0.5-15 min (Pulich et al., 1974; Batterton et al., 1978; Armstrong and Calder, 1978), to long term, i.e., 12-18 hours (Gordon and Prouse, 1973; T.R. Parsons et al., 1976~. The duration of incubation must be considered along with exposure time because, with long incubation, the response elicited is an integration of effect over the entire incubation period. Short term incubations (i.e., minutes) with Clark-type elec- trodes (dusk, 1978; Soto et al., 1975a,b) are recommended but are subject to problems of interpretation because of diurnal periodic~ty of photosynthesis and shock response. Long term incubations suffer from containment effects, and results are subject to changes arising from bacterial activity "Harris, 1978~. Termination of 1 C estimated photosynthesis by addition of DCMU (3-~3,4-dichlorophyll~l,l-dimethyl urea, a photosynthetic inhibitor) (Laceze and Villedon de Naide, 1976), neutralized formalin (Hsiso et al., 1978), or mercuric chloride (Trudel, 1978) needs further evaluation because cell lysis can occur when any of these methods is employed (Silver and Davoll, 19783. Cellular Constituents and Cell Structure Enzyme analyses and cell structural responses have received scant attention in hydrocarbon investigations. This is unfortunate because biochemical and electron microscope applications offer standardized methods which can be use- fully employed to study effects of petroleum hydrocarbons, particularly to determine the site and mode of action of these compounds. Techniques have included scanning electron microscopy transmission, electron microscopy, and membrane studies (Van Overbeek and Blondeau, 1954; Goldacre, 1968; Baker, 1970; Boney, 1970~. Electrolytes also leak from treated algal fronds (Reddin and Preudeville, 1981 ) .

160 Standardized methods have been applied to assess petroleum effects on adenosine triphosphate (Vandermuelen and Ahern, 1976; Armstrong et al., 1981), alkaline phosphatase and phosphodiesterase activity (Armstrong et al., 1981~. Culture Systems Laboratory methods for determining effects of petroleum hydrocarbons on phytoplankton populations require suitable culture media. Recipes for solid, liquid, or biphasic media, using enr iched seawater or def ined media, and for the requirements of individual species or groups have been published by Nichols (1973) for fresh water and McLachlan (1973) for seawater systems. Both "opens and "closed" containers have been used. Open systems, e.g., foam or cotton plug stoppers (Soto et al., 1975a,b), have the disadvantage of allowing the more volatile compounds to escape, thus reducing the hydrocarbon concentration during the course of an experi- ment (Rauss and Hutchinson, 1975; Dunstan et al., 1975; Vandermaulen and Ahern 1976~. Closed systems, which retard losses of volatile compounds, may also potentially limit growth and final yield from CO2 limitation, an effect not observed in seawater unless nutrient enr~ch- ment is very high (Duns ten et al., 1975; PUlich et al., 19743. Blankley (1973) discusses possible detrimental effects on the enclosed popula- tion arising from the composition and leachability of various types of materials used in the manufacture of stoppers and other types of closures. Both unialgal and axenic cultures of marine and freshwater phyto- plankton have been used in laboratory experiments to determine the effects of petroleum hydrocarbons. Mixed populations have not been studied. Thus, results obtained to date are indicative only of noncom- peting populations. Use of un~algal cultures has been criticized, especially for determining effects of hydrocarbons on physiological responses, because bacteria are present and can interfere. In any case, axenic cultures are required if physiological responses (dusk, 1978; Karydis, 1979; Soto et al., 1975a,b), enzyme analysis (Armstrong et al., 1981), or ATP (Vandermuelen and Ahern, 1976; Armstrong et al., 1981) are used as the indicator of stress . Almost all studies employing marine species have been carried out using liquid media in batch culture. Many investigators, notably in earlier studies, ignored nuts itional and irradiance prehistory of phytoplankton populations when determining effects of oil. Shifts in irradiance history, both intensity and photoperiod, as well as nutrient, temperature, and salinity regime of cultures, can elicit variable physiological responses . it is, therefore, or itical that cultures be maintained under similar growth and environmental conditions both tee for e and dur ing an exper iment. Sampling schedules should also be predicated on known diurnal rhythms or points on population growth curves. Both semicontinuous {i.e., turbidostat) and continuous (i.e., chemostat) culture systems can be employed to provide populations of known nuts itional and growth history. However, the extensive time involved in chemostat work can make th is approach less fees ible . cultures can be more helpful when working with several species, as opposed to the chemostat. Useful discussions of these methods have

161 been published by Goldman and Davidson (1977) and Murphy and Belastock (1980 ~ . A method for rapid screening for potential toxic, or inhibitory, effects of a particular petroleum compound has been employed by Pulich et al. (1974) and Winters et al. (1977~. Termed the algal lawn tech- nique, ~ cultures of the species to be tested are dispersed in a plate of molten agar, and a pad saturated with the substance to be tested is placed on the agar. Sensitivity is determined by a zone of inhibition. Usefulness of the method is 1 imited to those species that can withstand the relatively high temperature of liquefied agar (approximately 40°C) and are able to grow on an agar medium. Low temperature gelling agar (approximately 26°C) is now available and can ameliorate this problem. Accumulation and Depuration Radiolabeled pure petroleum hydrocarbons offer the most direct method for establishing uptake, accumulation, and release rates (Kauss et al., 1973; Soto et al., 1975a,b) . Improved and highly sensitive gas chromatography and combined gas chromatography/ mass spectometry have also been successfully employed to establish the presence of polycyclic aromatic hydrocarbons in epipelic diatoms. Population Responses Rate of growth or cell division, combined with generation time and f inal yield, can be considered integrators of factors influencing cellular metabolism. Population responses measured by visual counting methods also yield additional qualitative informa- tion on the ~condition. of the population (i.e., color , movement, loss of flagellae). Reviews, such as those published by Guillard (1973) and Sournia (1978), should be consulted for proper choice of counting cham- bers, cat Vibration, counting statistics, and 1 imitations of each method . In addition, electronic particle counters provide rapid results, thus allowing increased replication of both counts and experiments; but problems of coincident counts, Cal iteration for cell size, inter ference of nom iving matter, and counting of chain-forming species must be recognized and taken into account (Sheldon, 1978 ~ . In viva chlorophyll fluorescence is also easily measured and can provide a quantitative measurement of population increase, provided consideration is given to those factors which influence fluorescence yield, e.g., irradiance intensity, diet periodicity, dark exposure (see Kiefer, 1973; Loftus and Seliger, 19757. Enhancement of fluorescence by DCMU can provide an estimate of energy channeling within a cell and could be an indicator of potential photosynthesis, yielding a relatively quick and simple assay for detecting effect of petroleum products. Prezelin and Ley (1980), however, point out the inconsistencies of the method when it is used as an indicator of potential photosynthesis. Thus, further investigation is warranted. Zooplankton Field Methods F. ield methods are the same as for the phytoplankton . Contamination of the zooplankton being sampled by the net itself is a signif leant

162 problem. Also, care must be taken that the net does not pass through the surface slick and that it is rinsed with solvent between tows (R.C. Clark and Brown, 1977~. Gelatinous zooplankton require special methods. General ecological methods applied to zooplankton are also useful in assessing effects of petroleum. The least detailed parameters measured are biomass (Wiebe et al., 1973; Beers, 1976; omori, 1978) and total abundance (Edmondson and Winberg, 1971~. Live-dead counts may be done in conjunction with enumeration (Fleming and Coughlan, 1978; Seepersad and Crippen, 1978~. Recognizing the heterogeneous distribu- tion of zooplankton, species composition, age structure and sex ratios provides information on community structure, which may reveal changes more readily than biomass or total abundance. Species composition data permit calculation of diversity indices (Pielou, 1977), which may deal ine under pollutant stress (Copeland and Bechtel, 1971; Borowitzka 1972) or may not decline (Elmgren et al., 1980a,b). Animal production {Edmondson and Winberg, 1971) of the community or individual species may also change under pollutant stress. Biochemical composition of the zooplankton communities may change also (Samain et al., 1980~. However, biochemical composition will vary with temperature and nuts itional state, so that the effect of petroleum may be masked. Protein {Lowry et al., 1951; Packard and Dortch, 1975; Capuzzo and Lancaster , 1981), lipid (Marsh and Weinstein, 1966), carbohydrate (Dubois et al., 1956; Handa, 1966), fatty acid content (Morris and Culkin, 1976} , and digestive enzyme activities (Samain et al., 1980) per unit weight may be altered due to stress. Enzymes may also be induced to deal with toxic substances, such as the induction of mixed function oxidases in response to oil tJ.F. Payne, 1977; Walters et al., 1979), although not all species possess this capability. Interpretation of results is, therefore, difficult. In organisms with a mixed function oxidase, this system is important in metabol ic functions. Hydrocarbons may be taken up by zooplankton (Harris et al., 1977b; Corner, 1978; Spooner and Corkett, 1979), and hydrocarbon con- tent can serve as an indicator of oil pollution (Mackie et al., 1978~. Microscopic techniques for detecting oil contamination were employed by Conover (1971) and Polak et al. (1978), among others. In recent years, histopat~hological data (Yevich and Barsacz, 1977) have been used as indicators of long term sublethal stress. However, for the zoo- plankton community, there are few background descriptions of "normal. histological characteristics. Furthermore, there is no general consen- sus on which histopathological changes are reliable indicators of stress, although chromosomal studies of fish eggs have revealed that changes in chromosomal structure and mitotic division may be useful (Longwell, 19787. Laboratory Methods In laboratory studies, functional parameters, such as survival under both acute and chronic application of oil; recovery; physiological processes (respiration, excretion, reproduction, and growth); behavior (feeding and locomotion); and uptake, retention, and metabolism of oil

163 have been used to measure the reaction of an organism to oil. Survival has been measured in acute toxicity bioassays (24-96 hours) in both static or flow-through systems {see Methods of Assessing Toxicity of Petroleum to Marine Organisms section). Such bioassays are helpful in ranking oils in order of toxicity but are of limited value for eco- logical prediction "Wilson, 1975~. Chronic long term exposures are more useful because they allow detection of delayed mortality (Berdugo et al., 1977) and significant sublethal effects. Furthermore, physzo- logical processes of an organism are more sensitive indicators of stress. Many physiological tests can be car r fed out on board ship, as well as in the laboratory. Respiration can be measured polarographi- cally (Edwards, 1978; W.Y. Lee et al., 1978; Gyllenberg and Lundqvist, 1976), chemically by the modified Winkler method (Carritt and Carpenter, 1966; Cargo and Force, 1981), or manometrically (Capuzzo and Lancaster , 19813. Manometric techniques are limited, however, in that not all zooplankton can withstand confinement within the small volume and shaking required. In all cases, suitable precautions must be taken to ensure that zooplankton are not stressed in several ways simultaneously, e.g., by crowding and lack of food (Ikeda, 1976, 1977~. Ammonium excretion is usually measured calorimetrically by the method of Strickland and Parsons (1972) or Solorazano (1969), although other nitrogenous compounds, such as urea and pr imary amines, may also be excreted and, therefore, measured (McCarthy, 1971; McCarthy et al., 19773. For the small size zooplankton fraction, the 15N isotope dilution method can be useful (Caperon et al., 1979~. Respiration and excretion rate measurements can be combined as an O:N ratio, providing an indication of the biochemical substrate utilized as energy reserves (Capuzzo and Lancaster, 1981 ; S. Vargo, 1981 ; Mayzaud , 1973 ~ and provide some indication of disruption of normal energetic processes. Reproduction and larval development may also be affected by oil, and several parameters have been used to measure such effects, i.e., number of eggs (Ustach, 1977; Berdugo et al., 1977; Ott et al., 1978), embryological development and hatching (Donahue et al., 1977b; Tatem, 1977), and larval development and survival (Donahue et al., 1977a; Laughlin et al., 1978; Wells and Sprague, 1976; Nicol et al., 1977; Bryne and Calder, 1977~. Growth, as a sum of physiological processes, can be a good indicator of effects of oil. Measurement of growth can be a simple biomass determination (Edwards, 1978) or more complex assessments (Edmondson and Winberg, 1971), with carbon or energy budgets also proven useful (Edwards, 1978 ~ . Extens ive investigations have been conducted on zooplankton feeding (Conover, 1978) . Simple short term feeding exper iments can be carr fed out on board ship using i4C-labeled phytoplankton and the larger size fraction of zooplankton. However ~ with longer incubation r the 14C may be excreted, resulting in less reliable conclusions. Landry and Bassett (1982} have developed a method for evaluating grazing rates in whole water samples. In sits measurements have been made by Haney (1971) . More laboratory-or tented methods include the use of cultures of s ingle species of phytoplankton and zooplankton and microscopic counting techniques to evaluate those phytoplankton or zooplankton

