[Advances in Marine Biology] Advances in Marine Biology Volume 19 Volume 19 || Environmental Simulation Experiments on Marine and Estuarine Animals

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  • Adv . Mar . Bid . , Vol . 19, 1982. pp . 133-256

    ENVl RON M ENTAL SIM U LATlON EXPERl M ENTS ON MARINE A N D ESTUARINE ANIMALS

    J . DAVENPORT

    N.E.R .C . Unit of Marine Invertebrate Biology, Marine Science Laboratories. University College of North Wales. U . K .

    I . I1 .

    I11 . IV . V .

    VI .

    VII . VIII . IX . X .

    XI .

    Introduction . . . . . . . . . . Variability of the Inshore Environment . .

    at an intertidal estuarine site . .

    Development of Simulation Equipment . .

    A . Temperature and salinity fluctuations

    B . Rock pool physico-chemical conditions

    Regimes . . . . . . . . . . . Temperature Experiments . . . . . .

    B . Development . . . . . . . . C . Reproduction . . . . . . . . D . Adaptation . . . . . . . .

    A . Survival . . . . . . . . .

    E . Interaction with other factors . . Salinity Studies . . . . . . . .

    A . Survival . . . . . . . . . B Behavioural responses

    D . Growth . . . . . . . . . . E . Feeding . . . . . . . . . .

    G Oxygen consumption

    . . . . . . C . Reproduction . . . . . . . .

    F . Osmotic/ionic responses . . . .

    Oxygen Tension Studies . . . . . .

    Acknowledgements . . . . . . . .

    . . . . . .

    Pollutant Studies . . . . . . . . Conclusions . . . . . . . . . .

    References . . . . . . . . . .

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    I . INTRODUCTION

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    133 137

    139 143 145 160 166 166 175 177 179

    184 185 190 207 2008 209 209 230 233 236 240 242 242

    in2

    While the open sea is relatively stable in its physio-chemical makeup. indeed impressively so at depth. coastal and estuarine aquatic environments exhibit substantial fluctuations in physical

  • 134 J. DAVENPORT

    and chemical characteristics such as light intensity, turbidity, temperature, salinity and oxygen tension. These changes result from terrestrial, seasonal climatic, die1 and tidal influences. In coastal and estuarine littoral zones these various fluctuations, which interact in their effects upon organisms, are further complicated by periodic tidal emersion, with the consequent possibility of exposure of the littoral fauna to aerial and freshwater influences.

    Inshore waters and estuarine areas bear the brunt of mans impact on the seas; they receive most types of deliberately released pollutants (thermal, radiological, inorganic and organic) for eventual dilution and dispersion in the great bulk of the marine hydrosphere and are also affected by accidently discharged pollutants (mainly oil). Because pollutant delivery to the environment is often intermittent, and because the subsequent fate of pollutants is affected by tides, currents, variable freshwater run-off and various chemical or biological processes, it is most unlikely that marine animals will ever be exposed to sustained steady pollutant concentrations.

    For a variety of reasons animals of inshore waters have attracted rather more attention from experimental workers than have animals from the open sea. Obviously their availability and economy of collection have contributed to this, but their special attributes, evolved in response to the changeable nature of their environment have been particularly attractive to experimentalists. There are likely to be more challenging difficulties in understanding the adaptations of eurythermal or euryhaline animals compared with understanding their offshore stenothermal and stenohaline relatives. Similarly, i t is more logical to test the effects of heavy metal pollutants on inshore fish, crustaceans and molluscs, than on their deep water relatives.

    Despite a predominant interest in experimental material from changeable environments, the main experimental approach adopted in the study of the effects of various environmental factors on coastal and estuarine animals has been the steady state or direct transfer experiment (see Fig. 1 ) . Conventionally this approach is used to study the effects of one environmental factor in isolation. Animals are taken from their natural habitat and held in the laboratory under constant conditions which approximate to field conditions at the time of collection, insofar as knowledge of those conditions and available experimental facilities allow. With the exception of the environmental factor under consideration, all conditions are held constant throughout the experimental period.

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 135

    Experiment a I

    Accl imat ion

    Level n?

    Native I eve1 t t t Sampling procedures

    (for mortality, physiological

    changes etc.)

    t Collect ion

    of animals Transfer t

    Time I)

    FIG. 1 . Format of the steady state or direct transfer experiment

    The experimental animals are divided into groups. One, the control, remains at the acclimation level while the other groups are each transferred, usually directly, to situations where they encounter various levels of the factor being studied.

    This type of experiment is simple, does not require complex apparatus, and is easy to repeat at almost any marine laboratory in the world. With portable equipment, and the acceptance of some compromises in the degree of control over factors other than the one under investigation useful studies may be carried out in remote areas. The Lsteady-state experiment is, however, open to criticism on several grounds, particularly when the results obtained are extrapolated uncritically to field conditions (for example when assessing likely limits to distribution of the animals). The experiment essentially consists of suddenly changing the environmental level of a particular factor and then sustaining the new level, often for long periods. Commonly, any changes induced by the procedure are attributed to the new factor level, and the trauma involved in transfer is ignored. Yet in nature such a sequence ,of events never occurs. An example from salinity studies serves to illustrate this point. The mussel, Mytilus edulis (L,), occurs in a variety of brackish water areas. In the Baltic Sea, populations of mussels are found at sites where the salinity is much lower than in the open sea (down to

  • 136 J . DAVENPORT

    5%,; Theede, 1965), but the salinity levels are quite stable, varying little even on a seasonal basis, so the animals are never exposed to sudden osmotic shocks. On the other hand, many estuarine populations of Mytilus are exposed to severe tidal fluctuations in salinity (Milne, 1940; Cawthorne, 1979a). They encounter sharp salinity changes, but no particular concentration is sustained for very long, and the mussels, like many other bivalves, survive exposure to low salinities for a few hours by closing the shell valves, and retaining water of high salinity within the mantle cavity (Milne, 1940; Gilles, 1972; Hoyaux et al., 1976; Shumway, 1977; Davenport, 1979a). Consequently, the conventional steady state experiment does not represent the environmental situation of either the Baltic or estuarine mussels. This example also illustrates another pitfall of steady state experiments. Many animals have mechanisms for avoiding damage caused by short term exposure to sub-optimal or deleterious, environmental conditions. Thus, as discussed above, bivalve molluscs reduce contact with the external environment to a greater or lesser extent when the external salinity is low; analogous behaviour is found in barnacles, gastropods, burrowing worms and hermit crabs (Newman, 1967; Foster, 1970; Davenport, 1976; Shumway and Davenport, 1977; Davenport et al., 1980). Wells (1949a, b) suggested that ArenicoZu marina (L.) avoided exposure to unfavourable media by ceasing to irrigate its burrow, while recent work (Davenport, 1977; Davenport and Manley, 1978; Manley and Davenport, 1979) has shown that several bivalve species are capable of detecting heightened environmental copper levels and can close their shell valves, or at least reduce the rate of irrigation of the mantle cavity to avoid damage during a few hours exposure to this pollutant. It has also been known for a long time (Turner et ul., 1948) by workers interested in antifouling techniques that mussels can detect chlorine and close their shell valves to survive intermittent exposure to chlorinated sea water. All of these behaviour patterns are devices to counteract transient unfavourable conditions and evolved only for short term, notably tidal, periods. In the extended direct transfer type of experiment, animals are often inspected at daily or even longer intervals; by this time overriding respiratory or nutritional demands may have forced animals to abandon their avoidance behaviour and resume normal activity in conditions to which they would not usually be exposed.

    A refinement of the basic steady state experiment has been the multivariate approach pioneered especially by Costlow et aZ. (1960) and Alderdice (1963, 1972). In this type of investigation animals are

  • ENVIRONMESTAL SIMULATIOK EXPERIMENT8 137

    exposed to Combinations of factors (e.g. salinity and temperature, or salinity, temperature and oxygen tension, etc.) rather than single factor stresses. Obviously this approach is most useful for finding out how factors interact, and also for determining optimal laboratory or rearing conditions (Box, 1956) very rapidly. However, the experi- ments have also been used for the construction of multidimensional survival envelopes (e.g. McLeese, 1956) with the implication that these are valid for distribution in the field. The objections set out above for single stress steady state experiments apply equally to multivariate studies, with the additional comment that, if it is unnatural or unphysiological to expose animals to instantaneous single factor shocks, then it is surely even more artificial to impose sudden multifactor changes.

    Dissatisfaction with some aspects of the steady state experi- mental approach has spurred an increasing number of workers to develop apparatus to provide experimental regimes which more closely reflect the changeable nature of the environment, at least for one factor (e.g. temperature, salinity, oxygen tension) at a time. Initial steps in this direction were taken with temperature alone (e.g. Grainger, 1956; Khan, 1965). However, following the salinity studies of Tucker (1970a), several researohing groups have developed apparatus to mimic the salinity fluctuations of estuaries or coastal lagoons. Such salinity studies have been the most common form of environmental simulation experiment performed so far, although the project at the Netherlands Institute for Sea Research, Texel has been running for several years, and some thermal, oxygen tension and pollutant studies have also been performed.

    Since the literature devoted to such studies is still relatively compact, and because the recent advent of microprocessor tech- niques holds out the promise of relatively cheap and flexible apparatus to facilitate simulation studies, the time appears ripe for a review of the field.

    11. VARIABILITY OF THE INSHORE ENVIRONMENT

    It would normally be appropriate to replace the unphysiological aspects of steady state experiments by conditions of temperature, salinity or oxygen tension which animals are likely to encounter in nature. For realistic simulation studies therefore adequate inform- ation is required about the source and form of natural fluctuations ir!

  • 138 ,J. DAVENPORT

    levels of various environmental factors. For the sublittoral environment, whether coastal or estuarine, there is an extensive amount of information about temperature and salinities, simply because physical oceanographers interested in currents and mixing processes rely heavily upon these data in their calculations. Unfortunately much of this information is either somewhat inaccessible, or not well correlated with biological data. Also, except for a few estuarine studies (e.g. Sanders et al., 1965), little detailed information is available about short term (i.e. tidal or diel) changes in temperature or salinity. For the intertidal environment data is much sparser and often incomplete. For example, Southward (1958) showed that barnacles at Plymouth U.K. were exposed to a 16 deg C thermal change between tidal extremes in the summer; this observation was valuable, but gave no idea of the exact form of the temperature changes encountered by the animals over a 24 h period, although it seemed likely that temperature changes associated with emersion/immersion would be quite abrupt. Detailed information about the form and amplitude of changes in salinities and temperatures occurring in the intertidal zone over periods of as much as 24 h at different times of the year was virtually absent until the work of Cawthorne (1979a), itself indequate, being collected from only one particular estuarine site. An area of rather greater knowledge lies in the characteristics of the specialized rock pool environment, including fluctuations in oxygen tension and pH, which have been monitored by several workers (e.g. Stephenson et al., 1934; Pyefinch, 1943; Read, 1969; Ganning, 1971; Daniel and Boyden, 1975) and will be discussed in detail later.

    Most monitoring of polluted marine or estuarine environments has been carried out over weeks, months or years with emphasis being placed upon relatively infrequent chemical and biological sampling; the possibility or consequences of tidal or diel fluctuations in pollutant concentration have largely been ignored. However, the effects of thermal pollution, in the form of power station discharges, have attracted the attention of many researchers, and the first major review of the field was published as long ago as 1965 (by Naylor). Broadly speaking, power stations have two effects upon the environment. First, and most obviously, their discharges heat up the inshore waters nearby, the temperature increment decreasing with distance from the discharge point. In terms of fluctuations in environmental temperature this effect simply means that the fluctuations occur rather higher up the temperature scale than they would have done in the absence of the station. Secondly, power

  • ENVIRONMENTAL SIMULATION EXPEHIMESTS 139

    stations apply sudden thermal increases (usually of the order of 10degC in about 5s) to organisms entrained in the cooling water passing through the stations condensers. This latter effect can be very damaging, especially in summer when organisms are living closer to their upper thermal limits; Briand (1975) showed that biomass amounting to 1700 tons of organic carbon was destroyed in a year by passage through the condensers of two Californian power stations. The entrainment thermal shock has been simulated in the laboratory notably by Heinle (1969) and Diaz (1973, 1975); more work on both types of thermal effects caused by power stations would be valuable especially upon inshore tropical organisms which may be particularly vulnerable. However, it should be remembered that the thermal shock of entrainment is accompanied by great pressure increases as the cooling water passes through the narrow bores of the condenser. Also the cooling water may be chlorinated, to a greater or lesser extent, to prevent fouling and this may kill animals where thermal shock does not (Heinle, 1969).

    A comprehensive account of all types of environmental fluctuations which have been monitored by marine biologists would constitute a sizeable review in itself; the following two sections are derived from a few selected examples relevant to the simulation studies already carried out or in progress.

    A. Temperature and salinity Juctuations at a n intertidal estuarine site

    When Davenport et al. (1975) employed sinusoidal and abrupt salinity changes in their studies of larval salinity tolerances they were relying upon the work of Sanders et al. (1965), who monitored salinity changes in the Pocasset river, an estuary with a pronounced salt wedge effect, to justify the abrupt salinity regimes. On the other hand, measurements made by students of the University College of North Wales indicated that the salinity of the bottom water of the well mixed Conwy estuary fluctuated in a roughly sinusoidal manner (and often fell to near zero salinity). When these same idealized regimes were later applied to estuarine littoral organisms (e.g. barnacles, bivalves) some unease was felt about their validity. For example, were animals of marine origin such as Mytilus edulis ever exposed to fresh water even in estuaries? The work of Tucker (1970a) and Stickle and Ahokas (1974), the only other workers in the field at the time, wits of no assistance, since they too relied on simulations based (somewhat loosely) upon assumptions from sublittoral data.

  • 140 ,J. DAVENPORT

    Stickle and Denoux (1976) described tidal salinity fluctuations on an Alaskan shore but these only varied from about 8 to 25%,.

    With the later development of apparatus which could also simulate complex temperature changes (Davenport and Cawthorne, 1978), the problem of using realistic simulated intertidal regimes became more acute. A survey of literature revealed that, although a little information was available about temperature extremes during tidal fluctuations (e.g. Southward, 1958; Lewis, 1960,1963), nowhere did their data show the form of the fluctuations occurring between the extremes. Consequently, during the year from November 1977 to October 1978, monthly visits were paid to a site on the shore of the Conwy estuary in North Wales to monitor salinity and temperature changes over a 24 h period (Cawthorne, 1979a). The recording site was on the lower shore and chosen because it was quite close to the landward limit of common members of the coastal littoral fauna and flora and accessible at tides roughly midway between springs and neaps. Knight and West (1975) had shown that the estuary was well mixed, but the site selected was beside a narrow channel where turbulence was thought likely to produce especially effective mixing.

    Table I lists the epibenthic organisms found at the recording site (mobile or infaunal organisms were not considered since they might be able to swim or crawl away or might retreat into the substrate to avoid exposure to the full rigours of salinity and temperature changes recorded at a single point above the substrate). Temperature and salinity curves for two monthly recording periods are shown in Fig. 2; the periods of aerial exposure and timings of high water are also indicated. The results showed a great deal of variation in the amplitude and shape of the salinity profiles, despite being collected

    TAHLE I. LIST OF EPIHENTHIC SPECIES RECORIIEU WITHIN 5 METRES OF RECORDING PROBE ox THE LOWER SHORE OF THE CONWY ESTUARY (FROM CAWTHORSE, 1979a)

    Algae Fucus vesiculosus Fucus serratus Ascophyllum nodosum Ulva lactuca"

    Cirripedes Elminius modestus Balanus balanoides Balanus crenatus

    Molluscs Littorina saxatilis Mytilus edulis

    *Seasonel occurrence (summer/autumn) only.

  • E

    4

  • 142 J . DAVENPORT

    from the same point on occasions of similar tidal height. Changing seasonal and climatic influences, particularly where they affected the freshwater input to the estuary, were responsible for most of the variation. The maximum salinity range noted on a single tide was between 0 and 31%,, recorded twice during the 12 month study period; clearly a number of littoral organisms of marine origin may survive exposure to fresh water for brief periods. Maximum rates of salinity change during a tidal cycle varied from 7 to 18y&,/h. Temperature fluctuations were equally interesting. When the shore was covered with water, the temperature changes were associated with salinity changes. The amplitude of the aquatic temperature fluctuations appeared to be determined by the difference between river-water and seawater temperatures, and was not influenced by short term changes in air temperature even when these were quite extensive. Thus, in November 1977, when the seawater temperature was 7-4"C, and the river-water input was about 505C there was a fluctuation in water temperature between these levels, with rising salinity being associated with rising temperature. In April 1978, however, the freshwater input to the estuary was warmer than the sea, so that rising salinities were associated with falling tempera- tures. Generally, water temperatures changed slowly and amplitudes of fluctuations during tidal cycles were small, often of little more than 2-2 deg C. However, aerial emersion was always associated with far more abrupt temperature changes of much greater magnitude. The most extreme example of this occurred in May 1978 when the temperature a t the recording point rose by 12.5 deg C in 15 min on one falling tide. Typically, therefore, a single tidal cycle will expose epibenthic intertidal organisms to both gentle and abrupt temper- ature changes. During the 12 visits freshwater temperatures ranged from 1-0 to 19.5"C, seawater temperatures from 2.8 to 16*2"C, while air f,emperatures varied between 0-6 and 32.1"C. The maximum amplitude of thermal fluctuation recorded during a 24 h period also occurred in May 1978, and amounted to 18degC ( 1 4 ~ 3 2 C ) ; this agrees quite well with the observations of Southward (1958). Newel1 ( 1969) pointed out that temperature fluctuations in temperate intertidal zones were usually greater than in tropical areas where air i%nd sea temperatures were often reasonably close (Lewis, 1960,1963). Temperature fluctuations on the shore a t high latitudes have not attracted much study, although Davenport et al. (1979) suggested that eggs of the capelin MaZZotus viZ2osus (a salmonid teleost) laid on the sandy shores of Balsfjord, N. Norway might be exposed to die1 temperature changes of 25 deg C or more during the subarctic spring.