164 (Wells and Sprague, 1976) that have been eaten. Feeding on several species of phytoplankton, or a natural assemblage, can be measured using electronic particle counters (Berman and Heinle, 1980~. Fecal pellet production can also be used to estimate changes in feeding rates (Spooner and Corkett, 1974, 1979), but the test animals must produce fairly large, cohesive fecal pellets for this method to be practical. Behavioral responses, such as feeding and locomotion, comprise integrated physiological and biochemical processes. Behavior appears to be altered quickly when the animal is under stress and, therefore, shows promise as an indicator (Wilson, 1975; Olla et al., 1980~. However, not all behavioral responses can be easily quantified, and care must be exercised in selection of a behavioral response. Locomotion includes at least two components: rate and pattern of movement. Narcotization by petroleum can result in cessation of movement (Gyllenberg and Lundqvist, 1976; Wells and Sprague, 1976; W.Y. Lee and Nicol, 1977; R.F. Lee et al., 1978~. Changes in rate can be observed visually, but changes in the pattern of movement are more difficult to quantify. Video taping, coupled with computer analysis, shows promise (Lang et al., 1981), although methods currently employed are not suitable for species that accelerate rapidly. These methods are also time consuming and expensive. Locomotor y responses to external stimuli, such as light, gravity, and pressure, may also be used to assess effects of oil (Bigford, 1977~. Changes in locomotor y responses provide a qualitative, rather than quantitative, indicator of effects of oil, and are difficult to interpret in terms of permanence of effectts). Uptake, retention, and metabolism of petroleum in planktonic organisms have been studied using 14C-labeled hydrocarbons (Corner et al., 1976; Harris et al., 1977a,b; R.F. Lee et al. , 1981a) . Such measurements reveal rates of uptake, retention time, and presence or absence of ability to metabolize hydrocarbons. ACCUMULATION AND MODIFICATION OF PETROLEUM BY MACROORGANI SMS Field Exposure Methods Organisms can be exposed to petroleum in the field using any one or comb ination of water-soluble fractions, oil-in-water dispersions, surface slicks, oil-contaminated food, or oiled habitat. Table 3-6 lists examples of field exposure studies based on the physical form of the contaminant. Alternatively, field experiments can also be clas- sified according to method of exposure: (a) introduction of uncontami- nated organisms into contaminated areas (uptake studies) and vice versa (deputation studies), (b) sediment tray experiments where oil is mixed into a sediment in the laboratory and then returned to the field for a period of observation, (c) oiled enclosures, and (d) field surveys that compare environmentally exposed, wild samples with uncontaminated reference samples (monitoring schemes).

165 Introduction Experiments One method of exposure consists of transplanting small marine organisms from uncontaminated areas to contaminated areas. DiSalvo and Guard ( 1975) designed a mussel-exposure apparatus made of wide-diameter plastic PVC pipe. The apparatus containing the test organisms was placed on pilings below the low tide level or hung under floats. Other methods include suspending mussels and other shellf ish in mesh bags or cages (8.A. Cox et al., 1975; Whittle et al., 1978; Burns and Smith, 1978; Wolfe et al., 1981) or placing test animals in cages and sus- pending the cages off the bottom in the intertidal zone (Bender et al., 1977; Bieri et al., 1977, 1979) . Benthic organisms have been placed directly on contaminated substrates, either in open-bottom trays (Straw et al., 1976 ; J.W. Anderson et al ., 1978 ; Roesijadi et al ., 1978 ; Roesi jadi and Anderson 1979; Auger feld et al., 1980) or without enclosure (Lake and Hershner, 1977; Friocourt et al., 1981~. Sediment Tray Experiments Trays of contaminated sediment, either free of organisms for recruit- ment studies or containing selected bivalve mollusks or worms, arranged carefully by hand, were placed in intertidal beaches. The bottom of each tray was fitted with a screen to allow for natural tidal flow of seawater. In studies to determine the effect of oil-contaminated sediments on natural recruitment, sediment was collected and sieved in the field to standardized particle size. The sediment was exposed to three cycles of freezing and thawing in the laboratory to eliminate microorganisms, after which it was poured into trays and immersed in a large aquarium. The flowing seawater aquarium was equipped with a mechanism to provide simulated t idal draining of the seawater . When the water level was 1 cm above the sediment , a 4 % volume of oil was poured over the seawater, forming a uniform sl ick . The seawater was drained and the oil was allowed to remain on the sediment for 2 hours. The flour of seawater was r e instated and the excess, f loat ing o il was sk immed of ~ . Two additional tidal cycles were completed. The sediment-f illed trays were then placed in holes dug in the beach (Anderson et al ., 1978 ~ . Alternatively: the oil was mixed with the sediment prior to filling the trays. Coating of the sediment was achieved by adding a blender- prepared, oil-water emulsion to the freshly sieved sediment and mixing it in a cement mixer (J.W. Anderson et al., 1977~. Oil analyses should be done before, during, and af ter deployment in the f ield. Enclosure Experiments Bakke and Johnsen (1979) used sediment enclosures made of aluminum with tops of polyethylene sheets . Diver s embedded the enclosures to a depth of 0 .1 m in the sandy bottom. The polyethylene sheets were mounted while the frames were in place on the bottom. The benthic organisms in

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168 the enclosure were exposed to 30-35 L of an oil-in-seawater dispersion of Ekofisk crude oil. Experiments took place over a 9-month period. Oil can be poured onto the surface of an enclosure, such as a pond or a cordoned-off segment of a bay. Bottom-dwelling and free-swimming organisms are exposed to various forms of oil, depending on the natural or human-induced mixing conditions, tidal fluctuations, etc. (B.A. Cox et al., 1975; Lake and Hershner, 1977; Bieri et al., 1977, 1979~. The enclosure can be as large as an embayment, as in the case of a con- trolled oil spill (Blackall et al., 1981; Blackall and Sergy, 1981~ . Artificial Substrate Experiments In studies of epifaunal recovery, concrete construction br icks have been used as exper imental substrates to represent oiled rock habitats (Vanderhorst et al., 1981) . Bricks of uniform size, shape, and porosity are readily available in large quantities and are easily placed on a beach without requir ing a physical support system to keep them in place. In the study cited above, the bricks were preconditioned in flowing laboratory seawater for 2 weeks and then treated with a surface-borne slick of Prudhoe Bay crude oil to simulate repeated exposure of an intertidal rock for 5 days of tides. After another day in flowing, clean seawater, the bricks were placed in the intertidal zone for 1 month. At the end of the experiment, the animal species present on the top of each brick were identified and counted. Analyses of the oil (carbontetrachloride-soluble) adhering to the bricks were conducted using infrared spectrophotometry and glass capillary gas chromatography. Field Surveys A less well-control~ed method of study involves comparison of contami- nated wild populations with uncontaminated reference populations. Some large scale monitoring projects, e.g., Mussel Watch (National Academy of Sciences, 1980) and Oyster Continental Shelf Environmental Assessment Program, were intended, in part, to establish reference characteristics of petroleum contamination in various wild populations of marine macro- organisms. The information obtained has been useful in evaluating petroleum uptake, distribution, and discharge for selected organisms. The common problems associated with such methods are lack of scientific control over the wild populations and a lack of detailed knowledge of the history of contaminant-organism interactions. One difficulty in comparing hydrocarbon uptake data from chronically polluted areas with data from reference areas is filtering out the natural "noise" or variability in biological systems. Kwan and Clark (1981) described a system of pattern recognition analysis for ranking mussels collected at sites of differing contaminant input using paraffin hydrocarbon parameters. They were able to rank mussels collected from dif ferent locations in Puget Sound according to their degree of chronic and acute pollution exposure. In the case of acute petroleum pollution

169 (i.e., major oil spills), an extensive discussion on the philosophy and application of sampling for benthic and pelagic organisms was given by G.V. Cox (1980). Laboratory Exposure Methods General methods for preparing petroleum solutions for use in studying uptake and behavior modification of petroleum by microorganisms have been described. Our brief review is presented in the Preparation of Oil-Water Solution section. In addition to laboratory exposure of microorganisms to water-soluble fractions, dispersions, surface slicks, oil-contaminated food, and oiled habitats, intraperitoneal injection of pure hydrocarbons has been employed in a few studies, as a direct method of exposing individual animals to a known concentration of hydrocarbon, and is useful for studying pathways of metabolic conversion (Varanasi et al., 1979). Frequently, radiolabeled hydrocarbons containing tritium (3H) or carbon-14 ( 4C) are used, a method offer ing a sensitive and specific mode of detection, as well as a means of studying metabolite formation. Interestingly Roubal et al. (1977b) reported that it was not possible to correlate feeding experiments directly with rates of depletion of aromatic hydrocarbons or their metabolites from tissues in intraperitoneal injection studies in salmon. Table 3-7 lists the principal "state-of-the-art" techniques used for laboratory studies of uptake and effect of hydrocarbons by pelagic and benthic marine macroorganism (excluding marine mammals and birds) . Several of the methods described in Table 3-7 permit the application of dispersant-petroleum mixture and testing in a fashion similar to petrol- eum exposure. Initially, static laboratory tests were used to establish acute toxicity of dispersants in the absence of petroleum. Several methods developed for acute toxicity testing of dispersant-petroleum mixtures are also applicable for uptake. The "seas test (Norton et al., 1978) developed in the United Kingdom employs a propeller-mixing device inside a plastic cylinder inside a transparent plastic exposure tank containing the test animals. The oil can be added to the mixing vortex within the central cylinder by syringe or to the inner cylinder, prior to agitation, followed by syringe- delivered dispersant, a shor t waiting per iod, and f inally, initiation of mixing. This procedure has been used to expose free-swinuning organisms (e.g., the brown she imp, Crangon crangon) . The "beach test provides a means of assessing toxicity and uptake of dispersant on semimobile, intertidal organisms such as limpets (Norton et al., 1978~. The organisms are placed on transparent plastic test plates in flowing seawater until they have attached. After several days of conditioning in a simulated tidal seawater system, the plates containing the organisms are hand sprayed either with dispersants or oil. For toxicity tests, the number of limpets detaching immediately after exposure and at 24 and 48 hours after starting the test are counted.

170 In 4, ..- £ o Cal o U o :~: o A £ en - Ill g X C5 C: X o MU on o 4~ ~4 o 8 1 En c) A: ~4 At: U) e Ul e 0 0 a ~1 °x ~ :' 0 ~ ~; 0 £ · I: iD O ^ n u~as · a ~a _ ~__ ~ ~O O u4 C: -· ~·· U] ~: ~a, ^ ~ ^ ~ _ ~ _ u ~O ~a~ ~ ~JJ ~ ~: ~r a' ~ ~ ~2: ~: ~s ~a' a, a, ~. ~ ~· ~· _ ~ _ ~3 - 3 - d) ~· OO C) Z ~Z c ~ · - Y ~·- O c 1 0 _1 ~JJ C ~5 0 ~ ~ ~ U) ~O c a, ~ ~a, E ~ ~ o O ~m ~ `: o~ ~· n ~ ~n ~ ~ c ~ · ~0 ~ ~ ~ ~ c 0 -~ ~ X ~C ~ X C u ~n ~ ~r :3 ~ o 0 0 x ~ o-- ~n ~ e 3 ~ ~ 0 ~ ~ ~== ~ ~ ~ s ~ ~ ~ v `4 ~ ~O ~ ~O ~ ~ ~ 1 ~ O ~C ~ ~ ~ e~ ~ - V ~ X 5: ~ ~I Q. O U~ ~ ~n a ~s 0 c c ~ x 0 ~ ~ ~O ~ ~ ~ - ~ V N ~ a~ ~I O ~ a ~ . - X ~ U (U tQ (U O 1 a) 0 a 0 ~_4 cn ~ 1 Ql ~ ~a, . - C ~ . ~q 0 E ~ U] ~Ll tn u 0 ~n ~ c ~ ~ ~ u, 3 ~o ¢ ~n ~: ~ 0 ~ ~n ~ ~ Q. s 3 :' - ~ O ~ Q E ~ c ~ O s s v u ~ ~ ~ 0 ~ c ~3 ~ C ~· - ~ s ~ ~ s ~ 3 ~ ~ ~u, ~ sq ta Q u~ E U] ~O m ~u ~U m Z o. ~ s s s s s s s s s tn u ~u ~cn u ~u ~u ~u ~u cn u ~u ~u ~u ~cn u ~u Ql ~iQ I - 81 0-~ ~ _' ~._1 ,,' ,,' ~ ~CQ, O 1 1 ~ 3 ~531 O o - - 1 C ~O ~ ' 3 ,,, ~_ ~ e ~ ~ ~ ~ ~ ~ ~1 - - ~1 ~ O C C C ~1 U ~ ~ C.) 3 Q, ~ 0 0 - ~1 ~ ~4 ~O ~6) 3 0 3 ~ N 4) N 4) 0 41) ~ ~ O N ~ 3 3 ~d Q e ~e c C ~: ~: g 3 c ~C o ~ O=s u~ ~O 54 ~ 54 4~ ~ ~ ~ ~.C ~ ~ ~: ~ ~ 3 I ~ N I ~ D :^ E S I I I 1 3 . - ~ L1 3 O · · 0 O4 ~C) S:24 1 ~ I U U ~) C) ~· m 0 ~ ~ ~ Z Z Z ~m . ~ 5 ~,- ~,-1.- ~z O O .,' u o 0 3 4 ~' ~O h4 ~ ~Q, a) 0 0 ~ c 0 3 v ~JJ c ~ c E cco ~ x~ : ~a~ ~ ~ u' ~ 3 ~ s ~a) ~ ~P. r~s e ou ~Ou v ~ ~c s ~n 0 ~) ~dJ ~ (V ~) 0D ~O) 0 ~3 - C~ O N ~ 0 ~ 0 3N ~O CI c~ ~ ~ ~ 0c ~ O c) Q. - - E~ ~Q c ~c :' E0 ~O ~O ~ `: x`: ~s~ 0 ~ 3~ JJ ~Q c' Q ~ O ~ O ~0 ~ "1 ~a ~ - ~ 4~ ~~ ~ ~3 ~ ~ ~ ~ ~ ~ ~ ~ JJ ~o ~1 u ~ ~a, :, ~ ~ 3 3~ 3 eq ~c O ~O m 0 13 0 c ~1 , ,.~v 0-- ES 3^) fa > ~> ~tU -~1 U]U] U) ~C:U] U] O ·` ' S S ~D o 1 r~ ~ O O - 1 ~ a~ c: - - 0 UO 0 _ _ ~ ~ ~ 0 ~n E ~ 0 -4 ~ ~ ~ ~ ~ c 0 ~ ~ ~ ~ 0 0 c ~ ~ . ~ ~ 0 0 3 ~ ~o o _~ t~ C 1 ~ ~ ~ ~ 0 1 0 0 E ~ o