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 143

    B. Rock pool physico-chemical conditions

    Unlike the general estuarine or coastal shore, and contrary to popular opinion, intertidal rock pools are usually characterized by stable salinites (Orr and Moorhouse, 1933; Pyefinch, 1943; Ganning, 1971; Daniel and Boyden, 1975), although there may be vertical salinity gradients within them. Even when exposed to heavy freshwater inflow, the salinity at the bottom of littoral pools, left by the tide, is usually almost unchanged from that of sea water (Ganning, 1971; Davenport et al., 1980). This stability follows from stratification and generally poor mixing within the pools. On the other hand, layering and slow mixing contribute, with the metabolic processes of rock pool organisms, to substantial fluctuations in the temperature, pH and oxygen tension (PO,) of the pool water. Examples of such fluctuations are shown in Fig. 3. Maximum re- corded die1 fluctuations in temperatures in temperate areas vary from 10-15 deg C (Ganning, 1971; Daniel and Boyden, 1975) and so rock pool organisms may encounter amplitudes of temperature fluctu- ation approaching those associated with aerial exposure in other littoral organisms. However, as shown in Fig. 3, the temperature changes tend to be gentle rather than sudden. Similar changes were observed by Read (1969); Ganning (1971) indicated that such amplitudes of temperature fluctuation were not normally exceeded in tropical rockpools; in Barrier Reef pools Orr and Moorhouse (1933) recorded 10C diurnal changes. The work of the author (Davenport, 1979c) upon a Norwegian subarctic rock pool which fluctuated between - 8C (high salinity water beneath ice -see below) and 3C in spring, suggests that this is true at high latitudes also.

    These subarctic pools are also interesting because they break the pattern of stable salinities. Ganning (1971) was the first to note that freezing of the surface water in pools produced higher salinities in the underlying water, but this information was derived from rock pools in the Baltic splash zone where the maximum salinity recorded was only 14-3%,. However, Davenport (1979c), working on the shore at Tromso, N. Norway, where the sea is of normal salinity, found that salinities as high as 65%, were regularly developed on each tide beneath the ice formed in shore pools exposed to air temperatures below - 10C. The increase in salinity appears to take several hours, whereas the return to normal sea water salinities (32-34%,) probably occurs in a few minutes, when the ice is melted and the pools are flushed out by the relatively warm ( + 3 to +5"C) water of the incoming tide.

  • 1

    1 Pool 1 20

    E P

    26 24

    22

    20 18

    16

    14

    12 '08 10 12 14 16 18 20 22 24 02 0 4 06 08 08 10 12 14 16 18 20 22 24 02 04 06 08 08 10 12 14 16 18 20 22 24 02 04 06 08

    Pools submerged Periods of daylight 0 Pools exposed =Darkness

    Time of day ( h )

    FIG. 3. Diurnal variation in oxygen concentration (solid circles) and temperature (open circles) in six rock pools. From Daniel and Boyden (1975)

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 145

    The most striking short term changes which occur in the rock pool environment consist of massive fluctuations in oxygen tension. Both Ganning (1971) and Daniel and Boyden (1975) reported fluctuations in oxygen tension, between 3% and more than 300y0 air saturation in rock pools which contained large quantities of macroalgae or, in pools cut off from the sea for periods of days or weeks, dense populations of phytoflagellates. These fluctuations are basically with the highest values tending to occur when photosynthetic activity has peaked at around 1300 h or 1400 h. Minimum air saturation levels occur before dawn (approx. 0400 h) after respiration has proceeded in the absence of photosynthesis for several hours. However, tidal influences complicate this picture because immersed pools when subject to wave and current action will always contain water which is close to full air saturation, whatever the time of day or night.

    The oxygen content and pH of rock pool waters are closely related; both are affected by algal photosynthesis and respiration of the total ecosystem. Typically night time is characterized by low pH values (around 6-5-7-5) associated with oxygen depletion and CO, release, while daytime figures may rise to 9 or even 10 under conditions of oxygen hypersaturation (Ganning, 1970; Ganning and Wulff, 1969; Daniel and Boyden, 1975). Continuous monitoring of rockpool pH during tidal or die1 cycles appears not to have been carried out, although Ganning (1971) stressed the futility of occasional pH sampling. However, it seems probable that the form of pH fluctuations, like that of the changes in oxygen tension is made up of both gentle and abrupt components. As virtually no work has been carried out upon the direct effect of pH upon marine organisms, this would appear to be a fruitful field for laboratory simulation studies.

    111. DEVELOPMENT OF SIMULATION EQUIPMENT

    Simulation studies started with temperature investigations, the earliest of which appear to have stemmed from the interest of Grainger (1956, 1959) in varying rather than constant temperatures. Initially, experiments were simply direct transfer experiments in which observations were made immediately after the transfer (e.g. Grainger, 1956); a similar approach has been adopted by workers interested in power station entrainment thermal shocks (e.g. Heinle, 1969; Diaz, 1973, 1975).

    Khan (1965), in her work upon the development of the freshwater species Acanthocyclops viridis (Jurine), employed an irregular

  • 146 $1. 1)AVENPORT

    temperature regime (see Fig. 4), produced by keeping aquaria out of doors and occasionally switching immersion heaters within them on or off by hand. Monitoring of temperatures was continuous, but the analytical technique required to evaluate the effects of fluctuating temperature upon development, which followed from the work of Grainger (1959), was somewhat tedious. Also, by definition, experiments of this type cannot be repeated, and the approach does not appear to have been used subsequently. In any case, as is clear from Section 11, the short term temperature changes encountered in nature by marine and estuarine animals, although very variable, are not truly irregular, but are rhythmically related to tidal and die1 patterns of events.

    3 7

    0 2 4 6 8 Days

    Flu. 4. Irregular temperature regime employed by Khan (1965) in her studies on copepods.

    The bulk of temperature cycle studies have involved animals living in aquaria with no through flow, and exposed to uncomplicated temperature regimes. Thus Heath (1963) used square wave temperature profiles (wavelengths +I8 h, amplitude 10 deg C, while Hubbs (1964) used rather poorly defined changes. Linear increases and decreases over 24 h periods were employed by Thorp and Hoss

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 147

    (1975) and Widdows (1976). Costlow and Bookhout (1971) and Feldmeth et al. (1974) used die1 thermal regimes which consisted of linear changes in temperature, interspersed with periods of constant high or low temperature.

    Most of these investigations were .carried out with the aid of relatively simple equipment involving standard items such as time clocks, immersion heaters and motor-driven contact thermometers. Such apparatus has long been used for producing temperature increases for conditioning bivalves to produce ripe gametes (e.g. Loosanoff, 1945a; Loosanoff and Davis, 1950; Gruffydd and Beaumont, 1970). Costlow and Bookhout (1971) used rather more refined equipment; their experiments were performed in environ- ment cabinets with Honeywell temperature programmers. Such cabinets, which usually have light regime control as well, have been used for many years in botanical work, and are ideal for thermal studies upon small organisms (e.g. larvae) in small vessels, but may not easily be used with large animals or organisms needing a large exchange of water. Moreover, they are of no use in the simulation of fluctuations in other variables such as salinity or oxygen tension.

    Salinity simulation studies started with the work of Tucker (1970a) who imposed simulated tidal salinity cycles upon the gastropod, Scutus brevicutus (Montfort) to investigate its osmotic responses using the equipment shown in Fig. 5. Both she, and Stickle and Ahokas ( 1974) employed apparatus which essentially consisted of an aquarium which could be flushed out at a constant flow rate by water of a different salinity from a header tank. The salinity changes thus produced were of rather arbitrary form (see Fig. 14) and, in the case of Stickle and Ahokas study, were not accurately repeatable (Tucker gave no information on this point).

    In the light of these various defects, Davenport et al. (1975), in their descriptions of a salinity fluctuation apparatus which has since been used in several studies, set out the following design criteria which were thought essential for routine and comparative studies:

    1. Salinity must be repeatable, so that different species, or various larval stages of one species, may be exposed to the same salinity regime.

    2. Automatic repetition of profiles must be possible to allow the exposure of organisms to simulated tidal salinity fluctuations over long periods of time during feeding, growth or activity experiments.

    3. Substitution of one profile for another should be simple and must not involve adjustment which is difficult to reverse.

  • 148

    [XI ions mi-' Constant with

    time 1 Volume=Vml

    [c] ions m1-l ( a function o f

    time) Aquarium-,--

    < 2

    J. DAVENPORT

    -- [y l ions ml-' ( a function

    of time)

    - Constant pressure bottle

    Needle valve /

    FIG. 5. Experimental set-up used to obtain a predictable gradual dilution or concentration of a solution in an aquarium. The solution was continuously aerated and mixed by air being bubbled through a diffusion block. From Tucker (1970a).

    4. The design of the apparatus must allow salinity fluctuations of any form between the limits of fresh water and full sea water.

    5 . Flexibility in the time taken to complete a fluctuating salinity programme is necessary, so that both 24h tidal cycles and spring/neap cycles may be reproduced.

    6. The apparatus should be capable of delivery up to, say, 30 litres of water per hour, so that the response of several animals may be studied simultaneously.

    7 . The salinity of the outflowing water must be automatically monitored at all times.

    8. All factors except salinity should be kept constant, e.g. the temperature of the outflow should be controlled and the freshwater supply should be deionized (because the ionic content of fresh water can change with time and source).

    The apparatus described by Davenport et al. (1975) met all of these criteria, and is depicted diagrammatically in Fig. 6. Filtered sea water and deionized water supplies were fed into two polypropylene header tanks, the inlet of each tank being regulated by a domestic

  • ENVIRONMEKTAL SIMULATION EXPERIMENTS 149

    FIG. 6. Diagram of apparatus used to produce fluctuating salinity regimes. S.V., solenoid CC, conductivity cell. From Davenport et al. (1975).

    valve;

    ball cock valve. The header tanks were connected, by wide bore P.V.C. tubes each guarded by a solenoid valve, to a 200 ml perspex mixing chamber where the water was magnetically stirred. Mixed water flowed from this chamber through long heat exchange coils of P.V.C. tubing immersed in a thermostat and thence to apparatus containing experimental animals. The salinity of the mixed water was monitored by a platinum conductivity cell connectdd via a Carwyn Instruments* salinity monitor to a chart recorder. A control seawater supply was taken from the seawater header tank and also passed through the thermostat before delivery to apparatus containing control organisms.

    The form of the salinity regime was determined by the programmed opening and closure of the two solenoid valves. The * Carwyn Instruments, Pentraeth Road, Menai Bridge, Gwynedd, U.K.

  • 150 J . DAVENPORT

    p r a p m r n e r was a L.K.B. 11300 Ultrograd,* an instrument normally used to control the mixing of relatively small volumes of liquids at low flow rates to produce chromatography gradients. The programmer was the heart of the apparatus, since the rest of the equipment was similar in principle to the apparatus used by Alderdice et al. (1958) to supply water of steady low oxygen tension to Pacific salmon eggs. Recognition of the fact that the programmer could be modified to operate larger valves than those for which it was designed ended a long period of frustration during which other mechanical or electrical programmers were considered, and discarded by the author because of their complexity or lack of flexibility.

    The Ultrograd operated by photoelectric scanning of a rec- tangular screen, divided into black and white areas by a cut out piece of black paper, is shown in Fig. 7 . The black paper programme was held over the screen by a glass plate. Each time the scanning device crossed the border between the black and white areas the valve controlling the flow of one liquid opened while the other closed. Sea water flowed when the scanner was traversing the black areas. Scanning of the concentration axis took place every 7*3s, while scanning of the time axis was adjustable between 15 min and 16 days. When the scanner reached the end of its travel along the time axis i t automatically returned to the start of the profile and began again. The paper profiles were precisely located on the scanning screen, and near perfect accuracy and repeatability of salinity regimes was

    0 12 24 Hours

    Flu. 7. Diagram to illustrate cut-out scanning procedure of programmer. White area indicates paper removed. Arrowed path represents the track of the photoelectric scanner, but with the time displacement between scans greatly exaggerated. From Davenport et al. (1975).

    *L. K . B.-Produkter, ABS-161 25 Brommit 1 , Sweden

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 151

    T.L.

    18 12 6 0 Time (h)

    FIG. 8. Conductivity chart trace to illustrate calibration accuracy and repeatability of concentration programming (flow rate 100 ml/min). (1) Start of calibration programme, consisting of a stepwise increase in seawater concentration, each step representing 10% S.W. The seawater concentrations superimposed were obtained from seawater samples taken a t times corresponding to the midpoint of each step. (2) Start of programme to illustrate accuracy of programming. The regime produced consists of a drop in concentration from 100'$(o S.W. followed by an abrupt rise to 30% S.W. which was held for several hours. After an abrupt drop back to fresh water a 6 h sinusoidal rise back to 100yo S.W. was programmed. Solid circles and dashed lines represent the programmed concentrations; their positions on the chart trace were determined by the calibration data obtained from programme ( l ) .T .L. indicates the time lag caused by the space in the apparatus between the solenoid valves and the conductivity cell sensor. (3) Start of programme to illustrate repeatability by repeating the sinusoidal section of programme (2). From Davenport et al. (1975).

    demonstrated, as shown by the test results displayed in Fig. 8. Since the description of the apparatus was published in 1975 the design of the equipment has not been altered, except that the deionized water supply, which proved to be very expensive to operate, was first replaced by a distilled water source and later in 1977 by a tap water supply, the water being filtered and passed through a charcoal filter before being delivered to a 2000 gal. (9080 litre) storage vessel from which the salinity apparatus was supplied by centrifugal pumps.

    The L.K.B. Ultrograd is an expensive instrument, and this has led workers to use the same basic layout as Davenport et al. (1975), but with different (and hopefully cheaper!) programmers. Thus Atkins and Ritz (1977) described a salinity apparatus controlled by a programmer of their own design; this programmer also relied upon photoelectric scanning of a paper cut-out, was nearly as flexible as the Ultrograd, yet cost a great deal less. Hokanson et al. (1977) produced sinusoidal die1 temperature fluctuations with the aid of a cam

  • 152 ,J. DAVENPORT

    controlled programmer which operated two solenoid valves which controlled hot and cold water supplies. Spaargaren (unpublished), at the Netherlands Institute of Sea Research, also designed and built an ingenious salinity fluctuation apparatus during the period 1974-75 in which the solenoid valves were controlled by a modified bakers oven programmer! Instead of paper programmes, this apparatus em- ployed drums formed from thin copper sheet and cut to an appropriate profile. Spaargarens apparatus also had the refinement of feedback control; a conductivity cell recorded the conductivity of the water delivered by the equipment, and if this deviated from the programmed value, then one or other of the solenoid valves delivering salt or fresh water was opened to compensate. Such feedback control is also a feature of the latest microprocessor controlled salinity fluctuation apparatus at the Department of Oceanography, Southamptom University (Lockwood, personal communication), and the computer-interfaced, multifactor simu- lation equipment under construction a t the Institute of Marine Environmental Research, Plymouth, U.K. (Bayne, personal com- munication). Feedback control has its dangers though. Sensors must be totally stable and reliable or the experimenter will not be aware of errors caused by drift of the sensor response!

    All the equipment described so far was designed to change only one environmental factor (either salinity or temperature), although Davenport et al. (1975) had suggested that simple modifications of their salinity apparatus would allow the experimental possibilities listed below:

    1. Temperature fluctuations (of more complex form than those employed by Heath (1963) or Costlow and Bookhout (197 1)) ; produced by programmed switching between two seawater supplies maintained at different temperatures.

    2. Oxygen tension fluctuations; obtained by switching between deoxygenated and air (or oxygen) saturated seawater supplies.

    3. Fluctuations in food availability for filter-feeding species; produced by mixing filtered sea water and sea water containing algae.

    4. Pollutant fluctuations; obtained by mixing polluted sea water with pure sea water.

    In fact, although Ritz (1980) used the apparatus of Davenport et al. (1975) in slightly modified form to deliver water of fluctuating oxygen tension and salinity to intertidal amphipods, it was clear that completely anoxic conditions could not be accomplished with the

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 153

    apparatus as i t stood, since the minimum air saturation values attained by Ritz was 4%; temperature fluctuations could not be accomplished either, as long as water flowed from the thermally uncontrolled header tanks, through solenoid valves to a constant temperature thermostat.

    Because of these inadequacies, and spurred by a desire to be able to produce a greater variety of temperature regimes than attained previously, Davenport and Cawthorne (1978) designed and built apparatus which could deliver water of fluctuating salinity, temperature or oxygen tension. Its design is shown semidiagram- matically in Fig. 9 while some test results demonstrating its capabilities are displayed in Fig. 10. As in the earlier salinity apparatus (Davenport et aE., 1975), programming was accomplished

    P

    FIG. 9. Apparatus of Davenport and Cawthorne (1978). Key: CC = cooling coil: CT = contact thermometer, EC = experimental chamber, FW = freshwater header tank, H = heater, MC = mixing chamber, P = pump, Prog = Ultrograd programmer, S = solenoid valve, SW = seawater header tank, T = tap.

  • t f 0 Hours 8

    t 4 0 8

    0 s

    Cycle 1

    C y c l e 2

    Cycle 8

    0 8 Hours

    16

    FIG. 10. Apparatus test data from Davenport and Cawthorne (1978). (a) Repeatability of' temperature regimes. (b) Factor interaction. The results show a sinusoidal programme of8 h wavelength, high salinity being associated with low oxygen tension.

  • ENVIRONMENTAL SIMPLATIOX EXPRRIMESTS 155

    by a L.K.B. 11300 Ultrograd which switched between two solenoid valves located close to the bases of two rigid PVC cylinders, each about 1.5 m tall, and 2.3 m in diameter, sealed at the bottom but open at the top. Sea water could be supplied to both chambers from a header tank regulated by a float switch and solenoid valve; alternatively one of the pair could be filled with fresh water instead from a freshwater header tank. The water within each cylinder was circulated by a centrifugal pump, while its temperature was regulated to a value between extremes of + 3 and + 50C by a contact thermometer controlling both a 1.5 kW glass heater and the supply valve of a resin-coated copper coil, through which flowed cold ( - 3C) ethylene glycol from the laboratory supply. The cylinders and all piping were insulated with polystyrene and rubber foam. The tall cylindrical shape was adopted to provide sufficient pressure head to drive water flows of 300-1500ml/min, to minimize the distance between the temperature controlled water and the experimental vessel (which was also well insulated), and to reduce the water surface area for heatlgaseous exchange.

    The seawater or freshwater contents of each cylinder were temperature controlled and could be saturated with atmospheric air from a compressor, supersaturated by oxygen delivered from a gas bottle, or be deoxygenated by bubbling with oxygen-free nitrogen. Thus it was possible to deliver water which fluctuated in temperature, salinity, or oxygen tension either separately or in combination; i t was also possible to ensure that salinity fell while temperature rose, or that a falling oxygen tension was associated with rising temperature and salinity. However, factors could not be varied independently; the basic form of the factor fluctuations had to reflect the paper programme either directly or inversely. It was not possible to deliver water of slowly changing salinity and swiftly changing temperature.

    For simplicitys sake the above description has dealt with a programmer controlling one pair of solenoid valves, and one pair of cylinders. Obviously it is quite simple to control several pairs of valves and cylinders with one programmer; this is especially valuable with temperature work where a single shape of profile (e.g. sinusoid) may operate between different temperature extremes. In fact the apparatus described by Davenport and Cawthorne consisted of three pairs of cylinders.

    Obviously an apparatus delivering water of continually varying quality must be backed by continuous monitoring systems, which, in this case, consisted of platinum conductivity cells, thermistors and

  • 156 J . DAVENPORT

    oxygen electrodes. The apparatus has proved to be very useful in the thermal and salinity studies for which i t was primarily intended (e.g. Cawthorne, 1980). On the other hand, its use in oxygen tension fluctuation studies is still somewhat marginal as the water within such large cylinders required considerable quantities of nitrogen to maintain i t at zero oxygen tension. However, if a stripping column cylinder, of the type described by Fry (1951) and Alderdice et al. (1958) (i.e. tall, narrow and filled with glass chips to give a high water-gas interface surface area), were substituted, great economies in the use of nitrogen (or other gases) should follow, and long term or routine oxygen tension studies would be possible.

    During the period of development of the temperature/sal- inityfoxygen tension apparatus by Davenport and Cawthorne, it was discovered that the mussel, Mytilus edulis L., could survive intermittent exposure to concentrations of copper, which would be lethal if delivered continually (Davenport, 1977). These observations were extended by Davenport and Manley (1978) who determined the threshold concentrations of copper inducing shell valve closure in Mytilus. During these investigations it was realized that a pollutant delivery system which had the following characteristics would be desirable:

    1. It should be capable of supplying a flow of any steady concentration to experimental organisms.

    2. It should be possible to supply water of increasing (or decreasing) pollutant concentration (at various linear rates of concentration change) to allow the assessment of threshold concentrations for organisms behavioural responses.

    3. Mimicking of pollutant regimes occurring under field conditions should be feasible, where such information is available.

    The need for providing the first is not generally appreciated. With some types of conservative, unreactive pollutant (e.g. detergents, acid pollutants, phenols) i t is possible to perform steady state tolerance or mortality studies, in the manner of salinity or temperature tolerance investigations, by placing organisms in vessels filled with polluted sea water, lids being necessary to prevent the loss of volatile components. However, with pollutants which are accumulated from the environment or eliminated by organisms (e.g. some heavy metals, free chlorine, radionuclides, organochlorine compounds), such experiments can be faulty unless the vessels used are very large in relation to the organisms size. To illustrate this problem, consider a mussel placed in a 500 ml vessel filled with sea

  • ENVIRONMENTAL SIMPLATION EXPERIMEETS 157

    water contaminated with 0.5p.p.m. added copper. No matter how long the mussel remains in the vessel i t cannot take up more than 0-25mgm copper. However, a similar animal placed in flowing sea water containing 0.5 p.p.m. added copper and filtering 50 ml/min would be exposed to 36mgm of copper passing through its mantle cavity each day. Not surprisingly, in the former situation the animal would survive indefinitely, whereas in the latter, the mussel would succumb in 1-2 days (Davenport, 1977). Requirement 1 could be satisfied simply by dosing pollutants at a constant rate into a constant flow of sea water, and this approach was adopted by Manley and Davenport (1979). However, to meet requirements 2 and 3 as well, a more complex apparatus was required; this has been described by Manley (1980) and is shown in Fig. 11. Again a L.K.B. 11300 programmer was used, but in this case i t was coupled with a L.K.B. 11300 switch over two position solenoid valve as in the normal, low flow rate chromatography application of this equipment. Teflon tubes from two constant head vessels led to the two position solenoid valve. One of the constant head vessels was supplied by a pump with deionized water from a stock tank, the other with concentrated pollutant from a similar stock vessel. The overflow pipes of the constant head devices returned excess fluid to the appropriate stock tank to prevent wastage. Inert materials were used throughout the construction of the apparatus. Mixed deionized water and pollutant were delivered from the solenoid valve at a flow rate of 12 ml/min into a constant flow of sea water (approx. 500ml/min) and thence to the experimental animals. Reliable linear pollutant gradients were produced by this apparatus (see Fig. 12), and it has already been used in heavy metal studies (Manley, in preparation); experiments with changing pH levels and high salinities are also envisaged.

    Having described the simulation equipment developed so far, future trends in such apparatus are worth consideration. It seems certain that the present mechanically based programmers will tend to be replaced with microprocessors which may be linked to computer or desk top minicomputers. If the microprocessors chosen have sufficient memory capacity, then they hold out the possibility of enormous flexibility and complexity in apparatus performance. Four alternative directions in simulation studies appear likely with such equipment. First, i t would be possible to continue with simple idealized regimes of the single factor type used so far, but with the minicomputer/microprocessor combination allowing several of such experiments to proceed independently, with different factors and timing in different parts of a single laboratory. This would

  • 158 J . DAVENPORT

    T

    I I 1 - 7 I I I I I

    I I I I I I I I I I I I I

    FIG. 11 . Pollutant delivery apparatus; from Manley (1980). Key. A-animal chamber, C- constant head tanks (overflows returning to stock tanks), CW-unpolluted water a t constant flow, DS4is t i l l ed deionized water stock tank, M-mixing chamber, P- peristaltic pump, PS-pollutant stock tank, T-teflon tubing, UCTultrograd, UV- ultrograd valve.

    0 Time (minutes)

    FIG. 12. Test data from apparatus of Manley (1980). P-programmed pollutant concentration, A-measured concentration in animal chamber of apparatus shown in Fig. 11, M- concentration in mixing chamber.

  • ENVIRONMENTAL SIMULATION EXPERIMEKTS 159

    substantially reduce the cost of individual experimental set ups, each of which require a separate programmer at present.

    Second, programmes to duplicate precisely the complexity of longer term fluctuations in one factor might be written; this is an approach adopted by Lockwood (personal communication) in mimicking the salinity changes which occur during a complete spring-neap cycle in a particular estuary.

    Thirdly, it is now theoretically possible to model the fluctuations in several environmental physico-chemical factors independently, so that the natural environment at a particular site may be closely simulated in the laboratory. This approach, though expensive, is somewhat analogous to the construction of hydrodynamic models used in the study of the likely effects of proposed dams, barrages etc., and may be of especial use in the simulating of the effects of pollutant delivery to a real estuary. Apparatus of this type is under development at the Institute of Marine Environmental Research, Plymouth (Bayne, personal communication), but has apparently only been used in fluctuating salinity experiments so far (Livingstone et al., 1979). Obviously, though, if five or six factors (e.g. salinity, temperature, oxygen tension, pH, light intensity and algal concen- tration) are all being continually changed in more or less independent fashion, then it will not be possible to attribute responses of organisms to any particular factor, and the simulation can only have a background function against which other experiments are carried out.

    Finally there may be possible applications of the simulation techniques in aquaculture. Most rearing studies for commercially important marine organisms have concentrated upon the use of optimum steady conditions, following the principles and using the analytical techniques of Box and associates (Box and Wilson, 1951; Box, 1954, 1956). However, such conditions may not be appropriate in all cases. For example, Ling (1969), in his study of the large tropical prawn, Macrobrachium rosenbergii (de Man), reported that the adult prawns, which normally live in fresh water, migrate to estuaries during the breeding season and spawn at quite high salinities. The subsequent development of the young stages occur in brackish waters of various salinities. Given adequate information about the salinities associated with each larval stage of M . rosenbergii it would be technically feasible to duplicate these conditions in a hatchery/rear- ing establishment.

  • 160 J . DAVENPORT

    IV. REGIMES

    The main types of fluctuating temperature, salinity, oxygen tension and pollutant regimes employed so far in simulation studies are summarized in Figs 13, 14 and 15. From these i t is clear that there has been little standardization of regime form, although sinusoidal and square wave profiles (of tidal, 12 h or die1 wavelength) have been chosen independently by several workers. It is also obvious that all of the regimes displayed are of idealized form; none is a precise replica of real fluct$uations.

    Superficially the lack of standardization might seem undesirable, but close scrutiny shows that conventional steady-state experi- ments are equally chaotic in their design details; one researcher may use 2 deg C intervals in temperature tolerance studies, while another

    I I 1 0 24 48

    -- 0 24 0 24

    Hours

    FIG. 13. Examples of temperature regimes used in simulation studies. ( 1 ) Costlow and Bookhout (1971); (2) Thorp and Hoss (1975); (3) Hokanson et al. (1977); (4) Cawthorne (1979a).

  • EXVIKONMENTAL SIMCLATION EXPERIMENTS 161

    might employ 5 deg C intervals basically, but use 1 deg C gaps in critical parts of the temperature range. In similar vein, pollutant investigations have been extremely variable in the duration of exposure and range of pollutant concentrations employed.

    Precise mimicking of the conditions at a particular site, on a particular tide or day, would seem to have limited usefulness; i t would have little value in comparative studies and in any case, as would be expected, conditions at a single site can change markedly from day to day, and certainly alter seasonally. An exact simulation is therefore meaningless, but regimes generally representative of certain estuaries might well be selected.

    Some of the regimes chosen by workers have obviously been dependent on the apparatus used. This is particularly true of the

    - x 0 24

    1 0 24

    L 0) - - a a 0 24

    l'O1nl- 0 - 0 24

    nnn 0 12 0 12 0 12

    Hours

    nn 0 12 0 12

    Hours

    FIG. 14. Examples of salinity regimes used in simulation studies. ( 1 ) Tucker (1970s); (2) Stickle

    FIG. 15. Examples of pollutant and oxygen tension regimes used in simulation studies. ( 1 ) and Ahokas (1974); (3) Davenport et al. (1975).

    Davenport (1977); (2) Ritz (1980); (3) Ritz (1980).

  • 162 .I DAVENPORT

    salinity studies of Tucker (1970a), Zachary and Haven (1973) and Stickle and co-workers (e.g. Stickle and Ahokas, 1974; Stickle and Howey, 1975; Findley and Stickle, 1978), whose equipment produced an asymmetrical pattern of falling and rising asymptotic changes in salinity by alternately flushing out an aquarium with dilute and concentrated sea water. The concentrations produced were pre- dictable from the equations published by Wells and Ledingham (1940), who devised a technique for changing the salinity surround- ing isolated preparations from the polychaetes Arenicola marina L., Nereis diversicolor (Muller) and Perinereis cultrifera (Grube). However, precise duplication of salinity regimes, especially at different laboratories, would be difficult since fluid flow rates and vessel sizes were factors in Wells and Ledinghams equations. Since the data presented by Tucker (1970a) also showed that the regimes employed were not particularly closely representative of the environment from which her experimental animals were obtained (see Fig. 16), they seem to simply represent a somewhat arbitrary means of changing salinity in a relatively gradual manner. With the flexibility of more modern equipment there would appear to be little reason to persist with such an approach.

    during two tidal cyclesat the estuary

    sea water 8 4 1 , I I I I I l l 1

    0 1 2 3 4 Time, hours

    FIG. 16. Dilution of sea water in the environment and that produced in the laboratory. From Tucker (1970a).

    The thermal regimes used by Hubbs (1964) were restricted by apparatus limitations in a similar fashion. However, the temperature programmes developed by Costlow and Bookhout (1971) and since used in several studies by Costlow and his co-workers (e.g. Christiansen and Costlow, 1975; Rosenberg and Costlow, 1976; Christiansen et al., 1977a, b; Lucas and Costlow, 1979) appear to be

  • EKVIRONMENTAL SIMULATION EXPERIMENTS 163

    much more generally useful and repeatable. They also represent the major exception to the general use of square wave fluctuations in thermal studies. These programmes consist of a repeated sequence of 6 h at low temperature, 6 h of linear rising temperature, 6 h at high temperature and 6 h of falling temperature.

    The simple zig-zag die1 temperature changes used by Widdows (1976) appear not to have been used elsewhere, but were admirably suited to their design role in establishing whether the mussel Mytilus edulis could become acclimated to fluctuating temperatures. This work followed the earlier observations of Widdows and Bayne ( 197 1 ) that M . edulis could acclimate to changed but steady temperatures.

    Square wave and sinusoidal fluctuations appear to have par- ticular advantages, especially where both are used for comparative work. They are symmetrical and so do not obscure asymmetries in animals reponses. Square wave profiles represent the most abrupt means of changing between two levels of a particular factor, while sinusoidal curves are among the more gentle means of accomplishing such transitions. Natural rates of change must fall between these extremes. The essential feature of the sinusoidal pattern is that the return to the mean level accelerates in proportion to the deviation from the mean, just as tidal height varies sinusoidally with time. Moreover the field salinity data presented by Stickle and Denoux (1976), the rock pool oxygen tension measurement of Daniel and Boyden (1975), and the salinity and temperature results presented by Cawthorne (1979a) all approximate to sinusoidal curves.

    A further advantage of size and square wave profiles may be appreciated from the salinity regime programmes illustrated in Fig. 17. Sinusoidal ( P l ) and square wave (P2) profiles of similar wavelength and amplitude are shown. Because areas A1 and A2 are equal, the quantities of salt and water delivered at a given flow rate over a complete cycle are the same for both types of profile. This means that animals exposed to the P1 profiles will have access to the same total amount of salts and water during a simulated tidal cycle as animals exposed to the corresponding P2 profiles, but will not be exposed to the sudden osmotic shocks and long periods at low sea water concentrations characteristic of the P2 profiles. Similar considerations obviously apply to temperature, oxygen tension or pollutant regimes.

    If square wave and sinusoidal profiles or some other patterns of change are adopted in similar studies, there still remains the question of appropriate wavelengths and amplitudes. The situation for salinity investigations is relatively uncomplicated; except in bodies

  • 164

    100-

    so- r. vi 8

    0 .

    t,

    J. DAVENPORT

    7

    I 12

    Hdurs

    FIG. 17. Diagram to illustrate the two types of fluctuating salinity programme used. P1: this programme produces sinusoidally fluctuating seawater concentrations of near tidal frequency; P2: this programme delivers water of abruptly changing seawater concent- ration; A1 and A2 are equal areas; this means that animals exposed to either type ofsalinity regime will have access to similar total amounts of salt and water a t a given flow rate. From Davenport et al. (1975).

    of water cut off from the sea for periods of days or weeks (e.g. lagoons, salt marshes) which tend to change their salinity slowly, salinity fluctuations are normally of tidal wavelength. The only decision which then has to be made for laboratory simulation studies is whether a precise tidal wavelength should be mimicked or whether the more experimentally convenient 12 h wavelength should be adopted. So far 12 h wavelengths have been almost universally used (e.g. Stickle and Ahokas, 1974; Davenport et al., 1975; Livingstone et al., 1979), although Stickle and Howey (1975) used a tidal 12 h 25 min wavelength regime. This approach is also being adopted by Lockwood (personal communication). A few regimes of rather odd wavelength have also been used. Tucker (1970a) employed a short (4 h) regime which appears to have been a simulation of part of an estuarine tidal cycle, and therefore reasonably realistic. Findley and Stickle (1978), in their study of the haemolymph composition of the blue crab Callinectes sapidus Rathbun, used a 24.8 h wavelength salinity cycle variously described in their paper as tidal and diurnal, yet without field data to justify its double tidal duration. The amplitudes of salinity change employed in simulation studies will

  • ENVIRONMENTAL SIMULATION EXPNRIMESTS 165

    obviously depend upon the euryhalinity of the species being studied although amplitudes greater than those likely to be encountered in nature may be of value for comparative purposes in some investigations where the responses of euryhaline and relatively stenohaline species are being compared (e.g. Shumway, 1977a).