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c I: - :~ ~ - o ~ :^ Ed to · - ~ A) ·- ~ o · 1 1 Cal ~ ~ 1e JJ o A a, ~ - c r. or · - ~ 3- . 3 X 1 o ~D ~: - _ 1 _' 4) :5 _ 00 ~ _1 3 _ U) cSn rn ~ 3 U] O O ~ 3 O X Q. ~ 1 ~: O _ 3 a 1 ~a C) 1 C: o · - i., 0 a, JJ ~ O4 3 0 ~ ~ C) o oo o U' ~o c) s ~: 3 tn ~o a) o, C ~ ~de _ 3 C) ~ ~ so cn O a s tn . ~: . - ~E: 175 - _ o~ e . C) Q. ~4 ~: _ · ~ [4 ~ . 3 ~Q o X s v - a' _I c - ·~' ~: y ~ 3 - ~V ~D a' t- _ a~ _ _~ . 4 ~a 4 ~4 C O ~ C) =: O U O ~ O Q _ ~ b: O o 1 Ll C~ o . - o U' CD ~V ~ O ) C ·_1 C~ N G) :~ ~ ~V C ,,: S u, U) ~tn ~O _1 3 .,' O V ~ >~-~- ~- ~3 -~ ~ . - m 0~ 0 m 0 ~v Q, ~ ~c: 0 ~0 ~0 ~V S ~S 3 .C ~C ~N _' - - Ll ~ L ~4)~ :' V3 V 3 U · ~ O t`5 ~O Q, ~ P~04 P4 Z ~H Y ~: O V ~v V ~Ll C ' ~ `: O · - ~O ~ ~ ~ ' O ~ Q, ¢: U C ~C UC ~0- ~U O :^ O [: ~- ~ ~ :~ _ 3 ~~ ~ . - JJ ~ ~S V ~ ~ ~ c: _ _I ~S ~ ~- P. 0 ~0 : ~ tO _' ~ dJ ~ ~ U ~ U l~ _ ~_ X ~ ~ ~3 0 ~ _ ~ ~ ~ ~v~ -1 c) ~5 0 O ~ ~ ~ - O -- ~ ~ ~ OS JJ ~ ~- ~ C:- 4) 4~ ~ ~ ' ~ rJJ V 0 t: ~;J U ~rA - 3 ·' U: ~ ~ ~3 C: ~ - ~£ ~v ~C 0 ~ - eq ~5 t: ~ ~ O - X ~ ~ ~ ~C ~- V ~ O ~ ~ U ~ ~ =_ o 4J ~ V- JJ ~ 3 ~ ~ ~ ~ ~ £ ~ ~ S _' ~ S ~ O ~ ~O : ~ U ~ U) ~ Y 3 _ ~_~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - ~V ~O 3 ~ ~ ~ ~v _~ 0 · ~J S ~-I ~ C (1) ~- ~Q~ ~ ~ ~) ~ 3 3 ~v U, O _I 4~ ~_' ~ ~ _' C 3 3 ~ U) ~_ _ .~1 ~ _ -~1 , ~ U) U) O O O H C C tn ~ ~S -I C: .C O ~C ~ ~ ~ ~ _ l~v ~ C ~:: (IJ ~ ~1 1 0 V O ~ ~-I Ql~ - - `: ~ V C, O ~ 0 ~ ~V ~ S ~ ~I~ .O - ~ ~V- ~ J~ JJ -~ UO ~ 0 4) ~l ~ (IJ ~S N C 3 Q. ~ X S~ ~ ~ _. 41) 0 ~ 1 V- _ ~ ~ ~ .C ~ S C~ s:(0/ s: D :>' I I I I I I 3 ~ 1 O4 t,) {,) t,) Ca,) ~ ) C.) ~5: ~r Z : ~_I ~ ~ _I 3 o O 3 V S" ~ .,1 ~C ~ 3 O V ~.- O _1 ~ Ll 14 ~ O O CJ ~C-) _1 JJ ~ "rl o - - 3 ~r ~n .,' al Ul V ' - Q. ~`: ~ 0 - - _ O ~Q ~ ~ U] O N _ ~ O ~v L. S a, ~ a, 0 O~ O . - _ cn

176 c) `: a, ~: tn 4J C: o C) U] .,. o o .,. 4 54 a a1 U) D3 ~ "r4 ~ ~; o s ~: ~^ ~_ _ ~g ~r ~co a, cn ~`:: _ ~, ~0 ~ _t - - U) - c: _ ~·- · · ~· · ~ns a) ~L, ~a a ~Q ~: _ a, r~ ~. - JJ ~ a' g on ~- - ~U] - - ~X - & ct 3 0, o tQ eq h ~ Li ~n C) ._. ~n U] o q~ ~U] Q X ~ ^ O ~ ~ - ~s E~ a O ~ a) () Q a ,= ·_ U~ 1 U] ~4 :' U] Q X U~ ' :' Q) ~n cn O O CD o ~ X u~ a ..~ C) ~0 E 1 ~ 1 ~ ~o ~o . o 1 ~ ·- a,c: ~ O ~ v~ a · (} ooa, cn~ - . - C: ~ ~ ~C) m- - C~ · - '~ U~ ~ ~ ~ U: ·~ 0U~ 3 U) o o C) U] a, ~:>, ~ s n~ ·~4 v m 0 s Q. a' a, ~0 C: 1 ~ ~ C-) 3 ta) P. 4~ a, o 3 c 0 a, 53 E ~Q s ·~4 Ll a, 3 S ~3 ~ O O ~ :' ~c, o4 x m £ s aC ~ u, ~cn ~ ~u' u' u] h u) C C ~ ~ C ~ C _ ~a _ ~C 4S, ~ N ~N S S ~ X ~ ~ 4~ a' ~ ~ c s 1 1 1 1 1 C) C) ~ C) C) ~r er - - o U) ~^ <: · _' 3 - . .,. 0 tn .,. o '~ ~n 1 o D o ~n S: 0e U) o o C) ~: ,' a' a' 1 4 . - cn 0 a' o 3 . - :, ._. ~Q u ~u~ u ~n ~ s~ ~: s ~ c 1 r~ ~ 1 S c c L4 ~ ~s~ ~ ~ c _ ~ ~a a, ~n ~ - ~ o x o ~ c) ~ N C . - S C O I ~: JJ C ~I I 1 1 C: O C-) 1 1 X :~ a, c a, s l C) ~ .,, m 0 0 ~ S 3 ~I O ~- ~ S ' c 0 ~0 · - 0 :~ a) c ~, a O O =e ~ C c: u, ~ ~ Q4 c aJ ~O ~a' c ~ - - Q) C' -- Q. O S ~O C a, :> ~ O ~ ~ ~ - - ~ s · - ~ ra - - ~ ~x V S O ~ ~ u ~ ~ U) ~4 ~01 0 0 ~· - c ~u ~a, ~ ~ a, ~ ~c ~ ~ ~a' `: O ~ ~ I =~ a, O ~ = - ~ ~- ~ ·. 3 ~ ·- ~ Cn O ~ ~ ~O ~ s JJ ~ c c~ c £ ~ ~ :l ~ ~ ~ ~4 v ~ v ~o G~ , ~s - C: a ~ ou 3 Q ~ s ~ O c 0 ~ ~ Q. 0 ~ v ~ ~ cn e) ~ ~0 u~ s u. u, ~t.) tn ~ ~ 0 ~ 0 v - ~ t) £ c v ~ ~o. ~ ~ ~ se u ~ ~ E ~ Y ~: c ~ c ~ a~ ~ ~ ~~ ~ ~ ~ ~ 3 ~ - - ~ ~0 0 ~ ~ -4 0 t' ~c v 3 ~ Q. ~ 3 ~ Q, O ~ ~ ~ c ~ ~ ~ ~ eq £ E ~ ~Q O cs ~n ~ ~ ~0 ra ~ c ~ ~ ~- - 4~ ~ ~c: a, ~3 ~ ~ 0 a) 0 ~ ~ s ~ ~ ~ ~ ~ ~n ~ ~ u, O o ~ en S V ~ ~ V ~ ~ ~Q. ~ c: ~1 S eQ ~ 3 0 u ~ ~ O ~ ~ ~ ~ 0 0 D ~O O ~ O O ~ 4 ~ JJ ~s ~ c ~ ~ 3 0 4~ 0 ~ ~ ~ ee v 3 0 ~ v 0 en o~ ~ 3 O-- c ~ c: ~ ~n _ ~ 0 ~ ~V ~ C: ~ 0 u ~0 0 ~ ~u~ 0 tn )~ ~ - - ~ u, 0 ~x a u ~ u ~n u ~0 ~ ~a ~ ~a ~a ~:

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180 COMMUNITIES The success of surveys or monitoring depends upon careful attention to sampl ing des ign, methods, and analysis. Many investigators have descr ibed methods for sampling tHolme and McIntyre, 1971; A.D. Michael et al., 1979} and for measuring ecological variables useful in investigation of pollution (Gray et al., 1980 ) , as well as study design and analysis (R.H. Green, 1979 ~ . Sampling Methods Subtidal Habitats The trend in the development of sampling methods is toward ensuring complete capture of organisms within a unit area of seabed or habitat surface (Holme and McIntyre, 19711. Because grab samplers may not sample to a uniform depth under the area covered and may vary in depth of penetration, the preferred sampling device for soft substrates is a corer. In intertidal or very shallow water habitats, hand-operated corers are simple and efficient. In deeper waters, however, box-coring devices for sampl ing a larger sur face area to the desired depth in the sediment are required, especially in soft sediments. Large ships and heavy handling equipment are required to operate such devices. Spade box corers are the most commonly employed large volume cor ing devices and, properly equipped, can be used to collect comparatively undisturbed sediment columns to a depth of 20-50 cm or more. Frequently adequate penetration and core capture are cliff icult to achieve in sandy sediments. Larger, and consequently less common, benthic animals must be sampled by a trawl or dredge dragged over the bottom. Accurate and precise assessment of population density is, therefore, cliff icult. Relatively sedentary large animals may, indeed, be an important and s uscept ible component of the b iota . Stor t tows of dr edges and photo- graphic or televideo instruments or direct counts by divers or from submersibles are, currently, the most reliable methods for assessing such populations. Collection and adequate preservation of sediment samples for determination of sediment granulometry and petroleum hydrocarbons important. More samples can be collected than may be feasible to analyze, thus allowing for archiving samples, should they be needed for subsequent analysis. Samples are best collected as a vertical integral of the surface sediment to a prescribed uniform depth. However, some investigators prefer that cores be sectioned as f inely as possible and the sections be analyzed separately. If this is not possible, the uppermos t layer s are analyzed . For macrobenthos and meiobenthos, variability in sample processing can be a s ~gnif icant source of error . The very small mesh openings of sieves needed for separating animals from sediments reduce the loss of the target populations, but present some practical limitations. In the West Falmouth spill study, Sanders et al. (1980) used a mesh of 0.3 nun

181 and collected essentially all the adult macrobenthos. Historically, 1 . O-mm s ieves are used, but these may lose a s ~ zable por t ~ on of the macrobenthos. Furthermore, they are susceptible to variation in efficiency, due to length of sieving, amount of debris, etc. Careful use of a 0.5-mm mesh sieve results in capture of most of the adult macrofaunal taxa and is generally an acceptable compromise for studies in coastal and continental shelf habitats. In deeper water, where the macrofauna are small, the mesh should be 0.3 mm or finer. Meiobenthos , i.e. , metezoans which, as adults, pass through a 0.5-mm mesh sieve, require specialized sample processing procedures (Hulings and Gray, 1971) . They are commonly removed from sediment collected in a small diameter corer by elutr iation or density separa- tion. Animals elutr fated are retained on a sieve of omening size between 40 and 100 Am {commonly 63 um). The sensitivity of meiobenthos to petroleum is not yet well documented, largely because these constituents are difficult to identify. In general' only the abundance of a higher taxon (e.g., total nematodes) is assessed (Wormald, 1976; Grassle et al., 1981; Elmgren et al., 1980a,b). In cases of less than catastrophic pollution, it is probably more realistic to expect to observe effects only on individual species, rather than on the total density of a higher taxon. However, because of difficulties in species identification of the dominant meiobenthic taxon, Nematoda, few studies have actually been done to assess the effects of petroleum on dominant component species of the meiobenthos (Gier, 1979; Boucher, 1980~. Species and population analysis of subdominant taxa, in particular harpacticoid copepods, is, in general, relatively more easily accomplished. Intertidal Habitats Because of environmental changes which occur in the intertidal zone, including both rocky and sediment-covered substrates, stratified random sampling or sampling along line or belt transects through the inter- tidal zone is advisable (A.D. Michael et al., 1979~. For hard sub- strates, a line intercept method may be used to estimate populations. This approach to measurement of occupied surface area allows nondestruc- tive sampling and provides the advantage of monitoring the same individuals to assess long term effects on recovery. Although attention should be focused on the primary space occupied on structure-creating macroalgae and animals of rocky shores, asso- c fated epibiota should not be ignored. For example, although no effect of the Tsesis oil quill on the dominant alla FUCUS vesiculosus could be measured, most of the associated fauna, particularly the small Crustacea, were in fact adversely affected (Notini, 1980~. Microalgae 1 iving in intertidal sediments or on hard substrates are often important primary producers and may be subjects of concern if the habitat is affected by petroleum. Although their biomass can be approximated simply by measuring chlorophyll concentrations of surface sediments, chlorophyll concentration demonstrates, in nature, a high degree of temporal and spatial variability.

182 Vegetated Hab itats Biomass and productivity of marsh grass are best assessed by clipped quadrats placed in a random manner within habitat strata, e.g., high Spartina, low Spar tina, salt meadow, etc. (J. Michael et al., 1978~. Some benthic fauna can be sampled using corers and seines. Hand seines and baited traps should also be employed to sample marsh benthos. Epifauna, such as marsh snails, can be sampled along with the grass . However, the more difficult estimation of the burrower population, such as fiddler crabs, necessitates excavation of the substrate. Other physiological and functional measurements of salt marsh communities which may prove to be relevant In oil pollution studies, can also be made (Pomeroy and Wiegert, 19811. Sea grass biomass and production can be assessed, much as is done with marsh grasses, except that defoliated leaves are rapidly lost from the bed, complicating production estimates. Phillips and MCRoy (1979) described a variety of methods used to assess sea grass productivity and other aspects of the biota of the sea grass community. Because of. their ecological importance in trophic interactions and their suspected sensitivity to oil, the epibiota (including the diverse crustaceans) are best sampled by placing a fine mesh net over the grass and clipping the grass or collecting by suction methods. The concentration of the epibiota can be expressed both in unit area of seabed and biomass of grass. For mangroves, biomass and pr imary productivity of trees in the mangrove swamps are difficult to measure. Attention, instead, should be placed on measurement of the degree of defoliation and, subse- quently, on the recovery of defoliated trees. For faunal studies, the same conditions occur as in salt mar shes, except that the epibiotic community of trunks and prop roots may provide useful information. Coral Reefs Many methods used in oceanography can be adapted for the study of coral reefs (see also Chapter 5~; however, special consideration should be addressed to shallow areas, i.e., reef lagoons, reef flats, and algal ridges. Wave surge, shallow water depth, and tidal variations will dictate that specialized sampling methods be used. Stoddart and Johannes (1978) provide a detailed source of information for quanti- tative sampling of vat ious biological, chemical, and geological phenomena of coral reefs. Calcareous algae and scleractinian corals are major primary producers and are particularly important to study because they are more susceptible to toxic effects of oil pollution. In a recent review, Loya and Rinkevich (1980) discussed field and laboratory studies on the effects of petroleum in coral reef communities.

183 Analysis and Interpretation of Data Typical data resulting from benthic community sampling form a matrix consisting of a limited array of environmental variables and an imposing list of species counts for each of many collections. The approach often taken in interpretation of such data is to describe the distribution of a few abundant, or apparently diagnostic, species and relate these to environmental factors, thus effectively discarding a large portion of the data very often obtained at great expense. Alternatively, or in addition, an attempt may be made to simplify the complex array of data by computing a diversity index or some other statistic designed to represent the structure of the community, which discounts the majority of the information content of the sample, as well, including the fundamentally important information of what k inds of organisms were present. Thus, the results are difficult to interpret and the loss of information from such data reduction can have a s ignif i- cant effect on the conclusions of the study. Derived Community Indices Statistical methods for studying joint abundances of species, using probability distr ibutions fitted to abundance data and diversity indices, can be valuable for cer ta in appl icat ions . Of cour se, the underlying assumptions must be understood and, where possible, the conclusions tested by other means. Clearly, the statistical properties of some indices make them more useful than others, depending on the application. From a pragmatic point of view, a single number for certain types of inferences, or monitoring, might be desirable, as decision makers might prefer the parsimony of an index number. It is a well-established statistical fact, however, that one number or index may be insufficient for summarizing data; even so sample a distribution as the normal requires two numbers (a mean and a variance) for accurate descr iption. The bewildering complexity of data resulting from surveys of macrobenthos often prompts investigators to simplify results and report a derived index. Diversity indices have been particularly popular because of the presumed relationship between species diversity and environmental quality (Wilhm and Dorris, 1968), although Green (1979) and Smith et al. (1979a,b) state that this may not necessarily be a valid generalization. Diversity indices should not be used alone to assess impact but should be coupled with population or multivariate analyses that reflect qualitative community composition. Any index involves an inevitable loss of information, compared to the data from which it was calculated. In the case of species diversity measurement, the information lost includes the identity of the species in the community and the species history documented in the literature, both of which are characteristics of obvious importance in evaluating the consequences of any alteration in community structure. If diversity patterns are to be assessed, they

184 should be expressed primarily as species r ichness and evenness compo- nents, both of which are intuitively more meaningful. Graphical Representation of Community and Population Data The distribution of abundance in a biotic assemblage can often be more effectively represented graphically than by index. For population data, density plots and life stage histograms (sex, age) are particu- lar ly useful . A commonly used graphical approach in community analyses is to plot, as a logar ithmic ordinate, the density or biomass of all component species arranged sequentially from the most abundant to the least (Spies et al., 1980~. A different representation of the distribution of abundance involves plotting the frequency of species in geometrically increas ing s i ze classes of abundance (Gray, 1980; Gray and Mizza, 1979 ~ . Sanders et al. (1980) prepared s imilar graphical plots in the analy- sis of community variability with time by plotting not the abundance of each species, but a measure of species variability (e.g., coefficient of variation), representing species ranked by this variability. Such plots clearly represent the greater temporal population variability characteristic of disturbed communities (Figure 3-81. Multivariate Analyses Two samples can possess identical diversity indices and yield similar dominance curves, for example, and yet not share any taxa in common. Multivariate analyses, particularly numerical classification and ordina- tion, allow the simplif ication of complex data sets, wherein informa- t ion regarding the taxonomic compos ition of samples being compared is retained (Clifford and Stephenson, 1975; Boesch, 1977; Pielou, 1977; Orloci, 1978; Whittaker, 1978; R.H. Green, 1979; Orioci et al. 1979; van der Maarel, 19801. Too often practitioners will use a particular classif ication approach because it is available or was previously used. This frequently results in weak clustering and generally uninterpretable results (e.g., Bender et al., 19797. Criteria for the design of apropr late classification algor isthmi are given by Boesch (19773. Insight gained from application of numerical classification can be greatly enhanced by postclustering analyses, i.e., two-way or nodal analys is (Boesch, 1977; Straughan, 1980 ~ . Among ordination techniques, the most satisfactory methods for ecological analyses are reciprocal averaging (correspondence analysis) and nonmetric, multidimensional scaling (Hill and Gaugh, 19801. Ordination is generally more instructive than classification when the data are not too heterogeneous and when it is useful to view patterns as gradients, such as along a pollution gradient or in a time ser. ies . Multiple discriminant analysis (Green and Vascotto, 1978) is a useful technique which allows the investigation of environmental factors that best discriminate among previously defined groups (e.g., exposed to oil or not exposed to oil).

185 o 4.0 3.5 3.0 CC a: ,L 2.5 o Ad C' 8 2.0 1.5 1 .o 1 0.5 n I I 1 I I l cl; 1 I I l ~ 1 _: J I 1 l l l l l l 0 5 10 15 20 25 30 35 40 NUMBER OF SPECIES FIGURE 3-8 Plots of coefficients of variation for species populations in communities of macrobenthos near West Falmouth, Massachusetts. Stations are indicated where sediments were contaminated with No. 2 fuel oil. SOURCE: Sanders et al. (1980~. Species and Populations Certain benthic species exist in polluted environments or tolerate other disturbances. It is often appealing to interpret results of an impact assessment by examining the distribution of species known to be pollution tolerant, but great care must be taken in selection of a species, notably with regard to its identification and distribution. For example, Bender et al. (1979), in an evaluation of the effects of petroleum production in coastal Louisiana, observed that "if there had been a buildup of hydrocarbons or other pollutants, then one would expect a reduced benthic fauna and the appearance of pollution-tolerant species, such as Capitella capitata, in large numbers in affected localities." On the other hand, Sanders and Jones (1981) noted that in this case the benthos was of comparatively low density and was

186 characterized by pronounced dominance by two highly opportunistic species, the polychaet Spiochaetopterus oculatus and the bivalve ulinia lateralis. Gray et al. (1980 ~ pointed out that species such as Ca~itella respond to many forms of disturbance and concluded that the use of so-called indicator species is not rel table for ecological monitoring. Unfortunately, Capitella has been shown by Grassle and Grassle (1976) to comprise a group of sibling species, formerly thought to be one species. It is very difficult to separate members of Capitella capitata into their constituent species by morphology. Thus, in its composite, C. capitata, may not be a reliable indicator species even though its sibling species, individually, may prove to be finely tuned to different stresses. The opportunistic species cited by Sanders and Jones (1981) may be considered characteristic of predator-dominated estuarine habitats (Virnstein, 1977) such as those represented in coastal Louisiana. In general, it must be stated that reliance on one measure of change, disturbance, or stress is unwise and suites of measures are best employed because they offer greater reliability over the long term. Exper imental Approaches Beyond the Laboratory Exper iments conducted in the f ield or in mesocosms of fer promise in advancing knowledge of the effects of petroleum on marine benthos. For example, Grassle et al . (1981 ) followed the response of macrobenthos and meiobenthos in large volume mesocosms during and following chronic addition of No. 2 fuel oil. The behavior of the communities trans- planted in the control tanks followed the natural communities in the adjoining bay reasonably well and, by comparison of oiled treatments and controls, effects could be related to the buildup of petroleum hydrocarbons in the sediment. In another valuable experimental approach using enclosed spills, Bender et al . (1980 ~ described effects of weathered and unweathered crude oil on the benthos of a mesohaline salt marsh using controlled release of oil in intertidal communities. A different approach that has been pur sued is the colonization by benthos of azoic sediments placed in trays in petroleum-contaminated and control environments. Thus, rates of colonization and the composition of the recolonizing biota can be compared with azoic controls (Grassle and Grassle, 1974 ~ . Also, the settlement of larvae onto contaminated field plots is under intensive study (Vanderhorst et al ., 1981 ~ and settlement has been observed in the field on several occasions (Woodin et al., 1972 ; Straughan, 1971, 1972 ~ . The use of such exper imental approaches in the f ield for assessment of effects of petroleum on benthos greatly extends our ability to detect and understand these effects and, however, allows ache development and testing of valid hypotheses regarding effects {e.g., Spies and Davis, 1979; Spies et al., 1978, 1980~.