    Appropriate wavelengths for thermal fluctuation simulation studies pose problems. In terrestrial and freshwater investigations, diel temperature fluctuations have been used (e.g. Edney, 1964; Feldmeth et at., 1974; Hokanson et aE., 1977) and these make sense since warm days tend to alternate with cold nights. In marine, estuarine and intertidal environments the situation is more complex. In shallow coastal waters temperatures may fluctuate in diel fashion, but sublittorally in estuaries temperature changes are associated with salinity changes (Cawthorne, 1979a) and are therefore of tidal wavelength. In the intertidal zone temperature fluctuations result from a mixture of tidal and diel influences. Aerial emersion provides thermal shocks of tidal periodicity, but the direction and magnitude of the shock are determined by diel influences. Costlow and Bookhout (1971) employed diel temperature changes in studies upon larvae of the mud crab Rhithropanopeus harrisii (Gould); these would appear appropriate for the inshore environment near Beaufort, N. Carolina, where the experiments were carried out, but not for the Miramichi estuary in Canada where the species has been observed by Bousfield (1955). Sastry (1978) also exposed marine crustacean larvae to diurnal temperature changes which were justified by the field data of Hillman (1964).

    Oxygen tension and pollutant studies have been few in number. Ritz (1980) used both sinusoidal and square wave oxygen tension regimes of 12 h, near tidal wavelength. These were associated with 12 h wavelength salinity fluctuations with low salinity and low oxygen tension coinciding; this situation simulates an estuary where the freshwater input is organically polluted. From the work of Daniel and Boyden (1975) it would seem that rockpool oxygen tensions tend to fluctuate diurnally, but this pattern is distorted when the tide reaches the pool-an analogous situation to that described above for intertidal temperatures. Pollutant fluctuation experiments appear to have been limited to those of Davenport (1977) and Ritz (1980) who both employed square wave copper regimes of 12 h wavelength. These were rather arbitrary profiles intended to simulate a situation where animals were alternatively exposed to clean and polluted seawater on a tidal basis. No field data concerning short term pollutant fluctuations appear to be available.

  • 166 . I . DAVENPORT

    Before leaving the topic of regimes a little space needs to be devoted to the usefulness of linear profiles. Linear regimes consist of constant rate changes in factor level or concentration; an example of a linear pollutant concentration gradient may be seen in Fig. 12. Of course such changes, whether in temperature, salinity or other factors, are not likely in nature, but they are useful in accurately establishing threshold concentrations for behaviour or tolerance and studying the effects of different rates of change of factor level upon such concentrations. Linear regimes are therefore useful in augment- ing the data gained from the use of more realistic simulations.

    v. TEMPERATURE EXPERIMENTS The literature devoted to the study of direct and indirect thermal

    effects upon marine and estuarine animals is extensive and has been reviewed on many occasions. Relatively recent reviews include the excellent comprehensive articles of Kinne (1970) and Newell and Branch (1980) on invertebrates and Brett (1970) on fish.

    Of course much of this literature is devoted to single and multifactor steady-state experiments. Newell (1969) reviewed the effects of fluctuations in temperature on the metabolic processes of intertidal invertebrates, but the time scales of the experiments described were all closer to the seasonal pattern of temperature change rather than the short term tidal and die1 fluctuations which are of interest here. In any case all of the results described by Newell were derived from conventional steady-state experiments.

    The majority of fluctuating temperature studies on marine and estuarine animals have emanated from the Duke University marine laboratory at Beaufort, North Carolina, where they originated with the study of Costlow and Bookhout (1971). Other researchers have employed cyclic temperatures in their studies, but the animals concerned, mainly fish, have been freshwater (e.g. Feldmeth et al., 1974; Hokanson et al., 1977). However, as Kinne (1970) pointed out, the thermal reactions of marine and freshwater forms are often similar so such studies will be referred to where appropriate. For background information about the responses of freshwater fish to temperature, an earlier review by Fry (1967) is still most useful.

    A. Survival

    The upper and lower lethal temperatures of aquatic organisms have been measured in two ways. The less common method used by

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 167

    Tamura (1944), Tsukada and Ohsawa (1958), Southward (1958), Tsukuda (1960) and Crisp and Ritz (1967), involves gradually altering the animals ambient temperature and periodically sampling for mortality. The more widespread approach has been the instantaneous transfer steady-state technique which may be regarded as being analogous to the classic LD50 pharmacological assay method (Kinne, 1970). Lower and upper lethal limits are expressed as the temperature which kills 50% of a test population within a particular period-usually 24 h (LT5&24 h). Plotted temperaturemortality data are normally of sigmoid form (e.g. McLeese, 1956; Mihursky and Kennedy, 1967) and susceptible to Probit analysis which enable researchers to calculate LT50s with considerable precision.

    Thermal tolerance simulation studies performed so far fall into two categories. In the first are those which mimic the sudden thermal stresses imposed by entrainment of organisms in the cooling systems of electricity generating plants. Of course the designs of such experiments grade imperceptibly into the conventional steady-state experimental format, so a somewhat subjective decision has had to be made to decide which studies merit discussion here. It should also be stressed that damage of organisms by power station entrainment is rarely simply a matter of exposure to transient increases in temperature. Usually the combined effects of thermal stress and chlorination are responsible (Waugh, 1964; Heinle, 1969; Morgan and Stross, 1969; McLean, 1973; Muchmore and Epel, 1973; Hoss et al., 1975). There are also pressure fluctuations of considerable magnitude and heavy metal contamination of the cooling flow is not uncommon.

    Heinle (1969) exposed specimens of the copepods Acartia tonsa Dana and Eurytemora afJinis (Nordqvist) to sudden thermal shocks. He took considerable care to ensure that temperature increases took place rapidly ( < 20 deg C change within 5-10 min) but sustained the subsequent elevated temperatures for 24 h-an unlikely situation in the field where thermal discharge plumes lose heat to the atmosphere and mix quickly with colder water. Not surprisingly, both species survived a given temperature increase quite well if they were living in cold water, but not if their native water was warm. Thus, specimens of E . afinis living at 5C survived an increase of 20 deg C quite normally and even an increase of 25 deg C did not kill 50% of the copepods in 24 h. However, animals acclimated to 25C could not survive a 5 deg C increase for 24 h although rather revealingly there was no mortality during the first 4 h of exposure to elevated temperature.

  • 168 J DAVEXPORT

    Diaz (1973, 1975), in his studies on bivalve larvae, used a more accurate simulation of power station entrainment. In 1973 he worked with the larvae of the American Oyster Crassostrea, virginica (Gmelin) which were reared and maintained a t +25"C. To simulate entrainment the larvae were passed, at a concentration of 5-10 individual/ml, through a tube placed in a constant temperature bath. This ensured exposure for 5s to a particular elevated temperature. Three to four litres of sea water containing larvae which had received a thermal shock were then placed in a jar and cooled to + 25C over a period of 30 min. Similar experiments were performed upon larvae of the bivalve Mulinaria lateralis (Say) but with a basic temperature of + 20C. The experimental design appears to be quite representative of the thermal aspects of the power plant entrainment situation. Heating is almost instantaneous, as in condensers, while cooling is quick, as seems likely in rapidly dispersed thermal plumes. A similar experimental procedure has been adopted more recently by Sherberger et al., (1977) who have simulated the type of thermal shocks encountered by drifting aquatic insects which cross a thermal plume. The major results derived from Diaz' studies are displayed in Fig. 18 and Table 11. The data for Crassostrea virginica demonstrate that the deleterious effects of a brief exposure to elevated temperature may not be expressed for some time (4 days in this case). This could not have been predicted from steady-state experiments, and also casts a certain amount of doubt upon the usefulness of LT50-24 h deter- minations. The results for Mulinaria lateralis displayed in Table I1 indicate that susceptibility to thermal shock may alter with developmental state; 2-day-old larvae appeared to be relatively

    TABLE 11. MORTAI~ITIES o~ LARVAE OF Mulinia lateralis 48 H AFTER BKIEF Elrohr.rt~ 1'0 THREE ISC'KLSASES IN TEMPEKATVRE ABOVE 20C (SELEVTEI) FKOM THE DATA OF DIAZ. 1975)

    0 deg C' increase 10 deg C 15 deg C 20 dsg C

    Age of larvae (control) increase incrrase increase (days) yo mortality yo mortality yo mortality Yo mortality

    2 1.5 7.5 23.1 41.9 4 9.8 28.5 15.6 38.8 6 1.3 9.1 34.1 77.1 8 2.6 27.8 33.6 63.9

    10 1.8 2 0 9 36.7 42.7 12 1.6 23.8 27.0 44.8

    Mean 3.1 19.6 28.4 51.5

  • ENVIRONMENTAL SIMULATION EXPERIMEKTS 169

    0 2 6 10 14 18 22

    DAYS AFTER EXPOSURE

    FI(:. 18. Cumulative mortalities for 24 days of American oyster (Crussostrea virginicu) larvae exposed for 5 s to three increases in temperature above 25C when 3 days old. From Diaz (1973).

    unaffected by temperature increases, while 6-day-old larvae tolerated a 10 deg C increase particularly well but were affected badly by a 20 deg C increase. This species did not die until 2 days after a temperature shock. Delayed mortality is perhaps to be expected. Although proteins coagulate leading to cell death (Nassonov and Alexandrov, 1943) at high temperatures, most somatic cells can tolerate sub- stantially higher temperatures than the intact individual (Ushakov, 1968). For vertebrates, heat death of the individual generally seems to be caused by interference with the working of the central nervous system (Fry, 1967). Control processes appear to be less centralized in invertebrates and peripheral activity of bivalve larval tissues may persist for some time after the CNS has been irreversibly damaged.

    A more recent entrainment simulation study has been carried out upon larvae of a teleost fish by Middaugh et al. (1978). Much of this study was concerned with the effects of brief exposure to residual chlorine. However some investigations on thermal shock were conducted upon embryos and larvae of the euryhaline, eurythermal estuarine mummichog, Fundutus heteroclitus L. The animals were reared at 24C and exposed to 5 or 10 deg C temperature elevations lasting 7.5, 15, 30 or 60min before a return to 24C. Mortality was assessed 24 h later. All embryonic and larval stages showed increased mortality with both amplitudes of temperature increase. In embryos

  • 170 J . DAVEXPOKT

    longer exposure to elevated temperature did not increase mortality, though it did in newly hatched and 7-day-old larvae. However the latter point is somewhat academic; Middaugh et al. admit that only the embryos are likely to be entrained in nature.

    The second category of temperature tolerance simulation study encompasses investigations where cyclical temperature regimes have been employed. It has long been known that organisms often survive changing temperatures better than they do constant temperatures (Allee et al., 1949). However the earliest laboratory tolerance experiments with repeatable regimes upon aquatic organisms were those of Heath (1963) and Hubbs (1964). Heath was interested in the difference in critical thermal maximum (C.T.M. as defined by Lowe and Vance, 1955) temperature between species of cutthroat trout, Salmo clarki clarki previously acclimated to either constant or cyclic temperatures. Fish acclimated to diel square wave fluctuations between 10 and 20C survived to a significantly higher C.T.M. (29-77C) than trout acclimated to 15C (C.T.M. 29.06"C). Fish kept at a constant 10C had a C.T.M. of 27.63"C, while trout held at 20C had a significantly higher C.T.M. (29.88"C) very similar to that of temperature cycled fish. Hubbs (1964) showed that eggs and larvae of the freshwater teleost Etheostoma lepidum Baird and Girard survived diel fluctuations between 4 and 12C just as well as at a constant 9"C, yet constant exposure to 7C was lethal. Again this illustrates the enhanced survival often caused by thermal fluctuations, although it should be remembered that instantaneous, and usually un- physiological, thermal shocks may kill fish (Fry, 1957). The abrupt fluctuations used by Heath (1963) were small in amplitude and well within the thermal tolerances of cutthroat trout, while the temperature changes employed by Hubbs (1964) were quite slow (c. 2 deg C/h). More recently Feldmeth et al. (1974), working on the pupfish Cyprinodon nevadensis amargosae Eigenmann and Eigenmann, which inhabits shallow thermally unstable desert streams, have demonstrated an increase in temperature tolerance at both ends of the temperature scale induced by prior acclimation to diel temperature fluctuation regime (15-+35"C; 7 h a t low tempera- ture, 5 h of warming, 7 h at high temperature, 5 h of cooling et seq). These results conflicted with an earlier hypothesis of Brett (1944) which suggested that both upper and lower lethal temperatures were determined by the maximum temperature experienced during a diel cycle.

    How does prior acclimation to cyclic rather than steady regimes extend thermal tolerances in fish? Studies upon acclimation to steady

  • EKVIRONMENTAL HIMULATIOX EX PERIMEKTS 171

    temperatures in fish (e.g. Hochachka and Somero, 1968; Hochachka and Lewis, 1970; Moon and Hochachka, 1971) indicate that acclimation occurs by elaboration of isoenzymes uniquely suited to metabolic function at a particular environmental temperature. Feldmeth et al. (1974) suggest that the pupfish acclimated to cyclic temperatures simultaneously induce separate isoenzymes for both warm and cold conditions, thus gaining survival attributes not seen in fish acclimated to steady average temperatures. Unfortunately this type of study, where survival of extreme temperatures is tested after a period of exposure to moderate thermal fluctuations, appears to have been carried out only upon freshwater fish or migratory fish during the freshwater phase of their life cycle. There would appear to be no reason to expect marine fish not to have similar responses. Certainly a similar response has been demonstrated in a very different group-the terrestrial isopods. Edney (1964) worked on Porcellio laevis Latreille and Armadillidium vulgare Latreille. Isopods acclimated for 2 weeks to a diel regime of 12 h at 10C and 12 h at 30C had a maximum lethal temperature similar to animals acclimated to a steady temperature of 30C while the lower lethal temperature of the cyclically treated isopods approximated to that of animals acclimated to a steady 20C. In other words, the animals held in cyclical regimes had a total temperature range greater than that of isopods held at any single temperature.

    Cyclic temperature studies on marine and estuarine organisms appear to have started with the investigation of Costlow and Bookhout (1971) on larval development in the mud-crab Rhithropanopeus harrisii. These workers compared the effects of a variety of diel temperature fluctuations and constant temperatures upon survival and duration of larval stages of R. harrisii; the survival results obtained are displayed in Table 111. Clearly Costlow and Bookhout's approach differs somewhat from that employed in the freshwater and terrestrial studies described above. Instead of investigating how prior acclimation to modest temperature fluctu- ations affected tolerance of extreme temperatures, these workers studied the larval tolerance of the cycling temperatures themselves, some of which approached the lethal limits of the animals. Larvae were reared from hatching to the first crab stage at a constant salinity of25%,. Control larvae were kept at steady temperatures of 15,20,25, 30 and 35C. Experimental larvae were either exposed to 5 deg C fluctuations (15 + 20C, 20 -+ 25"C, 25 + 30"C, 30 + 35C and 35+4OoC) or 10 deg C changes (15+25"C, 2O+3O0C and 25+35"C). The picture that emerges from the results is rather complex. Survival

  • 172 J. DAVENPORT

    TABLE 111. SURVIVAL OF LARVAL STAGES OF R. ha?'riSii CULTURE]) AT 250- (FROM COSTLOW AND BOOKHOUT. 197 1

    Hatch to megalopa Megalopa to crab Hatch to crab

    No. of Tempera- meae ture ( "C) No. % No. Yo No. %

    150 15 87 58.0 32 368 32 21.3 42 1 20 380 90.3 35 1 92.3 351 83.4 442 25 389 88.0 359 92.2 359 81.2 434 30 288 664 252 87.5 252 58.1 100 35 5 5 0 2 40 2 2.0 400 1&20 253 63-2 230 90.9 230 57.5 400 2&25 337 842 306 908 306 765 400 25-30 329 82.2 292 88.7 292 73.0 400 30-35 362 90.5 334 92.2 334 83.5 100 3 5 4 0 0 0 0 0 0 0 650 15-25 503 77.4 437 869 437 67.2

    lo00 2&30 826 82.6 738 89.3 738 73.8 1150 25-35 733 63.7 525 7 1.6 525 457

    to the crab stage in the 15 -+ 25C cycling regime (67.2%) was better than the survival at a constant 15C (21.3y0) while the regime fluctuating between 25 and 35C was tolerated better (45.7% survival) than a constant 35C (2.0% survival). Again extremes of fluctuating temperature appear to be tolerated better than extreme constant temperatures. However, survival in the 20 -+ 25C regime was slightly poorer than at a steady 20 or 25C indicating that the same situation did not hold at less stressful temperatures. Finally the survival in the 30 -+ 35C regime (83.5%) was far better than that at either a constant 30 or 35C (58.1% and 2% respectively). This last result seems particularly anomalous since the survival of the 30 -, 35C regime represents the maximum survival to meta- morphosis for any of the experimental series of larvae. Costlow and Bookhout could only speculate that this regime might eliminate pathogenic organisms not killed by constant temperatures, or alternatively, that enzyme systems or physiological functions might in some way be particularly favoured by it. In a later study, Christiansen and Costlow (1975) were unable to duplicate this particular result with Rhithropanopeus harrisii so perhaps i t should be regarded as suspect. In a later study (though published earlier) Regnault and Costlow (1970) obtained results for the larvae of the shrimp Crangon septemspinosu Say. Broadly speaking the results were similar to those obtained for R. harrisii with extremes of

  • ENVIRONMENTAL SIMULATIOK FXPEKIMESTS 173

    temperature being tolerated better if the animals were given intermittent respite at less stressful temperatures.

    In subsequent studies, largely on R. harrisii, Costlow and his co- workers (Christiansen and Costlow, 1975; Rosenberg and Costlow, 1976; Christiansen et al., 1977a, b; Lucas and Costlow, 1979) have concentrated upon the effects of cyclic temperature on larval development and on the interaction of cyclic temperature with the effects of toxic compounds (heavy metals, insecticides). These studies will be discussed elsewhere in the review.

    As part of a broader study, Widdows (1976) showed that adult mussels, Mytilus edulis, could sustain filtration, and hence survive, in a cyclic temperature regime fluctuating between 21 and 29C. At a constant 29"C, however, filtration was halted and long term survival presumably impossible.

    The most recent study devoted purely to the effects of fluctuating temperature upon the survival of marine organisms is that of Cawthorne (1980). He studied nauplius larvae of the barnacles Balanus balanoides L. and Elminius modestus Darwin and compared their tolerances to: (a) steady temperatures, (b) temperature fluctuations of square wave form and 12h wavelength, (c) temperature changes of sinusoidal form and 12h near tidal wavelength. The data derived from this investigation are displayed in Fig. 19. They consist of sigmoid curves relating the maximum temperature encountered in a given temperature regime to the mortality induced by that regime. The LT50-24 h values calculated from these curves are set out in Table IV. These results show once again that survival a t extreme temperatures is better in animals exposed to cyclic rather than steady temperatures and resemble the data collected for a desert population of a freshwater teleost Gambustia a j jn i s a j jn i s (Baird and Girard) (see Otto, 1974). Additionally i t is clear that gentle (sinusoidal) salinity changes are tolerated better than abrupt fluctuations. Particularly interesting is the disparity between the two species in their response to fluctuating temperatures; although Balanus balanoides and Elminius modestus exhibit very similar upper lethal temperatures when exposed to constant conditions, E. modestus is much the more tolerant of high temperatures when the latter are encountered briefly or intermittently.

    Fry (1947) defined the limit of the zone of activity or thermal tolerance as the point beyond which an organism is unable to

  • TABLE I v . MAXIMITM TEMPERATURES ("c) CAITSINC: 50% MORTALITY IN CII tRIPRDE~AZIPI~IITS LARVAE EXPOSED TO VARIOUS TEMPERATIJRE REGIMES. 95% CONFIDENCT INTERVALS I N

    BRACKETS. (FROM THE DATA OF CAWTHORNE, 1979a, 1980)

    Type of temperature regime

    Species (a) Steady state (b) Square wave ( c ) Sinusoid ~~

    Balanus balanoides 32.2 ( 0 6 ) 345 (0.5) 36.6 (04) Elminius modestw 32.4 ( 0 9 ) 3 5 5 (0.5) 388 ( 0 4 )

    100- ( a )

    0-r I I I i 32 33 34 35 36 37 38 39 40 41 42 31

    Maximum temperature ("C

    Maximum temperature ("C)

    FIG. 19. Temperature of newly released nauplius larvae of (a) E. modestus and (b) B. balunddes. Circles indicate animals exposed to sinusoidal temperature changes of 12 h wavelength between 10C and various higher temperatures; squares indicate animals exposed to square- wave temperature changes; triangles represent animals exposed to steady elevated temperatures. Modified from Cawthorne (1979a).

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 175

    maintain its activity and survive indefinitely. This definition is clearly relevant only to constant thermal conditions which rarely occur in coastal and estuarine marine habitats.

    B. Development

    A large number of steady state investigations into the effects of temperature on development and growth in aquatic fish and in vertebrates have been carried out (see Gray, 1929; Sandoz and Rogers, 1944; Precht et al., 1955; Costlow et al., 1960; Costlow, 1967; Ong and Costlow, 1970; Alderdice and Velsen, 1971; Brett, 1970; Kinne, 1971; Lough 1975; Peterson et al., 1977 for some examples and reviews).

    It has long been known that development in most ectothermic animals is accelerated at higher temperatures (Belehradek, 1935), but the effects of varying temperatures are more complicated. Khan (1965) lists three possibilities, mainly derived from the freshwater studies of Grainger (1959):

    ( 1 ) Development proceeds at a rate to be expected from the results of constant temperature experiments. Here the alterations in temperature do not themselves affect the rate, and embryonic development is, for instance, speeded up by the expected amount if the temperature is raised.

    (2) Development does not proceed at a rate predictable from steady temperature studies. Here the alterations in temperature have an effect on developmental rate at the time they are taking place. This has been called an immediate effect (Grainger, 1959).

    (3) Like (2) development does not proceed at the expected rate because the temperature experienced in early development retards or accelerates subsequent development. This was described as an after effect by Grainger.

    Khans classification appears to have been ignored in cyclic temperature studies on marine and estuarine animals, as have the detailed and tedious methods she employed in her temperature studies on the freshwater crustacean Acanthocyclops viridis.

    Costlow and Bookhout (1971) found that developmental times (as distinct from survival) of larval stages of Rhithropanopeus harrisii were barely affected by fluctuating temperatures; development proceeded at a rate intermediate between the rates occurring at constant temperatures corresponding to the extremes of the tem-

  • 176 J DAVEXPORT

    50 40

    Hatch to crab

    Megalops to crab

    Hatch to megalops

    PI:. 20. Comparison of time required for development of larval stages of R. harriaii in the laboratory at constant temperatures and 5 and 10C daily cycles of temperature. From Costlow and Bookhout (1971).

    perature cycle (see Fig. 20). There was some indication that 10 deg C fluctuations slightly slowed development but this was certainly not the case with 5 degC changes.It would appear that larvaeof R.harrisil: are well adapted to temperature fluctuations which are a feature of their coastal/estuarine habitat. A similar situation was found for the larvae of the shrimp, Crangon septemspinosa by Regnault and Costlow (1970). In contrast, Sastry (1978) found that the duration of development of larvae of the sublittoral crab, Cancer irroratus Say was affected by fluctuating temperatures; zoeae developed faster and megalopae developed slower when exposed to 1O+2O0C or 12.5 + 17.5"C than they did at a constant 15C. However, both of these amplitudes of temperature fluctuation may be somewhat

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 177

    unphysiological, since Hillman (1964) recorded diurnal variations of only k2 deg C in the environment of these larvae.

    Recently, Lucas and Costlow (1979) have carried out a cyclical temperature study on some molluscan larvae-veligers of the intertidal and estuarine prosobranch gastropod Crepidula fornicata (L.). As for Rhithropanqeus harrisii growth under cyclic tempera- ture regimes was intermediate between that observed in veligers living at corresponding constant temperatures (see Fig. 21). Given that only a small number of investigations have been performed so far, this would appear to be the usual pattern for animals normally exposed to temperature fluctuations in the wild, and consequently well acclimated.

    C. Reproduction

    Although several studies have shown the importance of raised temperatures to the induction of spawning in bivalves (e.g. Loosanoff, 1945a; Loosanoff and Davis, 1950; Gruffydd and Beaumont, 1970) there appears to have been only one investigation so far in which the effects of fluctuating temperature upon reproductive processes have been monitored. Cawthorne and Davenport (1980) investigated the effects of square wave and sinusoidal tidal temperature cycles u,pon the release of larvae by adults of two common barnacle species, Balanus balanoides and

    /;:!:! 25O-3OoC I - E 300t 200 B OCOEP 2Oo-25OC / c C

    0 x

    OCOEP15-200C

    E ?! .- - -

    E 100

    P

    0 6 12 0

    Age (days)

    FIG 21 Crepnddaforntratn Growth of vehgers at varioub constant temperatures and 5deg (' temperature cycles of equal penodicity (COEP) Values are from measurements of 75 larvae From Lucas and Costlow (1979)

  • 178 . I . DAVENPORT

    250-

    200- I

    j 4 100-

    0,

    (b ) ( b l 500-

    r---,f&+ .......... k!-i,,i 400. r - - - - - - - - - ~ .~ .......... : #4-1 I ' !/I : I 'Y.1 .Sl$V ' Llkl ' I ! ! ~

    ' , ' , ' , ' 1 0 '

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 179

    produced by abrupt temperature changes would have advantages ecologically. Abrupt temperature changes of significant amplitude only happen in the intertidal habitat when immersion or emersion occurs (Cawthorne, 1971a). Because E. modestus closes its opercular valves and effectively isolates its mantle cavity in response to emersion, any enhanced hatching triggered by temperature change at this time cannot be expressed as release until the animal is reimmersed. Temperature changes associated with immersion gener- ally tend to be decreases in the summer and increases in the winter although die1 or climatic influences may sometimes modify this pattern. Because E. modestus breeds throughout the year a hatching response to sharp changes of temperature in either direction will always ensure that most larvae are released on a rising tide. This is particularly important in the estuarine habitat where rising tides tend to be associated with rising salinity. The experiments which led to the results recorded in Fig. 22 were carriedout in winter; it is noticeable that decreases in temperature were more effective in triggering heavy larval release than rising temperatures. Possibly this pattern reflects a degree of seasonal acclimation.

    D. Adaptation

    Precht et al. (1955) showed that ectothermic organisms held at a new temperature for some days show changes in the activity and concentration of some enzymes and other substances; these changes are often associated with changes in oxygen consumption. The initial stages of this process of adaptation were studies by Grainger (1956, 1958) who was interested in the immediate metabolic responses of various crustaceans to abrupt temperature change. Kinne (1964a) reviewed the then extant literature devoted to non-genetic adaptation using the various concepts of temperature acclimation proposed by Precht (1958) and since adopted by many workers (e.g. Alderdice, 1972).

    Not until the work of Widdows (1976) was the question of adaptation to cyclic temperatures tackled for any marine organisms. Widdows and Bayne (1971) and Bayne (1976) had previously shown that the intertidal mussel Mytilus edulis could adapt completely (Precht Type 2) in its filtration rate to gradual temperature changes between 2 and 20C. Obviously this allowed feeding activity to continue independent of season. Widdows (1976) showed that specimens of M . edulis acclimated their rates of filtration and oxygen consumption to cyclic temperature fluctuations (1 1 + 19C) by

  • 180 J . DAVENPORT

    reducing the amplitude of response over a period of about 2 weeks (see Figs 23 and 24). Compensation was only partial for oxygen consumption but complete for filtration rates, which became independent of the temperature changes. The oxygen uptake and filtration rates of animals adapted to cyclic temperatures did not differ significantly from the responses of mussels adapted to equivalent constant temperatures. This agrees well with the observations on growth and development of eurythermal organisms reported in the previous section. Widdows also showed (see Fig. 23) that acclimation to cyclic temperatures occurred over a similar period to constant temperature adaptation. Two suggestions to explain this were put forward. The first was that only a brief exposure to a new temperature was required to stimulate the initiation of the adaptive response, although presumably the stimulus had to be repeated at intervals to sustain adaptation. Alternatively, the mussel might integrate its response over the whole temperature cycle to become independent of thermal extremes. Finally, Widdows

    0'7[ I

    FIG. 23. Thermal acclimation of oxygen consumption by Mytilus edulis. (a) Rate of oxygen consumption in response to cyclic temperatures. TF (1 1-19C); 1 I T , 15"C, 19C. (b) Rate of oxygen consumption in response to constant temperatures, 11"C, 15"C, 19C. Mean1S.E. From Widdows (1976).

  • ENVIRONMENTAL SIMULATIOK EXPERIMENTS 181

    I I I I I I I I I I I I I 0 10 20 30

    Days

    FIG. 24. Thermal acclimation of filtration rate of Mytilus edulis. (a) Filtration rate in response to cyclic temperatures, TF (11-19C); 1 1 T , 15"C, 19C. (b) filtration rate in response to constant temperatures. 11"C, 15"C, 19C. Mean+S.E. From Widdows (1976).

    demonstrated that animals taken from relatively constant tempera- ture environments and tested immediately showed metabolic temperature-dependent responses, whereas mussels collected near a power station which imposed marked temperature fluctuations on them exhibited temperature-independence, thus confirming that the conclusions from the laboratory cyclic temperature studies were applicable in the field.

    In contrast to Widdows' results, Sastry (1978), who worked on larvae of the crab Cancer irroratus, found that oxygen consumption in animals exposed to cyclic temperatures (10 -, 20C) was shifted towards values characteristic of larvae living at 20C rather than towards those applicable to the mean value of 15C; Sastry suggested that enzymes which were inactive at 15C might become activated during temperature cycles which periodically exceeded 15"C, and that this activation might allow increased metabolic activity as suggested by Somero (1969). Sastry also showed that there were significant differences between the activities of various enzymes

  • 182 J. DAVENPORT

    (lactate dehydrogenase, malate dehydrogenase and glucose-6- phosphate dehydrogenase) in cycled and constant temperature crab larvae. However, the reasons for these differences were and remain obscure, particularly as there appeared to be no correlation with oxygen consumption.

    E. Interaction with other factors

    Since response surface techniques were introduced by Box and Wilson (1951), a large number of marine biologists have used such representations of multiple factor studies. In many of these investigations, and also in simpler two factor studies, temperature has been a variable (e.g. McLeese, 1956; Costlow et al., 1962; Crisp and Costlow, 1963; Alderdice, 1963; Forrester and Alderdice, 1966; Alderdice andvelsen, 1971; Ahokas and Sorg, 1977). The most recent major reviews of the field appear to be those of Kinne (1971) and Alderdice (1972), the latter being concerned mainly with the principles and mathematical techniques involved in response surface me thodology .

    Studies involving the interaction of cyclic temperatures with other factors (except for biological factors such as age or stage of development) have been few in number. So far no special method of analysis or presenting the results has arisen and no-one has yet had the temerity to draw conclusions from experiments in which more than one factor was cycled!

    Regnault and Costlow (1970) investigated the effects of cyclic temperature upon larvae of Crangon septemspinosa at two salinities, 20 and 30%,. The enhanced survival shown in response to temperature cycles by comparison with that exhibited at equivalent constant temperatures was more marked a t 30%, than at 20%,. Thorp and Hoss (1975) employed a much wider range of salinities (5 -, 35%,) in their studies upon adults of two species of grass shrimp, Palaemonetes pugio Holthius and Palaemonetes vulgaris (Say). Using a zig-zag die1 pattern of temperature change like that used by Widdows (1976), they obtained the data summarized in Table V. Clearly, cycling temperatures significantly depressed the survival of both species in low salinity (5%,) water. As a result of this study Thorp and Hoss stressed that more work was necessary to detect possible positive or negative effects of cyclic temperature regimes, particularly as they might have a bearing on environmental toxicity standards which have been based upon LC50 values obtained at constant, often optimal, temperatures. The investigation of

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 183

    TABLE V. MEAN PERCENTAGE SURVIVAL OF Palaemonetes pugio AND P. vulgaris HEM) AI' THREE SALINITIES AND THREE TEMPERATURE REGIMES FOR 21 DAYS (FROM THE RESULTS OF

    THORP AND Hoss, 1975)

    Salinity

    Temperature regime 5% 20% 35%

    1 . Cyclic 7-13C P. pugio 467 9 3 3 6 2 2 P. vulgaris 17.8 75.6 66.7

    P. pugio 800 93.3 77% P. vulgaris 64.5 84.5 75.6

    P. pugio 75.6 91.1 95.6 P . vulaaris 467 91.1 800

    2. Constant 10C

    3. Constant 7C

    Christiansen and Costlow (1975) lent weight to this advice since they found that survival of the larvae of the mud crab Rhithrqanopeus harrisii at low salinity (5%,), in contrast to that of the Palaemonetes adults, was better in a fluctuating temperature regime than had previously been demonstrated in an equivalent constant tempera- ture study (Costlow et al., 1966).

    In much of their subsequent work, Costlow's group have pursued the matter of the interaction of pollutants with cyclic thermal regimes. Rosenberg and Costlow (1976) studied the synergistic effects of cadmium and salinity combined with constant or cycling temperatures on larvae of R. harrisii and the eurythermal, euryhaline blue crab Callinectes sapidus. The multi-factorial design was complicated (63 different temperature/salinity/cadmium con- centration combinations were used for R. harrisii alone) and this, combined with the variety of larval stages used, generated a great mass of data which are rather difficult to compress. However, Fig. 25 gives a general picture for R. harrisii. At both the lower and upper ends of the temperature scale fluctuating temperatures apparently stimulate survival in all cadmium concentrations; this is particularly noticeable in the 30 + 35C temperature regime. On the other hand, to complete an already complicated picture, more recent studies upon the same species by Christiansen et al. (1977a, b) using combinations of temperature cycles and various concentrations of the juvenile hormone mimicking insecticides methroprene and hydroprene, failed to demonstrate any synergism with temperature.