187 FISH, SEABIRDS, AND MAMMALS Fish Some relatively unique methods have been used to measure effects of petroleum on fish. Because fish provide diverse and biologically complex subjects for study, complete coverage of every method applied to fish is not feasible. Acute toxicity tests provide good preliminary knowledge concerning upper tolerance limits of fish to petroleum hydrocarbons. Fish gen- erally respond rapidly to oil exposure, and therefore, bioassays need to extend only a few days in duration, in contrast to bioassays of many invertebrates requiring longer exposure periods to respond. Flow- through assays are preferable to static tests because of the high metabolic activity of fish and the resulting greater need for oxvaen , _ is replenishment and metabolic waste removal. The oxygen level critical for many species of fish, e.g., salmon require >5.0 ppm oxygen for maintenance. The LC50s obtained from acute assays can subsequently be used to determine hydrocarbon concentrations for determining sublethal effects. Methods for measuring hydrocarbon uptake, distribution in tissues, metabolism, and excretion are similar, in many cases, to methods used with invertebrates. However, methods unique for fish include those devised to determine the pathway of oil hydrocarbons metabolized by fish, and the metabolic potential of fish. Three exposure media are commonly employed to introduce hydrocar- bons of petroleum to fish: food. water. and sediment. TO determine . . . accumulation via the gut, contaminated food may be prepared for inges- tion by mixing the food with oil or oil components, which may be radiolabeled if the experiment dictates. The food containing the hydrocarbon may be fed directly, encapsulated in gelatin capsules and force fed (Solbakken and Palmork, 1980; R.E. Thomas and Rice, 1981), or fed by gastric ravage (Nave and Engelhardt, 19801. A useful method is to expose living, natural prey organisms to the hydrocarbons and allow the f ish to feed on the prey, but the hydrocarbon concentration in the food organisms is difficult to control. Exposure of fish to hydrocar- bons via water can be done by placing the fish in exposure solutions amended with radiolabeled or nonlabeled hydrocarbons or oil WSF for a given time interval, after which the fish are transferred to hydro- carbon-free water for deputation. The exposure medium may also be sediment mixed with hydrocarbon and placed in the tank with the fish. Internal hydrocarbon concentrations can be monitored by collecting subsamples of groups of exposed fish at predetermined intervals. The fish are sacrificed and samples of selected tissues and fluids, viz., 1 iver, gall bladder, muscles, and blood, are analyzed. Although extraction procedures vary, they permit separation of metabolites from parent compound (s) . For example, Nava and Engelhardt (1980) treated homogenates with methanol-benzene (1:1) for 24 hours on a rotator at 4°C, followed by centrifugation. ~ _ ~ tested separately for radioactivity to measure crude-oil-derived fractions and total metabolizes, r espectively. Another useful method The organic and aqueous chases were

188 involves digestion of half the sample in papain, to separate polar metabolites from nonpolar parent compounds, followed by extraction of carbon-14, employing 90 % formic acid over laid with 5-10 mL hexane (Roubal et al., 1977a,b). Total radioactivity can then be determined for the other half of the sample . Detection techniques include liquid scintillation counting, gas-liquid chromatography, and thin layer chromatogr aphy. Metabol ism in f ish occur s mainly through the inducible, mixed func- tion oxidase (MFO) system to form oxidized metabolites. These more polar derivatives are discharged by diffusion across membranes or con- jugated with serum components and excreted (Burns, 1976~. Induction of MFO enzymes, such a`; aryl hydrocarbon hydroxylase (AHH) can serve as sensitive physiological indicators of oil contamination {J.F. Payne and Penrose, 1975 ~ . MFO activity is measured in tissue homogenates by quantifying the conversion of a substrate to a metabolite, with different substrates requiring different methods for analysis. Individual methods are not covered in detail, but the following are some of the useful methods: cytochrome P450 (Omura and Sato, 1964 ), cytochrome be (Ernster et al ., 1962), NADPH cytochrome c reductase (Ernster et al., 1962; Masters et al. , 1967) , NADPH dichloropenolindophenol (Ernster et al., 1962), reductase (Masters et al., 1967), NADH cytochrome c reductase (Masters et al., 1967), aryl hydrocarbon hydroxylases (Dehnen et al., 1973; Depierre et al., 1975), aryl-4 monooxygenase (Lowry et al., 1951), and nitro reductase (Lowry et al., 1951) . Determination of the pathway and exoretion routes of petroleum hydrocarbons in fish has been accomplished (R.E. Thomas and Rice, 1981) by employing a split box arrangement, whereby a fish is fitted with a rubber dam attached posterior to the gills so that water could be sampled from sealed anterior (gills) and posterior (feces, urine) cha~nher s . Many different kinds of subletha.1 measurements can be made in fish exposed to oil, s~milar to those used with invertebrates. In general, fish are more easily excited than most invertebrates, and measurements of respiration, heart rate, blood cortisol levels, etc.,. must be done with this in mind. A group of fish, once sampled, may not be ready for another sampl ing for 24 hours or more. In general, sublethal measurements on fish have been done to measure stress and the energy utilized after exposure to hydrocarbons. For example, heart rates have been measured by implanting electrodes and recording EKG (Wang and Nicol, 1977~. Opercular rhythm and coughing have been monitored by measuring pressure changes in the buccal cavity (Barrett and Toews , 1978 ~ or by external electrodes (Spoor et al ., 1971; P. Thomas and Rice, 1979~. Energy reserves can be measured by determining rates of lipoqenesis (Stegeman and Sabo, 1976) or total lipid content. Growth rates are measured by recording weight and length (Moles et al., 1981~. Respiration can also be monitored by analysis of oxygen consumption rates, taking care to maintain proper PO2 levels in the water {Brocksen and Bailey, 1973 ~ . The ef feet of hydrocarbon on the stress response hormone, cortisol, also has been measured (P. Thomas et al., 1980) as well as the effects of petroleum

189 on ability of fish to handle stress, using stamina tunnels that assess the ability of fish to swim under measured water velocities (Beamish, 1978 ) . Growth of fish is relatively easy to monitor, but fish require long exposure times before significant differences can be detected, compared with controls. In contrast, energy budgets (scope for growth) provide a sensitive method for early detection of sublethal responses in fish, but are labor intensive. Energy input and output are determined by measur ing calor ic intake, respiration, and excretion. Scope for growth {P) is the difference between caloric intake and energy expended for respiration (Widdows, 1978a,b): P = C x [(F-E)/~1-E)F] - R C (car/day) = (Emg] food consumed/day) x calories/mg F = (ash-free dry weight/dry weight) of ingested food E = (ash-free dry weight/dry weight) of excrement R (car/day} = (mL O2 consumed/day) x 4.86 Effects on reproduction are measured by observing gamete condition tWhipple et al., 1981) and development of sexual dimorphism (Hedtke and Puglishi, 1980), and by quantifying egg fertility, egg survival, timing of hatch, and hatching success. Observations of behavioral responses yield information that may help 1 ink laboratory research with response and impact in the natural environment. For example, observations can be made on schooling ability in large circulating tanks fitted with a rotating gantry of fish (Partridge, 1982~. Food selection tests, allowing fish to choose between contaminated and uncontaminated food, will indicate preference for or against (or neither) contaminated food (Blackman, 1974~. Avoidance tests also can be done to determine whether fish prefer, avoid, or neither prefer nor avoid water containing hydrocarbons 1973; Weber et al., 1981) . The behavioral response of larvae is usually monitored by simple visual observation of given attributes Seab ir ds Exper imental Methods Much of the exper imental methodology for assessing oil effects on seabirds is derived from standard freshwater or terrestrial bird studies, although some specialized techniques have been developed (Peakall et al ., 1979 ~ . For example , techniques used for studying blood chemistry, growth, nutrition, respiration, and so forth are routinely appl fed to seabirds with equal success . However, there ar e difficulties in maintaining wild seabirds in captivity, especially adult birds, that have led researchers to use more tolerant species (e .g ., mat lard ducks) . Depending on the questions studied , the use of such substitute species can provide answers relevant to oil ing problems in seabirds.

19o Mar ine Birds The most meaningful information, especially on growth, nutrition, osmoregulation, and blood chemistry, has come from work done with seabirds. ~ , ~ ~ ~ I ~ seabirds or in the wild with nesting colonies. Not all seabirds are ~ ~ ~ Adult seagulls especially are very difficult to keep in good health. Nestlings, on the other hand, Here the aDuroach has been to work either with captive maintained that easily in captivity. including nestling seagulls, are much easier to handle, provided a good supply of food (fish) is available. Seabirds, however, can be unexpec- tedly choosy in their food. Young puffins have been raised success- fully in captivity by housing them in individual burrows, although their maintenance is very time consuming (Leighton et al ., 1983 ~ . Work with wild colon ies has largely involved e ither eggs and nestlings, or the young of burrow-nesting seabirds . Manipulating either eggs or young on the nest, in the wild, does not appear to cause any problems. While there may be some desertion of eggs earlier in the breeding season, the experience is that the adults are less likely to desert the nest after hatching. Work with fledglings depends on the species. Seagull chicks can be experimentally handled in the nestling stage (e.g., R.G. Butler and Lukasiewicz, 1979), but once they become mobile and begin to explore their surroundings outside the nest, they become difficult to handle. In this respect, burrow-nesting species such as puffins are much more tractable (e.g., Peakall et al., 1980a). Of the burrow-nesting species, the most useful have been the auks (puffins and black guillemots) and Leach's petrel (Peakall, 1980b; Ainley et al., 1981; C.H. Walker and Knight, 19813. Thus, in one study (Peakall et al., 1980b), nestling herring gulls (Laws argentatus) and black guillemots (Cepphus grylle) and adult Leach's petrels (Oceanodroma leucorhoal were ec,1 l ec ted and studied on islands of f the coast of Maine . Both the petrels and the guillemots were sampled directly in their burrows or taken from their burrows and brought into the laboratory. On the other hand, the gull nestlings had to be banded at the beginning of the experiments and spotted and captured at each sampling time. One major drawback with wild birds, and a criticism of some experi- mental studies, is the absence of nutritional history. While some seabirds show marked preferences for certain foods, many feed on a range of prey. Any variability due to such nutritional differences can be exacerbated in colonies near human settlements and industrial centers where the birds may, in addition, be exposed to a range of man-introduced chemicals (e.g., Knight and Walker, 1982a,b). These problems can be overcome, in part, by using suitable nestlings or fledglings raised in captivity, where nutrition can be controlled. Freshwater and Terrestrial Birds Frequently circumstances are such that wild seabirds are not readily available, and one has to turn to other species. Bird eggs, in terms of their susceptibility to oiling, are not highly species specific, and chicken-eggs, for example, could be used to study effects of oiling on hatching processes (e.g., Albers and Gay, 1982; Macko and King, 1980; Woo t ton et al., 1979~. However, when seabirds are not available, relevant results are likely to be obtained using either similar species or seawater- adaptable birds. In this respect, valuable results have been obtained

191 using mallards (e.g., Holmes et al., 1978; Goraline et al., 1981) . Although a mallard duck may not be as good an osmoregulator as a seabird, its capabil ity to induce the nasal gland and the osmoregulatory system makes this species a useful and readily available test animal for oiling studies of seabirds. Geographic Variations With increasing oil and gas exploratory activity in the polar regions, attention will in the future be directed to study- ing effects of oil on seabirds in cold climates. Fortunately, the common seabird species have a wide range in their distribution and also are found in more temperate areas. Thus, oil effect studies done on temperate species are probably applicable to northern forms, and data can be extrapolated with some degree of confidence and relevance. It is important to consider the added factor of temperature or cold stress in oiling studies of species of polar regions (e.g., Erasmus et al., 1981). Census Studies Probably the greatest hindrance to assessing the effects of oil on seabirds at the population level is the scarcity of information on population sizes. The most accurate census can be taken at seabird colonies during the breeding season. In both the eastern United States and Britain, these counts date back to cat 1900, yielding an 80-year census history. However, for the rest of North America and elsewhere, census records date back only to cat 1970 (e.g., R.G.B. Brown et al. , 1975~. Therefore, in most cases it is difficult to assess any apparent shift in colony sizes within the context of long term population trends. Even when there is a long history of counts, lack of standardized of methods often makes it virtually impossible to detect all but the largest changes. Fortunately, refinement in census techniques (e.g., Nettleship, 1976) in North America and Europe within the last decade will allow for statistically replicable census taking of colonies based on a system of standardized plots. Adequate census taking at colonies of burrow- nesting species such as storm petrels and many of the auks, especially when the species are also nocturnal, will remain extremely difficult. Such population estimates cannot take account of the large numbers of adolescent birds that may spend several years away from their colonies before returning to breed. Counting of birds at sea, from both ships and aircraft, has been developed and refined within the last years, and the approaches allow better statistical treatment of the relative abundance of seabirds in different zones of the open ocean, e.g., relative abundances over cold water continental shelves versus warm, offshore tropical seas. It is difficult to convert these figures into absolute estimates of the numbers of birds at sea and relate back to absolute number s in the colonies, While ship and, especially, aircraft surveys allow more extensive census tak ing, they also have cer Lain l imitations . Shipboard surveys can cover only relatively small areas and are difficult to repeat with