  • 184

    100

    80

    - 60 - :

    $ 40 Ln

    20

    0

    Salinit %o Temperature x) OC

    J UAVESPORT

    Hatch - Crab

    1 1 1 1 l l I / I I I 1

    20-25 25 25-30 30 30-35 35

    FIG. 25. Rhithropanopeus harrisii. The percentage of animals surviving from hatching to the first crab stage when exposed to various combinations ofcadmium, salinity and constant or cycling temperatures. From Rosenberg and Costlow (1976).

    VI. SALINITY STUDIES

    The greater part of the worlds aquatic environment consists of sea water with a salinity close to 35%,. The invertebrates in it are not under appreciable osmotic stress as their tissues and body fluids are approximately isosmotic with the external medium. It is generally held that marine invertebrates have a long marine ancestry without significant change in the composition of their seawater surroundings, except possibly over a geological time scale. For a recent discussion of the controversial question of the stability of seawater composition over geological time, Spaargaren (1978) should be consulted. Even marine fish, which have a lower ionic concentration in their blood than in the surrounding medium, are exposed to a constant rather than changeable stress.

    The fraction of the hydrosphere which is characterized by low, high or varying salinities is very small. Consequently, unusual or changeable salinities influence only a small proportion of the aquatic biomass. However, for reasons already discussed in the introduction, littoral, estuarine and other brackish water habitats have attracted the attention of many researchers. In such areas the controlling influence of salinity on distribution is often obvious, and the numerous attempts to correlate particular faunal types with certain salinity levels started nearly a century ago (e.g. Mobius and Heincke, 1883) and have persisted with considerable confusion and some acrimony to recent times (e.g. Kinne, 1964b; Khlebovich, 1969).

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 185

    Distributional, tolerance and metabolic data in relation to salinity have been reviewed by Kinne (1971) for invertebrates, and for fish by Holliday (1971).

    A great deal of this literature has been concerned with responses of animals to osmotic stress. The physiological mechanisms were first reviewed by Krogh (1939); more recent extensive reviews have been given by Potts and Parry (1964) and Gilles (1979).

    Studies containing elements of simulations of the real environ- ment have been performed by Wells and Ledingham (1940) who changed the concentration of the medium around their tissue preparations from euryhaline polychaetes, and by Haskin ( 1964) who exposed oyster larvae to changing salinities to see if increased swimming activity was associated with rising salinity. However, the rates of salinity change employed, and the form of the changes were quite arbitrary. The first real attempt to mimic natural fluctuations in the laboratory appears to have been that of Tucker (1970a) who investigated body weight and blood composition changes on the fissurellid gastropod Scutus breviculus. Since then a number of investigations at several laboratories have been performed, which may be subdivided in the following manner.

    A. Survival

    Three studies comparing the tolerances of marine or estuarine organisms to fluctuating rather than steady salinities have been carried out by Zachary and Haven (1973), by Davenport et al. (1975) and by Cawthorne (1978).

    Zachary and Haven (1973) were interested in the effects of fluctuating low salinity levels on survival and activity of the Oyster Drill Urosalpinx cinerea Say. Amplitudes of salinity fluctuations were small (roughly 2-3%,) and mean salinities ranged from 7.9 to 16*8%,. Drills were also held at various steady salinities ranging from 8 to 12%,. No investigations into the effects of higher salinities were performed as earlier workers (Frederighi, 1931; Galtsoff et al., 1937; Manzi, 1970) had shown that only salinities below 20%, were lethal. Zachary and Haven's experimental design and analytical methods were somewhat imprecise and cumbersome, and it is difficult to compare their fluctuating and constant salinity results. However, broadly speaking the drills were more tolerant of fluctuating conditions than of steady low salinities, particularly for the first 10 days of exposure.

    The study of Davenport et al. (1975) was carried out on larvae of

  • 186 J. DAVENPORT

    the scallop Pecten maximus (L.). Although larvae of the sublittoral stenohaline scallop were unlikely to encounter salinity fluctuations in their natural habitat, the results proved to be interesting. Larvae were exposed for 24h either to steady salinities or various 12h wavelength sinusoidal and square wave salinity regimes fluctuating between full sea water (34%,) and various lower salinities. At the end of the 24 h period the larvae were returned to full sea water for 18 h before being assessed for mortality against full seawater controls. The characteristics of these three types of salinity regime are detailed in Table VI, and the results shown in Pig. 26. The two fluctuating salinity regimes were much less damaging than constant exposure to lowered salinity levels and these results give a strong indication that conventional steady-state salinity tolerance studies can yield incorrect assessments of the likely salinity limits to distribution for a given species. Even more interesting is the difference between the effects of sinusoidal and square wave salinity regimes. Over a 24 h period, at a given flow rate, the total amounts of salt and water supplied to experimental animals were the same whichever cycling regime was used. On the one hand, larvae in the square wave regimes were exposed to abrupt osmotic shocks and prolonged periods at low salinity, neither of which were encountered by larvae exposed to the sinusoidal cycle. On the other, animals in the sinusoidal regime were exposed to full seawater conditions only momentarily unlike larvae

    T.4~31,~ VI. A COMPARISON OF THE FACTORS INFI,~NCIN(: AxImw IN THREE TYPES OF SALINITY REGIME EMPLOYED IN A LARVAL SALINITY TOLERANCE STWY UPOK Pecten

    maximus (FROM DAVENPORT et al., 1975)

    Regime type (all with 50% sea water minimum concentration)

    (b) Square (c) Steady (a) Sinusoidal wave state

    No. of osmotic shocks 0 4 2

    Period of exposure Negligible 12h 24 h

    per 24 h

    to minimum salinity

    delivered per 24 h by comparison with 100% S.W. control

    Percentage of salts 75% 75% 50%

    100% S.W. = 34%.

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 187

    100

    0

    0 50 100

    Minimum sea waterencountered (yo)

    FIG. 26. The effects of sinusoidal, abrupt and steady state salinity profiles upon larvae of Pecten mximwr. Triangles indicate steady state profile results; LC50 (24 h) = 49.7f0.4yO S.W.; Y = 13.9874181~ where c = yo S.W. Squares indicate abrupt (P2) profile results: minimum LC50 (24 h) = 357f04% S.W.; Y = 14.4544265~. Circles indicate sinusoidal (Pl) profile results: minimum Lc50 (24h) = 244&02% S.W.; Y = 7.918-0.120~. From Davenport et al. (1975).

    from the square wave profile which were in full seawater for 12 h in every 24. So what was the major factor in allowing enhanced survival in the animals exposed to the gentle sinusoidal salinity fluctuations; was i t the absence of osmotic shocks or the freedom from prolonged exposure to low salinities? To clarify this point Pecten larvae were exposed to the two special salinity regimes shown in Fig. 27. These represent gradual (P3) and abrupt (P4) introductions to a steady salinity (equivalent to 40% sea water). After 10 h of exposure to 40% sea water assessment for mortality commenced and was repeated every 2h until 18h of exposure had been completed. The results obtained are displayed in Table VII. It appears that a sudden osmotic shock, characteristic of the P4 type of regime, causes damage which leads to death after several hours. In contrast, the mortality of the larvae exposed to gradual osmotic changes (P3 regimes) rises very little after the minimum salinity is reached. It seems likely that if the change is gradual, animals which are particularly susceptible to low seawater concentrations die off rapidly leaving a residue of more tolerant individuals. It follows that for Pecten larvae, sudden osmotic

  • 188

    TABLEVII. AN ASSESSMENTOFTHE RELATIVECONTRIBUTIONS'I'O MORTALITY o ~ E s r o s r . ~ ~ ~ o Low SEAWATER CON('ENTI~ATIONS, AND SUBJECTION TO ABRUPT OSMOTIC SHOCKS 15 LARVAE OF

    Pecten maximus ( looyo S.W. = 33.5%,)

    (a) Results of experiments: values in relative percentage mortalities (corrected for control results).

    Hours of exposure to 40% S.W. Regime Experiment

    type order 10 12 14 16 18

    P3 1 18.99 23.32 1673 23.33 22.41 4 19.07 21.25 30.39 20.68 21.37

    P4 2 7.49 1503 30.38 5080 67.59 3 15.65 19.96 35.54 58.58 66.91

    (b) Results of analysis of variance: performed after angular transformation of above data.

    Variation in mortality Degrees of Calculated d.f. ratio for due to: freedom d.f. ratio P = 0.001

    (i) Differences between 1 393 21-0 regimes P3 and P4

    sampling times

    regimes and sampling times

    (ii) Differences between 4 25.2 11.3

    (iii) Interaction between 4 22.0 11.3

    (iv) Replication 10 ( 1 ) -

    0 I I I I I i 6h 10 12 14 16 18 c

    0 Exposure (h)

    FIG. 27. Diagram to illustrate the salinity programmes used to determine the relative importance of exposure to low seawater concentration and osmotic shocks. Arrows indicate sampling times. From Davenport et al. (1975).

  • ENVIRONMESTAL SIMITLATIOX EXPRKIMEN'I'S 189

    TAHLE VIII. MKIXAS MINIMUM LETHAL yo SEALZAT~W CONCEN'I'HATIOS~ ( 100~o

    TYPES 01" SALINITY RKGIME (WITH 95% CONFIDENCE LIMIT^) (FROM THE DATA OF S.W. =335%,) FOR NEWLY RELEASED CIHRIPKDE NAL-PLII EsI.osE1) FOR 24 H TO THI

  • 190 J . DAVENPORT

    TABLE IX. ORGANISMS KNOWN TO TOLERATE FULL RANGE OF SALIXITIES (@34%,) IN REGULAR SIMULATED TIDAL SALINITY CYCLES

    Species Source of information

    Barnacles Balanus balanoides Balanus crenatus Balanus improvisus Elminius modestus

    Cerastoderma edule Crassostrea gigas Mya arenaria Scrobicularia plana Anadara senilis

    G a m m r u s duebeni

    Bivalves Mytilus edulis

    Amphipods Marinogammarus marinus

    Crabs Carcinus maenas Fish Blennius pholis Algae Fucus serratus

    Enteromorpha spp Ulva lactuca

    ~~ ~ ~

    Davenport (1976) Davenport (1976) Davenport (1976) Davenport (1976)

    Shumway (1977) Bettison (unpublished data) Bettison (unpublished data) Bettison (unpublished data) Bettison (unpublished data) Djangmah et al. (1979) Ritz (1980) Ritz (1980) Davenport (unpublished data)

    Davenport and Vahl (1980) Dickson (1978) Dickson (unpublished data) Dickson (unpublished data)

    B. Behavioural responses

    Reduction of tissue contact in response to adverse external salinities is common in sessile or slow moving benthic intertidal or estuarine organisms (Kinne, 1971). Cronklin and Krogh (1938) and Milne (1940) showed that the mussel Mytilus edulis, reduced contact by closing the shell valves. Since then similar mechanisms have been demonstrated for a variety of invertebrates including other bivalves (e.g. Freeman and Rigler, 1957), gastropods (e.g. Segal and Dehnel, 1962; Avens and Sleigh, 1965) and barnacles (e.g. Barnes and Barnes, 1958; Foster, 1970). Several mobile nektonic invertebrates and fish have been shown to detect deleterious salinities and to be able to select more favourable or even optimal conditions (e.g. Bull, 1938; Gross, 1957; Baggerman, 1960; Jansson, 1962; McLusky, 1970; Davenport, 1972a; Bettison and Davenport, 1976). However, there is insufficient information on the form of short term salinity changes encountered by such animals in nature because their behaviour may allow them to avoid environmental fluctuations partially or completely. In consequence, laboratory simulation studies have largely been restricted to studies of planktonic animals or of sessile or slow moving benthic organisms.

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 191

    Studies on planktonic animals have been mainly concerned with oyster larvae (Crassostrea spp.) living in estuaries. There has long been controversy about the means by which the larvae of estuarine animals maintain their upstream position in estuaries prior to settlement. Three stances have been taken by various researchers. First, some authors believe that oyster larvae are simply transported by currents, and exhibit no differential vertical position with tidal stage (Loosanoff, 1949; Andrew, 1954; Korringa, 1952). Second, Pritchard (1953) stated that in a two layered estuarine situation, oyster larvae would only have to maintain a benthic position to stay in high salinity water and move upstream. Finally, there are those workers who have suggested that oyster larvae can detect increased salinity levels and become more active in response to them, thus leaving the bottom and being swept upstream on the flood tide (Nelson, 1912; Haskin, 1964; Wood and Hargis, 1971). The work of Hidu and Haskin (1978) appears to be the latest in this sequence. These investigators observed larvae swimming in a salinity cell (Haskin, 1964) supplied with water rising in salinity a t about 0*5%,/h (somewhat slow in most real estuarine situations). The results obtained were rather confusing, particularly as considerable differences in swimming speed between small and large larvae were demonstrated. Reference to the study is included because Hidu and Haskin suggest that the trials should be extended to large scale experimental water columns which could simulate estuarine water column conditions and in which in addition to temperature and salinity, pressure and quantitative aspects of light could be controlled-an ambitious and expensive aim which would require more information about short term changes in estuarine conditions than is available at present!

    Cawthorne and Davenport (1980) working on the larvae of the barnacles Elminius modestus and Balanus balanoides approached the problem of the vertical position of estuarine planktonic organisms from a different viewpoint. Unlike oyster larvae, newly released barnacle nauplii need to be carried downstream towards the sea to survive. Also they cannot isolate themselves fram low salinities by closing up and descending to the bottom, as may bivalve larvae. It seemed likely that nauplii might react to falling salinities by ceasing to swim and subsequently sinking into higher salinity water down in the water column. Once in high salinity water their survival would be assured as they would tend to remain within it. For the settlement cyprid stage in estuaries the situation is somewhat different. Cyprids can isolate themselves from deleterious salinities by closure of the

  • 192 J . DAVESPOHT

    carapace valves and would benefit by sinking in low salinity water so that they were not carried too far upstream before settling. Cawthorne and Davenport, using linear falling salinity regimes of realistic rates of salinity fall derived from the field study of Cawthorne (1979a), obtained data which support these hypotheses (see Table X). Larvae of the generally more euryhaline E. modestus kept swimming in rather lower salinities than those of B. balanoides. This difference in response is less marked between the cyprids of the two species. Interestingly, the salinities which prevent swimming and result in the nauplius sinking are quite close to the lethal limits of both species as predicted from the sinusoidal salinity cycle studies of Cawthorne (1978); cessation of movement occurs a t salinities much lower than those causing mortality in steady-state studies.

    Investigations into the behaviour of sessile organisms exposed to simulations of estuarine salinity regimes started with a study of the responses of barnacles by Davenport (1976). A novel method of continuously recording activity in barnacles, derived from the impedance pneumograph technique of Trueman (1967), was used (see Fig. 28) to monitor opercular valve and cirral movements in barnacles exposed to sinusoidal, near tidal salinity regimes fluctuating between full sea water and 20% sea water (33.4%, and 6.7%,). The proportion of time that the animals were active in each hour was noted, together with the limiting seawater concentrations required to induce the cessation of activity or permit its recovery. Such inactivity in barnacles at low salinities has been described

    TABLE x. RESPONSES OF BARSAVLE LARVAE TO FALLIN; SALISITIEh ( MOIHPIED FROM CAWTHOKNE AND DAVENPOI~T, 1980). MEAN SALIKITIES (WITH 95% CONFIDENCE IST~CRVALS) COKRESPOSDINU TO CESSATION OF SWIMMING IN 50% OP LARVAE, AT RATES OF SALINITY FALL

    ~h SHOWN

    A. Elminius modestus Stage Nauplii (stage I ) Cyprids

    Rate of change 16.75 4.81 16.75 .8 1

    Critical salinity

    B. Balanus balanoides

    ( W h ) 8.8 f 0.9 6.6 f 0.3 9.0 k 1.3 8.3 f 0.9

    Stage Nauplii (stage I ) Cyprids

    Rate of change 16.75 4.81 16.75 4.8 1

    Critical salinity 12.3f1.0 9 3 k 1 . 0 109+1.0 9.1k1.1 (YJh)

  • ENVIRONMENTAL SIMlTLATION EXPEKIMESW 193

    Shielded cable

    Minutes B

    +it--r-, r I 1 +t -$&a,-.-*. Ealanus rrnprovrsus-Conwy h-h Ealanur improvisur-Baltic

    Balanus crenatus

    -W-YcY-IC-jjJw( Elminius rnodestur

    Ealanur balanus

    1~~~~~~~~~~~~~~~~~~~ / Eaianus harnerr

    w-j Balanus balanoides

    F I G . 28. (A) Arrangement for activity recording. (B) Examples of activity records. Two sets of records for B. improuisus are displayed to demonstrate that the recording technique is equally applicable to small Baltic specimens (2-5 mm basal diameter) and large Conwy animals (15-20 mm basal diameter). From Davenport (1976).

    somewhat confusingly as salt sleep by Barnes and Barnes (1958). Examples of activity patterns are shown in Fig. 29; critical seawater concentrations for several littoral/estuarine balanomorphs appear in Table XI. From this table it may be seen that the responses of Balanus crenatus Bruguikre, Balanus balanoides and Elminius modestus to salinity fluctuations are very similar, despite the lower shore distribution of B. crenatus. This last species appears to be barred from higher placement on the shore because of its inablity to close its opercular valves in response to aerial emersion; certainly i t is not because of sensitivity to low salinity. It is also noteworthy that the responses are remarkably symmetrical, with activity stopping in falling salinities at about 20% and being resumed at much the same seawater concentration when the external salinity rises again.