192 regularity. Ships necessarily avoid hazardous zones of shallow water, close inshore, where seaboards and sea ducks are often abundant. Aerial surveys, on the other hand, can cover all marine zones along sampling tracks with repeated regular ity. Several surveys have produced absolute estimates of bird numbers at sea. Unfortunately, absolute identifica- t ion of birds from the air is not always possible. For example, many of the species of alcids, seabirds especially vulnerable to oil pollu- tion, cannot be identified while they are in the air, a serious limita- t ion to aer ial surveys of f both the West and East coasts of Nor th America (e.g., R.G.B. Brown, 1980, p. 13) . In addition, the problem of ground-truthing aer ial surveys , i .e ., both census number and bird identification, has not yet been solved satisfactorily for most species. Finally, aer ial surveys are restr icted by range and safety requirements of the aircraft, as well as by expense. Oil-induced mortality of seabirds is difficult to establish with certainty, (R.G.B. Brown, 1982~. For several reasons, all mortality figures are gross underestimates because they are based mainly on the number of birds that drift ashore. Most oiled bird counts are based on shoreline surveys, including a body count per kilometer and extrapola- t ion to the length of oiled beach. However, many slicks never reach land and birds encountering such offshore slicks are never enumerated. Similarly, many oiled birds sink and drown before reaching coastlines. One estimate (Hope-Jones et al., 1970) suggests that only 20% of oiled birds reach land; the percentage is thought to be smaller for west Atlantic waters, where prevailing winds tend to carry corpses of the birds out into the Atlantic (R.G.B. Brown, 1982~. Marine Mammals Aside from of] itself, other activities and by-products of oil explora- tion and exploitation (e.g., noise, debris, shipping movements) can affect marine mammals. Because of their movements on and through the sea surface, marine mammals are highly vulnerable to contact with oil . Notwithstanding these concerns, probably less is known of how oil affects marine mammals than any other group of marine organisms. Only recently has work begun to address this issue experimentally, i.e., with suitable controls, instead of relying on field observations and news accounts, which may be conflicting and, in some cases, imprecise. There are several reasons for this gap in understanding, mainly that the behavior, physiology, etc., of marine mammals are not well under- stood even without the added dimension of effects of oil. A1SO, many of these animals are too large, or otherwise unsuited, for captive or f ield studies. There are marked differences among the 9 groups of marine mammals, which comprise a total of more than 130 different species. Thus, it is difficult to select representative marine mammals or to measure effects of oil, noise, and other factors on each species. Because research on marine mammals attracts public interest, experimen- tal studies are vulnerable to public criticism. Nonetheless, much can be learned of how oil affects mar ine mammals if intelligent choice of study animals and appropriate clinical methods is made.

193 Toxicological Studies Conventional toxicology studies , e . g ., establ ish ing acute lethal toxicities, on mar ine mammals are not popular and receive wide public attention and Or iticism. Also, such tests provide little information on how oil interacts with mar ine mammals because such tests are rarely designed to provide the information that is needed (e.g., Sprague, 1971) and only a small number of animals usually are available. Abundant information gathered for other species is available and can be used to derive conclusions concerning the toxicity of oil for marine mammals (Geraci and St. Aubin, 1982~. Standard clinical methods, coupled with chemical analytical tech- niques, can be used with good success to examine uptake and metabolism of petroleum hydrocarbons in marine mammals, with relatively little harm to the animals. For example, controlled oil uptake studies have now been done on captive marine mammals, using small doses of radio- labeled hydrocarbons subsequently detected in biopsied tissue, blood, urine, and feces {Engelhardt et al., 19777. Similarly, the physio- logical impact of oiling on these animals can be monitored and studies done using standard clinical methods (Geraci and Smith, 1976; Engelhardt, 1982 ~ . Surface Oiling Contact of oil with the skin or hide of marine mammals can cause problems for the animals. Traditionally, direct immersion procedures have been employed. There are several disadvantages with th is approach . Rarely are enough animals available to yield statistically signif icant results. Only one petroleum product can be tested at a given time and exposure time on the skin cannot be easily controlled. Again, such methods are subject to intense public criticism. An alternative approach that is promising is analogous to skin allergy tests for humans. The method is relatively harmless to the test animal, permits controlled testing of a variety of compounds, and yields far more information {Geraci and St. Aubin, 1982~. It has been used repeatedly on captive and live-stranded cetaceans and provides information on biochemical and ultrastructural changes in skin cells. Such an approach is particularly appropriate for skin-sensitivity studies of large mammals such as the mysticetes. Oil Avoidance Information on the ability of cetaceans to detect and avoid surface oil can be obtained from direct observations in the field or using trained captive mammals. Both have provided some data, but much more informa- tion is needed, since some of the observations reported to date are contradictory, pointing out the difficulties in extrapolating from experiment=] sin captivity" studies to responses in the field. Certain cetaceans, such as the bottlenose dolphin Tursiops truncates, can be

194 readily trained to detect and respond to surface oil slicks as limited as 1 mm thick, apparently by echolocation (T.G. Smith et al., 1983, Geraci et al., 19837. This approach, apparently harmless to the test animals, provides useful information, specifically on oil avoidance and in general on echolocation. Possibly this approach could be extended to other captive species to determine whether f indings for bottlenose dolphins can be extrapolated to other odontocetes. Greater difficulty is met in (1) obtaining similar observations from wild marine mammals in the field and (2) extrapolating from the experimental data to oiling incidents in the wild. Observations have been made on wild gray whales swimming through natural oil seep areas off the coast of California. The advantage to this approach is that observation of several parameters related to behavior under natural oiling conditions can be made, including aerial surveys. However, under such conditions it is difficult to obtain a large number of observa- tions on any single test animal. Also, one has little control over the amount or chemical composition of oil to which the animal is exposed. Interference With Feeding Little information is available on interference with or blocking of feeding in oiled marine mammals. Potential fouling of the feeding mechanism in baleen whales has gained attention. However, direct oiling of the baleen apparatus in live animals, whether in the wild or in captivity, is subject to public scrutiny. The problem can be attacked by seeking evidence of oil contamination of the baleen apparatus in stranded or commercially hunted specimens and by carrying out studies of isolated baleen in specially designed water flumes (Geraci and St. Aubin, 1982~. The latter offers signif icantly greater control over experimental conditions and , with appropr late modif ica- tions, should be useful for studies of subtle effects of oiling on the baleen str uctur e . Spills of Opportunity In the f inal analysis, valuable information can be gathered from systematic studies carr fed out dur ing accidental oil spills at sea, both by direct observation of mammal behavior in oiled waters and by gather ing autoptic data on stranded oiled animals (e.g., J. Parsons et al., 1980) . Only limited information is available concerning the behavior of marine mammals found in the vicinity of an oil slick, whether at sea or stranded on shorelines. Stranded animals provide useful information on effects of the physiology and, therefore, samples should be collected and analyzed to confirm results of studies done antemortem. In the past, potentially useful information was lost or missed because sys- tematic reporting and collection of samples of stranded marine mammals during spills was not done. To correct this, and perhaps reduce the need to work with live animals in captivity, federal and regional

195 officials should collect and freeze, where possible, whole carcasses, or at least be familiar with methods for collection of skin, blubber, blood, visceral organ, and stomach content samples. CYTOGENIC AND MUTAGENIC METHODS A wide range of cytogenic and mutagenic assays are now available, with both new developments as well as improvements on existing techniques being reported in the scientific literature (Stich and San, 1981~. Most have been developed for bacterial or mammalian cell lines. Very few are available for use with marine tissues or cell lines. The methods are essentially designed to measure or score chromo- somal abnormalities, i.e., cytogenic or cytologic aberrations, and mutational changes, i.e., inheritable changes in nucleic materials in daughter cells. Chromosomal Aberrations cytogenetic methods have not been used extensively for marine systems. Thus, it is not surprising that chromosomes have not been examined to any extent, with respect to petroleum pollution. Chromosomal aberrations can be structural (breaks or gaps} or can involve changes, either in the number of chromosomes or chromosome sets, resulting from faulty separation or division. Consequences of such aberrations are generally deleterious, not beneficial or neutral. Abnormal chromosomes are especially prone to further misdivision and breakage, resulting in additional deficiencies and duplications. Chromosome aberrations in somatic cells are involved in some stage of tumorigenesis. When the aberration occurs in the germ line, it is transmittable to the next generation, provided it is com- patible with reasonably normal embryo development. Unbalanced genetic complements (duplication for one segment or deficiency for another) usually lead to death early in embryonic development. Certain recipro- cal translocations can result in sterility in both animals and plants (Russel and Matter, 1980; Sparrow and Woodwell, 1963~. The methods described with respect to marine species are treated in detail in the literature (Hollaender, 1971, 1973, 1976; Hollaender and deserves, 1976; Kilbey et al., 1977~. Direct Examination of the Chromosome Karyotype Direct examination of the chromosome karyotype is practical only when the organism has a suitable complement of chromosomes, that is, of a given size range and number. The direct chromosome method can be applied only to certain tissues and cell types that lend themselves well to the spreading of individual chromosomes and possess a good mitotic index. Organisms with such karyotypes can provide detailed, qualitative information on chromosome mutation. Even when species with

196 less than ideal karyotypes have been studied intensely, careful study of the chromosomes has been productive, as for example in the mouse and human systems. Marine Invertebrates Little cytogenetic work has been done on marine invertebrates. Nonvertebrates present special problems as to method and source of tissue to be examined. Marine invertebrates and tem- perate and cold water fishes have significantly lower rates of mitotic turnover, in general, than mammals and certain terrestrial plant tissues, and their gametogenesis is seasonal. Since chromosomes are directly studied cytogenetically only in selected stages of mitosis or meiosis, difficulties ar ise in obtaining sufficient numbers of cells for chromosome analysis on a year-round basis. Externally fertilized, freely developing zygotes, however, offer a good source of material. Among the invertebrates, the Ostreidae, species of which are com- mercially valuable, possess 10 pairs of chromosomes. The chromosomes of several oyster species have been well described, and a full karyotype analysis has been published for the American oyster Crassostrea virginica (Longwell et al., 19671. The chromosomes and freely spawned, externally fertilized eggs of shellfish make them ideal subjects for studies of oocyte chromosomes in meiosis, chromosomes of the male in fertilization, and early developing embryos (Longwell and Stiles, 1968~. Hatchery cultivation of the oyster in aquiculture makes this shellfish attractive for such work, especially since oysters play a significant role in nongenetic assays of water quality. Recently, the marine polychaete worm Neanthes arenaceodentata was reported to have an ideal karyotype for direct chromosome study. Also, Neanthes has been used to examine sister-chromatic exchange (Pesch and Pesch, 19801. Plants A significant amount of cytologic information is available from the examination of terrestrial plants, notably meiosis in the develop- ment of the male gamete (microsporogenesis), along with root tip meristem cells. Fish In general, fish possess large numbers of small chromosomes. Although fish comprise the largest of the vertebrate groups, the chromosomes of only a few species have been counted or described, and even less are marine. Complete chromosome karyotypes have been characterized for only about 2-3% of the 20,000-23,000 living species of fish (Chen, 1969; Gold, 19797. In contrast, 30% of the living species of eutherian mammals has been studied, some extensively. Recently there is renewed interest in the chromosomes of fish with respect to studies of their evolution, particularly in the Salmonidae and also with respect to genetic eng ineer ing appl ications for the breeding of aquaculture strains (Gold, 1979; Ojima, 1980~. Basic cytogenetics and gene mutations of certain groups of fresh- water fish have been studied tSchroder, 1973; Ojima, 1980), and a few freshwater fish have been shown to possess ideal karyotypes, desirable for direct studies of chromosome mutation, viz., the mud minnows Umbra