    Symmetry of such responses to salinity appears to characterize sessile animals which are well adapted to pronounced salinity fluctuations. Thus, intertidal barnacles (Davenport, 1976), bivalves (Shumway, 1977a; Shumway and Youngson, 1979; Djangmah et al.,

  • 100 100

    W L

    c 0

    ? c 0

    50 50 :: 8 v) E

    c

    8

    0 00

    100

    L - W 0

    50

    8

    00

    0 12 Hours

    24

    FIG. 29. Activity in Conwy estuary barnacles. (a) Balanus crenalus, (b) Elminius modestus, (c) Balanus improvisus, (d) Balanus improvisus in more extreme salinity regime. Dotted line =presents salinity regime; other symbols represent mean values with 95% confidence intervals. From Davenport (1976).

  • ENVIRONMENTAL SIMULATION EXPERIMEBTS 195

    TABLE X I . CRITICAL SALINITY LEVELS FOR INTERTIDAL BALANOMORPH BARNACLES EXPOSED TO A SINIJSOIDAL SALINITY REGIME FLUCTUATING BETWEEN 3340,b A N D

    6.7% (SIMPLIFIED AND MODIFIED FROM DATA OF DAVENPOHT, 1976)

    Mean salinities ( X ) (a) Inducing activity (b) Inducing activity

    Species cessation onset

    A From Menai Strait Balanus crenatus 23.3 Balanus balanoides 23.6 Elminius modestus 22.0

    21.7 23.3 19.5

    B From Conwy estuary Balanus crenatus 1 9 1 19.4 Elminius modestus 204 24.4 Balanus improvisus No Activity Cessation

    1979) and the lugworm Arenicola marina (see Shumway and Davenport, 1977) all show symmetrical activity responses (see Table XII). On the other hand, sublittoral or poorly adapted lower shore forms, such as offshore barnacles (Davenport, 1976), scallops (Bettison, unpublished data), sea anemones and sea squirts (Shumway, 1978a, b), and hermit crabs (Davenport et al., 1980) all exhibit asymmetrical responses. In these activity ceases at salinities similar to, or even lower than, those inducing inactivity in better adapted forms; activity is not not resumed until much higher salinities have been attained.

    The source of the asymmetry appears to vary from species to species. In specimens of the hermit crab Pagurus bernhardus (L.), which do not resume activity after exposure to low salinities until they have been in full sea water for some time (Davenport et al., 1980), the probable reason is that their sense organs are isolated from the environment when the crabs retreat into their shells in response to lowered salinity (Shumway, 1978~) and register a rise in external salinity only after enough salt has diffused into the retained water within the shell (Davenport et al., 1980). On the other hand, forms such as the deep water balanomorph Balanus hameri appear to be rendered comatose by exposure to low salinities 2nd are then unable to resume activity until a period spent in high salinity water has revived them (Davenport, 1976).

    From Table X I and Fig. 29 it may also be seen that the salinity reactions of the barnacle Balanus improvisus Darwin are different from those of other intertidal barnacles. In a regime fluctuating between full sea water and 20% sea water specimens do not cease

  • 196 J. DAVENPORT

    activity. Even when exposed to fluctuations between full sea water and pure fresh water they only close their opercular plates and stop cirral activity at salinities below S%, (see Table XII). This result agrees quite well with the laboratory experimental results of Foster (1970). However, Foster also suggested that estuarine (Tamar) B. improvisus closed its opercular plates at about la%, in nature. This suggestion arose from blood osmolarity readings taken from freshly collected animals. He presented no direct evidence to support this, and the recent study of Cawthorne (1979b) has since confirmed that B. improvisus does not become inactive at salinities above 10%. Fyhn (1976) presented data which suggests that the species, unlike other balanomorphs, is capable of osmoregulation. This would help to explain the unusual degree of euryhalinity which allows B. improvisus to penetrate far into the Baltic Sea where other barnacles cannot survive.

    TABLE XII. ACOMPARISON OFTHE RESPONSESTO SALINITY OFBalanuScrenatus FROM DIFFERENT SOURCES EXPOSED FOR 24 H TO SINUSOIDAL SALINITY REQIMES (12 H WAVELENGTH) FLUCTUATING BETWEEN 33.4 AND 6.7ym (CALCTJLATED FROM DATA OF

    DAVENPORT, 1976)

    1st activity 1st activity 2nd activity 2nd activity cessation onset cessation onset

    Source salinity salinity salinity salinity

    1 . Conwy 19.6k 1.3 191 f3 .5 18%+ 1.4 19.6k3.6

    2. Conwy estuary 212f2.1 21.2k 1.8 20.6+ 1.8 232k 1.0

    estuary

    but held for 1 month in full S.W. (33-4%0)

    3. Menai Strait 244f2-6 21.05 1-7 2 2 2 A 1.5 22.4k 1.3

    Values are mean salinities with 95% confidence intervals.

    Davenport (1976) also showed that the salinity responses of barnacles were susceptible to alteration by non-genetic adaptation (i.e. acclimatization-Kinne, 1964a). This phenomenon is illustrated in Table XI11 for B. crenatus. From these data it may be seen that the Conwy estuary barnacles cease and start activity at lower salinities than B. crenatus from the Menai Strait, where barnacles are normally exposed to full seawater conditions. However, Conwy B. crenatus held for 1 month in full sea water could not be distinguished in their responses from Menai Strait specimens, so had clearly adapted.

  • ENVIRONMENTAL SIMULATION EXPERIMESTS 197

    TAHLE XIII. CKITI~AI, SALINITIES FOR BI.:HAVIOURAL RESPONSEX OF WELL h A P 1 ' R i ) INTERTIDAL ORGANISM^ (TO NEAREST ym) IN SIML-LATEI) TIDAL CY('LES

    Activity cessation Activity onset Species salinity salinity LSource of data

    Barnacles Balanus crenatus 23 22 Davenport (1976) Balanus balanoides 24 23 Elminius modestus 22 19 Balanus improvisus 8 8

    Bivalves Mytilus edulis 14 12 Rhuinway (1977a) Crassostrea gigas 20 13 Modiolus demissus 21 20 Shumway and

    Youngson (1979)

    Anadara senilis 15 15 Djangmah rt al. (1979)

    Polychaetes Arenicola marina" 19 19 Shumway and

    Davenport (1978)

    "These values are for water overlying a substrate characterized by high interstitial salinities in which the lugworms were burrowed.

    Much of the interest in the study of Davenport (1976) lay in comparisons with previous steady-state work on intertidal barnacles by Foster (1970). Foster made most of his observations after barnacles had been held in media of constant concentration for 24 h. For Elminius modestus he reported that activity was present down to 50% sea water (i.e. 17%,) in Menai Strait specimens, and 42% sea water (14%,) in Conwy animals. Apart from the fact that his technique gave no indication of salinities corresponding to activity resumption in rising salinities, these concentrations are significantly lower than the values reported by Davenport (1976) (see Table XI). It seems likely that Foster's animals were active in lower concentrations partly because they may have had longer to adapt- though the results of Cawthorne (197913) largely deny this possibility-but mainly because respiratory needs tended to override the closure response to lowered salinity when such seawater concentrations were held for as long as 24 h. Foster also found that Elminius died after 30 h continuous immersion in 25% sea water (8*5%,), but in both laboratory simulation experiments (Davenport, 1976) and field studies (Cawthorne, 1979a), it has been established

  • 198 J . DAVENPORT

    that the species can and does survive regular exposure to much lower salinities.

    Finally, Davenport (1976) collected some data which suggested that the salinities including closure in intertidal barnacles might be affected by rate of change in external salinity prior to closure. Cawthorne (1979b) used linear falling salinity regimes of various rates of change to clarify this point. He established that in B. crenatus, B. improvisus and E . modestus the seawater concentration corresponding to shell closure was independent of the rate of fall in salinity except at unphysiologically high rates of salinity change. In contrast B. balanoides did show a sensitivity to rate of salinity change. Animals from the Menai Strait exposed to a 48 h duration drop from 33.5 + O X , ceased activity at 24*9%,; with increasing rate of salinity change this value rose to a peak of 27.6%, for a salinity drop over 7.5 min. Corresponding figures for Conwy specimens were 23.6 and 26-3%, respectively. Interestingly, Cawthorne ( 1979a) showed that retained mantle fluid salinities in B. balanoides adults exposed by the falling tide in the Conwy estuary were about 26%,-again indicating that simulation studies give a realistic picture of responses in habitats characterized by short term fluctuations in physico- chemical conditions.

    Comparable data concerning the salinities which induce shell valve closure in bivalve molluscs have been collected for several species (see Table XII), but the most detailed studies have been carried out on the common mussel, Mytilus edulis, a prominent member of the fauna of estuaries. Milne (1940), working on mussels from the Aberdeenshire Dee, showed that the salinity of the water retained within the mantle cavity of mussels exposed to pronounced tidal fluctuations in salinity never fell below 21%. He also noted that the retained mantle fluid salinity was rather higher in areas characterized by severe salinity changes than in places where the changes were less extreme. Similarly, Shumway (1977a) found that the mantle fluid osmolarity of mussels which had closed in response to an abrupt salinity profile fluctuating between 33*5%, and O X , was significantly higher than the corresponding value found for mussels exposed to a regime which changed sinusoidally between 33.~5%~ and fresh water.

    Together, these observations suggested that the adduction response of Mytilus to declining salinities might be at least partially dependent upon rate of change of salinity, as it is in the barnacle B. balanoides. Davenport (1979a) found that this was indeed the case, but, as is evident from Fig. 30, there was no simple relationship

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 199

    A

    1 I I 1

    0 1 I I I 1 0.1 1 .o 10 100 lo00

    Rate of fall in medium salinity (%o/h)

    FIG. 30. Mytilus edulis. (a) The effect of rate of change in external salinity, prior to shell valve closure, on the mantle fluid salinity maintained after closure. (b) The effect of rate of changes in salinity on the external salinity inducing complete shell valve adduction. Symbols represent mean values with 95% confidence intervals (for six animals). From Davenport (1979a).

    between external salinity at the time of shell valve closure and the salinity of the mantle fluid found inside after closure. Although the salinity corresponding to valve closure fell with increasing rate of external salinity change, retained mantle fluid salinities actually rose! This led to the discovery that the isolation of the mantle cavity of a mussel from the external environment is not produced simply by shell valve adduction, but results from a three-part sequence of reactions. First, it became clear from the data presented in Table XIV that the salinity of the retained mantle fluid is primarily determined by closure of the exhalant siphon; once this siphon is closed, effective irrigation of the mantle cavity ceases. A t this time lateral and frontal ciliary activity upon ctenidia stops or slows

  • 200 d . DAVENPORT

    Taur,~ XIV. SAIJSITY LEVKLS Assocumu RTI'H THE COMPONKNXS OY THE Isoixrrox RESPONSE: OF Mytilus edulia Exros~i) TO FAI.I.ING SAI.INITIW (FIWM DAVENPOKT, 1979a)

    A. Summary

    Salinity ?Ao) o j Salinity External salinity (x0) mantle fluid after

    regime (time (mean values with 95% conjdence intervals) complete closure (min) taken A t exhalant A t inhalant At complete (mean values with f r o m 33.5 to siphon siphon shell valve 95% conjdeme

    O%,, closure closure closure intervals)

    3.75 25.3 & 2.2 21.8 f 3.4 10.7 2.9 3 0 6 k 1.2 7.5 25.6 + 0 7 204 & 3.3 17.5 f 3.7 29.2 f 1.2 15 25.9 t 0.9 19.3 f 2.5 17.4f1.5 28.9 & 1.2 30 28.4 k 1.7 24.9 f 3.3 18.8 3% 31.0f09 60 24.8 k 1.3 23% f 1.9 20.2 f 3.7 29.7 f 1.6

    B. Example (335%, -+ Oo/, in 3 7 5 min)

    External salinity ( X ) A t exhalant A t inhalant At complete Salinity (%,) of

    siphon siphon shell valve mantle Puid after Animal no. closure closure closure complete closure

    1 26.0 25.5 8.5 31.5 2 23.5 19.5 140 295 3 22.0 19.0 7.0 2 9 0 4 27.5 23.0 12.5 31.5 5 2 6 5 18.5 12.5 30.5 6 2 6 0 25.5 9.5 31.5

    (Sleigh, 1962; Ajana, 1975; Davenport and Fletcher, 1978). When the external salinity has dropped further the inhalant siphon shuts; later still the shell valves close. Once the valves are firmly adducted, the mantle fluid salinity remains unchanged for many hours (see Fig. 31). Of course there had to be some advantage gained by Mytilus from this behaviour pattern which is quite prolonged under conditions of slowly changing salinity and therefore contrasts strongly with the immediate total closure of Mytilus to tactile stimuli. Since M . edulis can close its valves in seconds when touched, why does it close in piecemeal fashion over a period of many minutes when exposed to slowly falling salinities? The answer to this question is evident from the results presented in Fig. 32 and Table XV.

    Mantle fluid oxygen tensions were monitored while mussels were exposed either to abrupt drops in salinity (regime A) or to slowly

  • ENVIRONMENTAL SIMULATION EXPERIMENrS 20 1

    I 1 0 8 16

    Hours after closure

    20 1 ,

    FIG. 31. The salinity of the retained mantle cavity Auid of mussels (Myti lus edutis) exposed to a sudden fall in external salinity (335%, + O X , in 7.5 min) to a sustained freshwater level. Symbols represent mean values with 95% confidence intervals (for six animals). From Davenport (1979a).

    falling salinity (regime B). This was done by slow circulation of mantle cavity fluid through a low volume external circuit which included an oxygen electrode. In the abrupt salinity regime closure of siphons and valves occurred almost simultaneously, and the mantle fluid oxygen tension fell rapidly as the animals tissues took up oxygen. In the slowly falling salinity regimes the tripartite sequence of closure was prolonged; the mantle fluid oxygen tension fell more slowly and irregularly. It must be remembered that although sessile estuarine osmoconformers benefit osmotically by isolation from the environment when external salinities are low, they also incur penalties. Food may not be collected, nor gases or metabolites exchanged during the periods when isolation is effectively complete. In Mytilus once the exhalant siphon is closed, the animal may continue to gape for periods determined by the rate of external salinity change. During this period of gaping the mantle fluid salinity does not fall much (perhaps 1 -4%,) because of poor exchange with the exterior, but its oxygen tension takes much longer to fall than in animals which close suddenly. Several factors are probably responsible for the slower rate of oxygen depletion in partially isolated, gaping mussels. First, the mantle cavity of a gaping mussel is physically larger than that of a tightly closed one and hence contains more oxygen; this factor will operate until final shell valve closure. Second, diffusion of oxygen may take place through the inhalant siphon if open or across the exposed mantle edge if closed.

  • 202 J. DAVENPORT

    EIV E I V 1 50

    50

    E l V

    -Y

    E l V

    -+it

    E I V I I 1

    EIV E I V

    EIV

    j:'i E I V

    A B A B

    -- 0 l o 1

    Time (h)

    -7 0 1 0 1

    Time (h)

    FIG. 32. Mantle fluid oxygen tensions in six mussels exposed to sudden (a) and slow (b) falls in external salinity (bottom diagram). E corresponds to exhalant siphon closure, I to inhalant siphon closure and V to complete shell valve adduction. Drawings between traces show the size and shape of each of the six mussels studied (scale bar = 5 cm) and the positioning of the hypodermic needles used in the oxygen tension measurement circuit. The values superimposed a t the end of each oxygen tension trace correspond to the salinities of retained water samples taken a t the end of each experiment. From Davenport (1979a).

    Finally there may be small bulk movements of water between the interior and exterior because of small movements of mantle, valves, or foot. Supporting evidence for this last suggested factor lies in the irregular form of the oxygen traces derived from animals in the slow (B) salinity regime and displayed in Fig. 32. Davenport suggested that, at the Conwy estuary site studied by Cawthorne (1979a), this behaviour pattern would gain mussels living there an extra hour of available oxygen each day-a worthwhile bonus for animals which

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 203

    TABLE xv. THE EFFWT OF RATE OF FAIL I N EXTERNAL SALINITY U P O N THE DHIINE OF MANTLE FI,UID OXYGEN TENSION (PROM DAVENPORT, 1979a)

    Time interval (man) between closure of the exhalant siphon and mantle fiuid oxygen

    tension falling to 15 mmHg

    , , , Animal no. 7.5 min (33.54%,, S) 2 h (33&00/, S)

    1 29.5 45 2 19 49 3 18 33 4 255 47 5 32 69 6 24.5 47

    Mean 24.75 48.33 S.D. 5.56 11.64

    are deprived of an external oxygen source for up to 10 h per day because of aerial exposure.

    There are other implications from this study. Several physiol- ogists have relied upon data from propped open bivalves, in which the shell valves are held apart by glass tubes or plastic wedges (e.g. Pierce, 1971; Shumway, 1977a; Costa and Pritchard, 1978) implying that propped open animals are in full tissue contact with the environment. Costa and Pritchard (1978) even postulated that the mussel was a short term osmoregulator on the basis of such studies, despite the extensive earlier work which had shown Mytilus and other marine bivalves to be osmoconformers (e.g. Lange, 1963,1970; Lange and Mostad, 1967; Pierce, 1970, 1971; Pierce and Greenberg, 1972,1973). My results showed that a mussel could largely isolate the mantle space simply by closing the exhalant siphon without shell valve adduction. Hence the tissues of propped open bivalves are not in intimate contact with the external medium. This indication was confirmed in full in a further study (Davenport, 1979b) which demonstrated that M . edulis was an osmoconformer even in the short term.