197 limi and Umbra pygmaea (Kligerman et al., 1975; Kligerman and Bloom, _ 1977; Hooftman and Vink, 1981~. Pr ime tissue sources of f ish cells for chromosome analysis are epithet ial cells of gills, f in, scales, and cornea and hematopoietic tissue of the anterior kidney, testis, intestine, and the early embryo (Demon, 1973; Wol f and Quimby, 1969 ~ . Lymphocytes of circulating blood in fish do not, in general, respond well to mitotic stimulation in vitro, as do those of human lymphocytes. However, there have been some reports of success using this method. It has been conf irmed by autoradiographic observation that the intestinal epithelium of fish is a typical cell renewal system (Hyod~Tagoch i, 1968; T . S . Johnson et al ., 1970 ~ . The yolk-sac membrane of pelagic f ish eggs collected from sea surface waters in plankton provides good material, in the early egg development stages, for direct chromosome study (Longwell and Hughes, 1980~. Larval tissues of fish may prove more useful than those of adults because of a higher mitotic turnover. Marine Mammals Direct examination for chromosomal aberrations in mammals is usually done using tissue culture cells, many lines of which are of embryo origin, and hematopoietic cells of bone marrow, or lympho- cytes of circulating blood stimulated to undergo a simple mitosis in vitro. However, restrictions and difficulties in sampling marine maT'unals create special problems for cytologic studies, somewhat analo- gous to the human, for which special methods to circumvent these diffi- culties had to be developed. Future studies could well be done on tissue-cultured cells employing biopsies, aborted fetuses, and maternal or fetal membranes and monitoring by cultur ing samples of circulating blood as is done in human studies. Birds Methods for chromosome analysis are available for avian embryos and can be applied to marine birds. Chromosome analysis of chicken embryos has been proposed as a method for monitoring of chemical- induced mutagenicity (Bloom, 19781. The major difficulty faced in studying marine birds is collection of samples in sufficient number to obtain statistically significant results. Work with affected bird populations after major oil spills would provide some opportunities for this cytogenetic work if its potential significance were recognized. Micronucleus Test and Related Mitotic Assays This test has found extensive use in mammalian and plant studies. With proper modifications, it appears to be a promising approach for marine tissues (Schmid, 1976, 1977) . In mammals the test is usually applied to the polychromatic erythrocyte of the bone marrow. Several labora- tor ies have shown the micronucleus test comparable in sensitivity to direct chromosome examination, as well as offering substantial savings in time and cost (Kliesch and Adler, 1980; Jenssen and Ramel, 1976, 1980 ) . The test was developed specif ically for chemical mutagenicity (Countryman and Heddle, 1976 ~ and has since been used effectively to

198 measure cytogenetic aberrations in plants (Ma , 1979 ), rat spermat ids (Lahdetie and Parvinen , 1981 ), mammalian l iver cells , prenatal mammals (Stoyel and Clark, 1980; Cole et al., 1981), and mammalian meiosis (Lahdetie and Parvinen, 1981) . This assay, or modif ications thereof, may be adapted to studies of adult, larval, and fish embryo in bioassays of petroleum and dispersants, in monitoring and in field studies of the effect of oil spills, to certain invertebrate tissues of sufficiently high mitotic turnover, or to marine mammals. For several years, Soviet researchers have measured the effects of incorporated radionuclides on fish embryo cells by scoring micronuclei of cells in mitosis, telophase bridges of translocated chromosomes, and laggard or fragmented chromosomes, as well as micronuclei in nondividing or interphase cells (AEC, 1968, 1972~. This approach differs from the micronucleus test applied to mammalian bone marrow only in that it is used on dividing as well as nondividing cells, and scores also mitotic events leading to micronucleus formation as well as micronuclei. Using such a suite of cytological criteria, Longwell and Hughes (1980) measured mitotic-chromosome abnormalities in developing eggs of the Atlantic mackerel collected by plankton in the New York Bight, employing both the embryo itself and the yolk-sac membrane of the egg. Hagstrom and Loaning (1967) used mitotic disturbances, among other factors, in a cytological study of the effect of lithium on the develop- ment of the sea urch in embryo. Sea urchin eggs have been proposed as general test ob jects in pollution bioassays, oil, and related studies (Kobayashi, 1974; Lonning, 1977 ~ . Dominant Lethal Gene Mutat ions This test is a measure of heritable chromosomal abnormal ities in the male germ line which kills the embryo early in development. The basis for the test is the incompatibility of gross chromosome changes with successful development. Recent cytogenetic studies in mice and ham- sters have quantitatively related induced dominant lethal mutation frequencies to the incidence of broken chromosomes at early cleavage metaphases of the embryo (see Rohrborn and Hansmann, 1977~. It is likely that the absence of one or more chromosomes of the several chromosome pairs is a prime cause of genetic death of many early embryos (Russell and Matter, 1980 ~ . For all its shortcomings, especially when the female is treated, the dominant lethal test has been widely applied in the past as the only practical mammalian in viva test for heritable mutation in the germ line. The production of dominant lethal mutations has been studied in Tilapia tHemsworth and Wardhaugh, 1978; Wardhaugh, 1981), the guppy (Woodhead, 1977), trout (Newcombe and McGregor, 1967), and medaka (Egami and Hyodo-Tagachi, 1973~. Embryo deformity was usually manz- fested initially during gastrulation. The large majority of morpho- logically defective embryos were nonviable. Further information on the applicability of the test has come from studies on fish sperm treated with known mutagens, e.g., carp, rainbow trout, poled (TSoi, 1969; Tsoi et al., 1974) . Fertilizability of the sperm was not affected; however, effects were expressed at subsequent developmental stages.

199 The dominant lethal gene mutation assay, notably cytogenetic modifications of it, appear to be applicable to marine species that are culturable through at least gastrulation, particularly for the oyster where it can be combined readily with cytogenetic examination of early cleavage chromosomes. Sister-Chromatid Exchange Staining methods yielding differential staining of the two chromatics of the chromosome allow detection and scoring of sister-chromatic exchange events in a modification of the chromosome spread procedure used to detect chromosome aberrations. This type of test procedure has some of the same limitations as the direct chromosome examination procedure, but fewer metaphase need be examined. It is attractive, however, as comparative studies of chromosome aberrations and sister-chromatic exchange often show the latter to occur in the absence of chromosome breakage. In cultured Chinese hamster cells, sister-chromatic exchanges were observed at considerably lower concentrations of mutagens than those needed to induce aberra- tions (Natarajan et al., 1976~. Sister-chromatid exchange has proved very sensitive for the detection of many mutagenic chemicals, both in viva and in vitro, including those, such as the polycyclic aromatic hydrocarbons, that require metabolic activation to be mutagenice In vitro (tissue culture) applications of this method are much more easily conducted than in viva, even though the assay has been suc- cessfully employed in viva using some plant species (Kihlman and Kronberg, 1975), the chick embryo (Bloom and Hsu, 1975), and two species of mammals (for example, Stetka and Wolff, 1976~. Barker and Rackham (1979) successfully applied sister-chromatic exchange to cultured cells of the freshwater fish Ameca splendens. When the response to mutagens was compared to that of cultured mam- malian cells, fish cells seemed less sensitive. This method has also notably been applied to the freshwater mud minnow (Kligerman and Bloom, 19771. Valentine and Bishop (1980) found intraperitoneal injection of two known direct mutagens to induce dose-dependent increases in sister- chromatid exchange in intestinal metaphase chromosomes of the same fish. S. ister-chromatid exchange was also tested on 8- to 10-day-old embryos of the marine polychaete worm Neanthes. Effects of a known mutagen, Mitomycin C, exhibited the expected dose response at the level of sensitivity exhibited by mammalian systems (Pesch and Pesch, 19801. Sperm Mutation Test Certain mutants In mice and particular mouse strains are regularly characterized by abnormal shapes of the mature sperm heads and tails. It has recently been determined that irregularities in shape of sperm heads are produced by known mutagens, but not by nonmutagens. AS a

200 result, sperm abnormalities have been proposed as an assay for muta- genesis. Such a test would have the advantage of providing direct evidence for gene toxic activity within the germ cells in viva {Sobers, 1977). The sperm-shape abnormality assay, which appears to detect gene, not chromosome, mutation as in the dominant lethal gene mutation test, has been used to detect mutagens, carcinogens, and teratogens tHeddle and Bruce, 1977; Topham, 1980a,b; Wyrobek and Bruce, 19783. Recently, Bruce and Meddle ( 1979 ~ reported an excellent overlap in the mutagen detection capacity between the Salmonella bacter ial assay and the abnormal sperm assay. Polycycl ic hydrocarbons are positive . Effects on the progeny of mice with induced mutations affecting sperm head shape have been assessed by measuring the incidence of abnormal sperm in offspring (Topham, 1980a). The sperm of several fish has been described (Ginsberg, 1972) and these compare favorably to mammalian sperm, with respect to usefulness in the sperm mutation test. Oyster sperm are somewhat smaller but can be scored for abnormal ities rapidly using the light microscope . Hollstein et al., (1979) point out that this test could be of value in monitor ing genetic effects of environmental and industr ial chemicals, along with cytogenetic examinations of oocytes and spermatocytes. The sperm mutation test should be applicable to field, laboratory, and monitoring studies of mar ine f ish and mollusks, provided mutation therein also affects abnormalities in sperm morphology. In Vitro Cell Transformation Assays employing cell transformation (Hollstein et al., 1979) are carcinogenicity tests rather than tests of chromosome mutagenicity. Also, they require considerable technical skill, experienced judgment, and time and demonstrate a strong correlation between carcinogenicity and mutagenicity. Petroleum-induced abnormal tissue growth in crabs (Albeaux-Fernet and Laur, 1970), bryozoans (Powell et al., 1970), and f ish (Kezic et al., 1980) has been reported. Typically, the test concerns changes in morphological nature and division rate of cultured cells after incubation with the suspect chemical. Colonies of transformed cells are sometimes demonstrated to be malignant when injected into host animals. unlike their nontrans , formed, precursor cell types. For example, polycyclic hydrocarbons were reported as having both cytotoxic and transforming effects on Syrian hamster fetal lung cultures (Richter-Reichhelm et al., 1979) . S imilar studies might be conducted on cultured f ish cells . In a modification of the in vitro transformation assay, mammalian embryos were exposed to chemicals in viva before culturing (DiPaolo et al., 1973~. An analogous test can be devised for intact , freely spawned early embryos of marine species, the larval development of many of which is adversely affected by petroleum hydrocarbons (National Research Counc il. , 19 75 ~ .

201 Mutagenesis Assays Methods for measuring mutagenic potential in the strictest sense, i.e., those that score changes in phenotype, are based largely on the use of specialized bacter ial strains, certain yeasts, and mammalian cell lines. Among the bacteria most commonly used are Salmonella typhimurium (Ames et al., 1975) and Escherichia colt. The latter, E. cold WP2 , was used successfuly to detect mutagens in mussels from the polluted Lagoon of Venice {Parry and Al-Mossawi, 1979) . Best known, however, is the "Ames test," based on a number of specialized S. typhimurium mutant test strains that revert readily, via point mutations, to wild type upon exposure to mutagens. The Ames Assay The Ames assay is rapid, easily applied to a wide range of potential mutagens, and extremely reproducible. It consists very simply of mixing one of the several tester strains that are available with soft agar and an amount of the test substance, pr for to plating the mixture onto a histidine-deficient (minimal medium) plate. The soft agar contains sufficient histidine to allow approximately two divisions of the S. typhimurium tester strain. After the histidine has been utilized, the culture ceases growth, except for spontaneous mutants which continue multiplication (ca. 50/109 cells). However, if the culture is exposed to a mutagen (e.~., W or benzopyrene), the number of mutants increases, increasing the colony count, ideally in a direct dose-response proportion. In many cases when the response to a suspect mutagen is negative, positive response can be elicited by the inclusion of a microsomal extract (S9) of rat liver in the bacteria/soft agar mixture. The explanation is that some compounds are not mutagenic themselves, e.g., benzofa) pyrene, but mutagenicity resides in their epoxide derivative. Usually such "activations is done using the S9 fraction of induced liver homogenates obtained from animals exposed to a known mutagen. A positive response can also, in some instances, be enhanced by pre- incubating the Ames tester strain with S9 soft agar mixture before overlaying onto the minimal medium plate. For applications and limitation of the Ames assay, the literature should be consulted, viz., Nagao et al. (1977), Muller et al., (1980) , and Klekowski and Barnes (1979~. It should be pointed out that , apparently , some complex environmental mixtures contain antimutagenic components, as in the case of acetone extracts of lyophilized Mytilus edgily tissue from the Dutch coast, which were reportedly able to completely abolish the mutagenicity of benzota~pyrene in the Ames test (Mattern et al., 1981, cited by Odense, 19821. Modif ications on the Ames Assay Recently B jorseth et al . (1982 ~ incorporated the Ames test into thin layer chromatography (TLC), by layering the mixture of soft agar,

202 bacterial tester strain, and S9 directly onto a chromatographed TLC plate. Thus, mutagens or promutagens requiring activation and occurring in the different fractions (TLC spots) were identified directly. A second modification involves use of a liquid incubation medium employing blue in plates with 50 or 96 wells, allows statistical evaluation of the mutagenic response by scoring the number of acidified wells (Hubbard et al., 1981~. A third version, the host-mediated assay, involves injection of both the suspect mutagen and the Ames bacter ial tester strain into a suitable host (mice, rats) (Frezza et al., 1982~. The advantage of this modification is that the mutagen is "activated" naturally by the host S9 system instead of by commercially prepared S9 in a test tube mixture, a method found useful in detecting mutagens in mussels collected from a lagoon in Venice. Other Systems A limitation of the Ames test is that, while it appears generally to be good for detecting mutagenicity using bacteria, a possible result does not always correlate with mutagenicity in animals. Thus, eukaryotic cells, i.e., yeasts or mammalian cell lines, are more applicable for detecting mutations based on chromosome damage (e.g., Von Borstel, 1981; McCormick and Maher, 1981~. Little work has been done with marine algae, but one approach potentially applicable to marine phytoplankton is that of irandermeulen and Lee (1977 ), who employed Chlamydomonas reinhardtii to examine the potential mutagenicity of several oils. Applicability to Petroleum Studies One hindrance to the exper imental testing of mutagenic effects of petrol eum ~ and other substances ~ on mar ine plants and an imals is the lack of genetically uniform ~ inbred) test organisms . The availability of h ighly inbred animals would permit testing a smaller number of animals, yet yield higher precision. Some strains of freshwater fish species are being bred for such purposes, but the marine research needs have not yet been met. No single test of mutagenicity or cytogenic aberrations employing a single tissue or life stage can be expected to provide full information on chromosome mutation risk. Uptake, metabolism of the chemical, and transport will all affect the response measured using a particular tissue, cell type, or phase of the life cycle. Also, the sensitivity of chromosomes to mutation will vary according to stage of mitosis or meiosis. Unfortunately many marine species have less than ideal chromo- some complements for direct, detailed study of individual chromosomes, i.e., karyotyping. However, the newer, indirect measures have the advantage over s impler tests on prokaryotes in that they measur e d irectly the effects of a substance on chromosomes in the eukaryotic cell and organism of concern and are not merely predictors of mutagenic potential.

203 ECOSYSTEMS An ecosystem study is that which assesses interacting habitats, involves determining pr imary and secondary productivity, standing crops of organ- isms in major compartments, key species within those compartments, and the cycling of carbon and nutr tents through those compar tments as a function of sunlight, temperature, current regimes, and biological and physical-chemical interactions over at least one annual cycle and, for some questions, over a time scale of decades. Assessment of the impact of oil on an ecosystem should include measurements relating changes in the biological components and functions of those components with changes in chemical composition of the oil and its metabolites and reaction products. All types of oil over several orders of magnitude of concen- tration will have impacts (Hyland and Schneider, 19761. Field studies have the advantage of examining the natural ecosystem, even though there may be disadvantages, such as a lack of necessary background data and/or suitable controls, uncertainty as to what should be measured and over what period of time, as well as considerable expense. Mesocosm studies offer the advantages of controlled experi- ments with a greater representation of the ecosystem and are, in general, less expensive than field studies. They offer particular value for the study of chronic spills, rather than acute events. From accumulated experience with oil spills (Gundlach and Hayes, 1978; Glemarec and Hussenot, 1981; Sanders et al., 1980), vulnerability indices have been developed. These indices have been applied to the A8K>CO Cadiz oil spill in Brittany, to the lower Cook Inlet, Alaska, and to the Peck Ship spill in Puerto RiCo (Gundlach and Hayes , 1978 ; Michael et al ., 1978 ; Davis et al ., 1980 ~ . The approach compr ises geomorpho- logical aspects, species assemblages, and persistence of oil. The residence time of oil clearly is an important factor in eco- system analyses. The residence time of oil in the water column, in general, is in the order of hours to days, unless new discharges occur, whereas oil residence time is much more prolonged for the benthos. Recovery time may be in the order of hours to days for phytoplankton and zooplankton, depending on the extent and duration of the event. Fish eggs and larvae may be destroyed in the immediate vicinity of an oil spill during the short period of time that oil concentrations may be lethal. However, it should be pointed out that there is no known instance of an oil spill covering an entire spawning area for a particular fish species, although it is possible that, for localized coastal spawning populations, such as salmon and herring, this could be a net result. The admonition regarding the changing composition of oil as it weathers and is degraded must be repeated (see Chapter 4~. As the oil is altered, its toxicity will also change, and this is important in assessing ecosystem effects. Even in areas of chronic hydrocarbon input, effects of hydrocarbons on the ecosystem will be difficult to differentiate from inputs of nutrients, metals, and other organic compounds. Habitats most impacted by an oil spill, assessed by the persistence of oil in that habitat and according to vulnerability indices (Gundlach and Hayes, 1978), are the

204 ones that should be studied over the long term. Most often, these will comprise benthic studies of depositional areas on the continental shelf, in estuaries, or in salt marshes. Mesocosms are useful principally for studying ecosystem inter- relationships of plankton and small benthic invertebrates of the sort that are normally collected in core samples. However, mesocosms are not able to provide complete simulation of the entire ecosystems, i.e., including f ishes and large benthic epifauna. Yet they are especially effective for analyzing problems of chronic pollution, where plankton interrelationships and the effect of perturbations of the plankton on benthic fauna are important and need to be monitored. Mesocosms are less useful for studying oil spill situations because only in few cases have ser ious effects on plankton been demonstrated. An important future use for mesocosms may well prove to be in the simulation of ecosystem effects in bays and estuaries subject to inputs of multiple pollutants such as petroleum, other organic contaminants, and trace metals. The potential synergistic effects these various substances may produce are poorly understood at the present time and merit attention. ~ variety of experimental procedures will be required, since the relationships are too complex to be dealt with definitively by field investigations alone. Studies of the plankton and a portion of the benthos can be care fed out in properly scaled mesocosms. Mesocosm experiments recently have been reviewed in a volume edited by Grice and Reeve (19821. At the Marine Ecosystems Research Laboratory (MERL), mesocosms have been scaled to mimic a temperate pelagic estuarine water column with a heterotrophic soft-bottom benthic community in order to conduct studies on the fate and effects of pollutants in estuaries (Pilson et al., 1979~. In experiments with No. 2 fuel oil, unexpected changes In biological compartments in the water column and benthos were detected quantitatively (Elmgren et al., 1980a,b). It is possible these changes would have been only qualitatively perceived in field studies of oil spills (E1mgren and Frithsen, 1981~. In addition, the biogeochemical behavior of individual hydrocarbon compounds, including their role in degradation and the products thereby produced, can be traced and budgeted through the ecosystem when mesocosms are employed tHinga et al., 1980; Gearing and Gearing, 1982a,b; R.F. Lee et al., 1981b). Mesocosm experiments, however, are not a panacea. In fact, over time scales of a metazoan generation or longer, proper dynamic seal ing may be impose ible. RECOMMENDATIONS One of the difficulties in assessing the significance of many of the laboratory petroleum exposure studies reported dur ing the past decade, and in compar ing results obtained by different labor ator ies, has been the lack of standardization of methods. Each group of investigators has proceeded to develop methods to su~t its own needs, abilities, and goals. Consequently there is no universally acceptable set of experi- mental parameters, such as test species, seawater medium, temperature,

205 duration of exposure, contaminant concentrations, mode of contaminant introduction, means of determining rates of uptake or loss, and measure- ments of physiological change. Experimental parameters that can be used under given conditions have been described (Becker et al., 1973; G.V. Cox, 1974, 1980) and are useful to consider in connection with the design of the experiment. The following represent some specific recommendations ar ising out of our considerations . 1. Bacter ia and fungi provide the major mechanisms for oil degradation. Exper iments with higher plants and animals must take into account the fact that organisms under study interact with a constantly changing substrate, as a result of concomitant microbial activity . Substrates altered by microbial processes may be more toxic than the original oil. 2. The demonstration of oil pollution effects on prank sonic communities is hampered by the constantly changing spatial and temporal distribution of plankton in the environment. The enormous resources required for adequate sampling and analysis of postspill prank sonic communities usually render such large scale studies prohibitive. Resources should be focused on community species composition and biomass determinations. 3. Both enzyme analyses and studies of structural distortions of cellular components in response to hydrocarbon contamination are promising areas of research that, as yet, have received little attention. Furthermore, histopathological methods for detecting long term sublethal stress suffer from a lack of background descriptions of normal histological characteristics. A general consensus should be developed as to which histopathological changes best serve as reliable indicator s of stress . 4 . The use of both semicontinuous and continuous culture systems, necessary to provide populations of known nuts itional and growth history but heretofore untried for studies of hydrocarbon effects, should be given greater attention. Fluorescence enhancement in phytoplank ton by DCMU is a promising area of research but requires further investigation. 5. Benthic plants and animals are often the biota most in contact with deposited petroleum and consequently should be studied in relation to petroleum effects. As benthos occur in such a variety of habitats, each varying in susceptibil ity to oil contamination, studies on benthos should focus on areas where contamination may persist, such as depo- s itional areas in coastal wetlands, estuar ies and continental shelves . After an initial survey of community composition, resources should be focused on populations of specif ic taxa. Summary indices such as species abundance, diversity, taxa ratios, and total biomass (density) should not be used as sole measures of community health. The prevail- ing use of isolated species diversity indices should also be dis- couraged. 6. In soft-bottom assemblages subject to an acute oil spill, ben- thic crustaceans should be studied because of their known sensitivity to perturbations, including petroleum contamination, and their trophic importance. Chronic, low level effects in such habitats, especially on

206 _, ~ growth and reproduction, should be studied with long lived, low mobility species when a~railable In hard substrates, pr imary space occupiers, key species, or str ucture-bu ilding ~ framework ~ species should be the major taxa studied. 7. Whenever possible, acute toxicity bioassays should utilize flow- through exposure systems of proven design. Weathered and photooxidized oils should be investigated. Toxicity of petroleum metabolites produced by mar ine bacter ia and animals should be evaluated. Major emphasis in laboratory effects studies should be placed on life-cycle and chronic sublethal effects. Studies of effects of petroleum on reproduction, b ioenergetics, and histopathology should receive greater attention. 8. Suites of physiological and biochemical diagnostic tests should be developed, val idated, and adopted for f ield use for man itor ing impacts of pollution and recovery in the mar ine environment. Long term monitoring, while desirable before a spill, is not a necessary prerequisite for impact assessment of acute spills. Control (reference) sites are requisite for comparisons, even if baseline data exist. Whole-ecosystem studies of the fates and effects of oil or its constituents are desired and can be effectively conducted in meso- cosms . Such studies may reveal unexpected effects on phytoplankton, zooplankton, and benthic fauna not observable under field conditions. Determinations of the rates of disappearance and biodegradation of oil compounds in the water column and in the sediments can also be made in mesocosm experiments. It is important also, however, to be aware of problems in dynamic physical scaling. A pressing need exists for the development of suitable field experiments to confirm many of the results obtained in the laboratory. The goal is the capabil ity to predict the impacts of chronic and acute petroleum contamination on the various levels of biological organization (cellular, organ, organism, population, community, ecosystem, and biome) in the mar ine environment. REFERENCES AEC. 1968. Problems of biological oceanography. AEC-TR-6940. National Technical Information Service, U. S. Department of CoIIunerce, Spr ingf ield, VA 22151. AEC. 1972. Marine radioecology. AEC-TR-7299. National Technical Information Service U.S. Department of Commerce, Springfield, VA 22151. Ackman, R.G., and D. Noble. 1973. Steam distillation: a simple technique for recovery of petroleum hydrocarbons from tainted fish. J. Fish. Res. Board Can. 30:711-714. Adlard, E.R., L.F. Creaser, and P.H.D. Matthews. 1972. Identification of hydrocarbon pollutants on sea and beaches by gas chromatography. Anal. Chem. 44: 64-73 .

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This comprehensive volume follows up and expands on an earlier National Academy of Sciences book. It is the result of an intensive multidisciplinary effort to assess the problems relating to petroleum-derived hydrocarbons in the marine environment. Specifically, it examines the inputs, analytical methods, fates, and effects of petroleum in the marine environment. The section on effects has been expanded significantly, reflecting the extensive scientific effort put forth in determining the effects of petroleum on marine organisms. Other topics discussed include petroleum contamination in specific geographical areas, the potential hazards of this contamination to human health, the impact of oil-related activities in the northern Gulf of Mexico, and the potential impact of petroleum on fisheries.

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