    Thirty years ago Wells (1949b) postulated that parts of the complex burrowing behaviour of the lugworm Arenicola marina prevented the irrigation of the worms burrow with noxious water. Arenicola is moderately euryhaline, but cannot survive in water of less than loo&, constant salinity, and even that concentration only applies to the Baltic Sea populations; it is the classical example of an

  • 204 J. DAVENPORT

    osmoconforming polychaete (Schlieper, 1929). In estuaries the lugworm may be found on mud flats which are washed with virtually pure fresh water at some stages of the tide although the interstitial salinity almost certainly remains high and varies little (Kinne, 1971 ). Shumway and Davenport ( 1977) used simulation equipment to confirm Wells hypothesis for salinity responses. Lugworms were placed in seawater-laden sand in a vessel of the type shown in Fig. 33. Sea water flowed over the top of the sand from the variable salinity apparatus and the worms activity was monitored by a sensitive pressure transducer connected to a fine capillary tube thrust deep into the head shaft of the Arenicola burrow. When the animal had settled, sinusoidal or square wave salinity fluctuations between 32 and 9*6%, were programmed for the water overlying the burrow. In both types of regime the worms responded to low salinity by becoming quiescent at the bottom of the burrow until the salinity rose again. The promptness of the salinity response to abrupt decreases in salinity is evident from the traces shown in Fig. 34.

    T

    FIc:. 33. Sand-filled box with A . marina in burrow and glass capillary, T, positioned in anterior end of burrow. Arrows indicate direction of water flow. From Shumway and Davenport (1977).

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 205

    Results from worms exposed to sinusoidal regimes established that a salinity of about 20%, both induced inactivity in falling salinity regimes and triggered a resumption of irrigation in rising salinities. Periodically lugworms would test the water above the substrate by briefly pumping water (see Fig. 35). However, they quickly became inactive again if the salinity was still low. Presumably low salinity water drawn into the burrow during testing rapidly equilibrates with the higher concentration of the interstitial water of the substrate. As will be discussed in more detail in the next section, the control exerted over body fluid osmoconcentration as a result of this behaviour is quite remarkable and comparable with that attained by the closing responses of intertidal barnacles and bivalves.

    A few studies have been performed upon animals which are less likely to encounter regular salinity fluctuations. In three separate studies Shumway (1978a, b, c) studied specimens of the coelenterate Metridium senile (L.), the ascidian Ciona intestinalis L. and the hermit crab Pagurus bernhardus exposed to simulated tidal salinity cycles. For M . senile and C. intestinalis fluctuations between lOOyo and 30% sea water were used although some specimens of the former species did not survive this; P. bernhardus experiments were performed with regimes which did not drop below 50% sea water; more extreme regimes were lethal. It has to be pointed out that only one of these three species, C. intestinalis, is known to extend into estuarine areas (MacGinitie and MacGinitie, 1968). The other two are littoral rather than estuarine, and the anemone M . senile is confined to the lowest intertidal levels and below. It is difficult to envisage its ever being exposed to salinities as low as the lo%, minima employed by Shumway, and certainly not for 6 h at a time. Large P. bernhardus are found only in the sublittoral, while the littoral population consists of small crabs usually living in rockpools. Davenport (197213, c, d) showed that the species were moderately euryhaline, that the small intertidal specimens, unlike their large offshore relatives, were capable of volume regulation and that the soft abdomen of the hermit crabs played a part in resisting the effects of low salinity. However, a recent field and laboratory investigation by Davenport et al. (1980) suggests that the species rarely encounters salinities below 25%, and then only briefly; Davenports earlier studies and the investigation of Shumway (197th) almost certainly exposed the hermit crabs to far lower salinities than they ever meet in nature. The reason for this is simple; Shumway (1978~) stated that tide pools are subject to dilution by rain and terrestrial run off. While this is true of very shallow pools, perhaps up to 5cm in depth, and

  • 206 J. DAVENPORT

    100 % sea water

    \ A \

    \ U 1 min \

    B

    30% sea water

    FIG. 34. Changes in A . marina activity when exposed to a 30% seawater minimum abrupt salinity change, and the animals position in the U-tube after activity had eased . From Shumway and Davenport (1977).

    30 % sea water

    FIG. 35. (a) Trace recorded during sampling excursion of A . ma~ina exposed to decreased salinity and (b) the position of the worm in the glass U-tube a t the time of sampling. From Shumway and Davenport (1977).

    also of the surface layers of deeper ones, the bottom water of most littoral pools of the type inhabited by hermit crabs remains high and stable (Pyefinch, 1943; Ganning, 1971; Daniel and Boyden, 1975; Davenport et al., 1980).

    Given that there are some reservations about the severity of the regimes used by Shumway, her studies do extend our knowledge of the convergent evolution of the behavioural responses of benthic osmoconformers to salinity. Metridium reduced tissue contact with

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 207

    the external medium in low salinities by expelling water from the coelenteron, retracting the tentacles and finally contracting the whole body to become as small as possible (see Fig. 36). Ciona ceased its regular squirting activity and tightly closed its siphons to prevent entry of water for filtration whenever the external salinity fell to about 20%,; this left only the relatively small surface area of the tough, thick and leathery and probably impermeable outer skin of the sea squirt in contact with the external medium. Hermit crabs retreated into their shells when the seawater concentration fell by about 25%; Davenport et al. (1980) used linear salinity regimes to pinpoint the critical concentration for Pagurus at 20*5-22.5%,, a value unaffected by rate of salinity change prior to retreating. All three species showed pronounced asymmetry of response indicating their relatively poor level of adaptation to estuarine conditions.

    1 I 0 6 I2

    Hour.

    PIC:. 36. The effect of fluctuating salinity on activity in M . senile: all animals are drawn to the same scale. From Shumway (1978a).

    C. Reproduction

    The only work so far concerned with the effects of simulated tidal salinity regimes on reproductive processes is that by Cawthorne and Davenport (1980), which consisted of a study of larval release in the intertidal barnacles Balanus balanoides and Elminius modestus. Unlike cyclic thermal regimes which revealed differences between the two species (see Section VC), the salinity fluctuations elicited similar responses from both species. As might be expected &he bulk of larval release occurred at high salinities (e.g. Fig. 37) in the range of the adults activity. At low salinity, as during aerial emersion, the adult barnacles close their opercular plates. Consequently larvae which hatch within the adult mantle cavity during the period of isolation cannot be liberated until external conditions become favourable again and cirral activity is resumed. The results obtained from animals exposed to both square wave and sinusoidal regimes

  • 208 J . I)AVESPOHT

    PIC:. 37. Larval release from adult specimens of Balanus balanoides exposed to a sinusoidal salinity regime fluctuating between full sea water (hour 0,12 and 24) and fresh water (hour 6 and 18). From Cawthorne and Davenport (1980). Symbols represent mean values with 959 confidence intervals. Dashed line indicates salinity profile.

    indicated that a small number of larvae are released at salinities below those associated with activity in the adults. It has been reported (e.g. Davenport, 1976; Cawthorne, 1978) that the mantle cavity is not totally isolated under these conditions. Pneumostonie formation (i.e. parting of the soft lips of the opercular flaps to form a small hole) and testing (Crisp and Southward, 1961) offer opportunities for larval release in these species a t low salinities.

    D. Growth

    It seems likely that studies of growth during exposure to salinity cycles in sessile invertebrates such as bivalves and barnacles would be fruitful since the extremes of distribution of such organisms in estuaries are probably determined not simply by salinity tolerance. Growth rates may be equally important and will be determined by the proportion of time that the animals are able to collect food and exchange gases or metabolites. At the time of writing no published information is available although such studies are in progress on Mytilus (Gruffydd, personal communication) with the aid of the laser diffraction growth measurement technique of Stromgren ( 1975) which allows daily growth measurements to be performed on young mussels.

  • ENVIRONMENTAL SIMULATION EXPERIMEKTS 209

    E. Feeding

    Again little information is available at present although the papers of Davenport and Vahl (1979) and of Vahl and Davenport (1979) explore the impact of salinity fluctuations upon the feeding strategy of the intertidal teleost Blennius pholis L.; this work will be discussed in more detail in a later section. Also Bettison (in preparation) has shown that filtration rates in bivalves such as Mytilus and Crassostrea gigas Thunberg are enhanced after periods of shell valve closure at low salinity in simulated estuarine salinity cycles. Matthiessen (1960) had previously shown that feeding rate decreased with decreasing salinity in the bivalve M y a arenaria L. Obviously changing external and internal ionic composition in animals, particularly osmoconformers, exposed to fluctuating salinities are likely to affect physiological processes such as nerve conduction, ciliary activity and circulatory efficiency, which will themselves in turn influence feeding.

    F, Osmoticlionic responses

    1, Extracellular jluid composition

    Kinne (1964b) suggested that investigations into the effects of salinity as an ecological factor should include studies of the consequences of fluctuating patterns of salinity. Six years later Tucker (1970a) carried out the first laboratory study of tidal salinity fluctuation on the prosobranch gastropod Scutus breviculus. This species is normally marine but penetrates the Heathcote- Avon estuary in New Zealand to a limited extent. Tuckers preliminary field salinity measurements showed that the environmental salinity of S. breviculus fluctuates only between 34 and 29%, and values below 34% persisted only for about 1 h during each tidal cycle. Her laboratory simulations reflected these conditions and consisted of a sequence of 1 h at 34%,, 30 min falling to 29%,, 30 min at 29%,, 30 min rising to 34%,, followed by 2 h at 34%, (see Fig. 38). Tucker was interested in the osmotic and ionic responses of the gastropod. First she showed that S. breviculus was an osmoconformer, becoming isosmotic over the salinity range of 25.5-34%, in about 8 h. Using her estuarine simulation she showed that the haemolymph concentration changes were damped by comparison with those of the external medium (see Fig. 38). Thus, whereas the salinity of the external

  • 210

    e 2 -

    84

    J. DAVENPORT

    1 : :---;Sea water concentration

    - I l l 1 1 1 I I I I I

    medium fell by 15% during the simulated salinity cycle, the prosobranch's haemolymph concentration fell only by about 8%. Such damping was first remarked upon in polychaete worms by Wells and Ledingham (1940). Also there was a time lag or retardation (hysteresis) of haemolymph osmotic changes compared with the medium concentration changes. Damping and retardation are presumably caused because osmotic uptake of water and outward diffusion of salt through the integument and other membranes takes time. From dimensional considerations damping and hysteresis will tend to be more marked in larger animals. Since Tucker's study an excellent mathematical treatment of the damping and time lag effects of tidal salinity cycles has been written by Spaargaren (1974). Obviously damping and time lags are responsible in large measure for the enhanced survival of many animals (except perhaps small larval forms) exposed to fluctuating rather than steady salinities; if the body fluids do not have time to equilibrate fully with the extremes of external salinity fluctuations, then the animal concerned will survive conditions which would be lethal if sustained. Tucker also measured the haemolymph concentrations of various cations; her results are displayed in Fig. 39. From previous work it would seem likely that hydrated sodium and potassium ions would pass through membranes more readily than larger hydrated calcium and magnesium ions (e.g. Dakin and Edmonds, 1931; Webb, 1940; Conway, 1956, 1960). Koizumi (1935) showed that rates of cationic penetration through the skin of a holothurian Caudina chilensis (Gould) could be arranged in the following expected sequence:

    K + > N a + > C a 2 + > M g 2 + .

  • ENVIRONMENTAL SIMIJLATION EXPERIMENTS 21 1

    x-x Sodium ---- Potassium

    I I I I I I I I I I I I 0 I 2 3 4 5

    Time, hours

    PIC:. 39. Changes in the concentrations of cations in the blood of animals subjected to changing seawater concentration simulating environmental conditions. (Each curve mean of values for 22 animals). From Tucker (1970a).

    From the results displayed in Fig. 39 and from some additional direct transfer experiments Tucker established that the sequence for Scutus was as follows:

    Ca2+ > K + > Na+ > Mg2+. In other words, the rates of movement of potassium, sodium and magnesium were as expected if their rate of permeation through the integument was determined by the size of the hydrated ion. However the loss rate of calcium in lowered salinities was much greater than expected. Tucker could only offer various speculations to explain this discrepancy. Enhanced membrane binding of haemolymph calcium at low salinities seems to be the most plausible of her suggestions. Similar unexpected and unexplained anomalies about the behaviour of calcium have been reported in subsequent studies on other animals. In a later related study Tucker (1970b) showed that the nerve conduction velocity in Scutus was reduced during periods of low salinity exposure; changes in ionic concentration and/or ratios, particularly of the divalent cations were probably responsible for this phenomenon.

    Since Tucker's work several similar studies have been performed on various invertebrates and fish, usually employing rather more generally applicable salinity regimes. For convenience these studies may be subdivided into three categories: (a) those concerned with osmoconformers, (b) those investigating osmoconformers which possess behavioural responses to salinity and (c) those concerned with osmoregulators.

  • 212 J . DAVENPORT

    (a) Osmoconformers. Stickle and Ahokas (1974) monitored the perivisceral fluid composition of three species of Pacific echinoderms, Pisaster ochraceus (Brandt), Cucumaria miniata (Brandt) and Strongytocentrotus drobachiensis (Muller) exposed to simulated estuarine conditions. Binyon (1966) had stated that echinoderms were an exclusively marine stenohaline group. A few asteroid starfish do occur in stable low salinity areas such as the Baltic and Black Seas, but these they have penetrated over long periods of geological time and so their essential intolerance of salinity change remains. However, Loosanoff (1945b) showed that the starfish Asterias forbesi (Desor) tolerated very dilute seawater for brief periods. Armed with this information and some field data which strongly suggested that the three echinoderms under investigation did encounter pronounced salinity fluctuations on the Alaska coast, Stickle and Ahokas were able to justify their use of simulated tidal salinity fluctuations between 30%, and lo%,. As may be seen from Fig. 40 the perivisceral fluid concentration changes were much damped by comparison with those of the external medium; environmental osmolarities fluctu- ated between 900 and 300 mOsmoles kg- while the minimum body fluid concentration in all three species approximated to 650 mOsmoles/kg. Shifts in concentrations of chloride, sodium, potassium and magnesium tended to follow the form of osmotic changes, but calcium levels in the perivisceral fluid exhibited a pattern of change very different from the other ions. In P. ochraceus and S. drobachiensis the calcium levels declined with lowered salinity, but more rapidly than would be expected from the size of the hydrated calcium ion (see Table XVI). However, calcium levels did not recover when the salinity rose again. Similar findings were reported for several bivalves by Shumway (1977a). In contrast, the calcium concentrations of C . miniata hardly changed at all until salinities started to rise after the period of low salinity exposure when calcium levels actually rose above the initial values. No explanation was offered for these phenomena. Stickle and Ahokas apparently did not appreciate that the sequences of ionic loss rates in falling salinities (estimated from their data and shown in Table XVI) were, in all three species, different from those expected from a simple consideration of hydrated ion size. In a later study on molluscs, Stickle and Ahokas (1975) remarked on deviations from the expected sequences in the animals studied. Such interspecific differences merit further study-clearly losses of cations are not determined solely by the concentration gradient between the extracellular fluid concen- tration and the exterior together with the porosity/permeability of

  • ENVIRONMENTAL SIMCLATION EX PEKIMENTS

    900

    800

    I 700-

    p 600- - 0 - .I

    E 500- - L g 400- D

    300-

    200

    100

    213

    ( 0 1 -

    -

    - -

    1 1 1 1 1 1 1 1 1 1 1 1 1

    95

    90 g

    85 = 0 0 n

    80 E ::

    75 2 L

    70

    0 1 2 3 4 5 6 7 8 9 1 0 1 1 12 Hour

    FIG. 40. (a) Osmolarity of the ambient sea water of P. ochracew (open circles), S. drobaehiensis (open squares) and C. miniata (closed circles). (b) Perivisceral fluid (above) and yo body water (below) values for all three species (symbols as in (a)). Vertical bars represent 95% confidence intervals. Modified from Stickle and Ahokas (1974).

    the integument. Since the work of Tucker (1970a, b) and of Stickle and Ahokas

    (1974) three very similar studies have been performed. Stickle and Ahokas ( 1975) studied amphineuran and prosobranch molluscs, Stickle and Howey (1975) investigated an oyster drill Thais haemastoma (L.), while Shumway (1977b) investigated four more

  • 214 J. DAVENPORT

    TAME XVI. SEQUENCE O F CATIONIC LOSS RATES IN THREE SPk:.:('IES OY ECHISO1)ELLMS (ESTIMATED FROM THE FIGURES OF STICKLE ANI) AHOKAS, 1974)

    Species Loss rate sequence

    Piaaster ochraceus Cucumaria miniata Strongylocentrotus

    drobachiensis

    'Ca2+ concentrations in the haemolymph of C. miniata did not decrease in falling salinities and actually increased in the rising salinity area of the salinity regime.

    species of echinoderms, all asteroid starfish. Damping of changes in body fluid osmotic and ionic concentrations in comparison with external salinity fluctuations was demonstrated in all of these animals, but apart from various interspecific differences little novel information was gleaned. The study by Stickle and Howey (1975) showed predictably that the body fluid concentrations of the oyster drill T . huemastoma exposed to diurnal 24h wavelength salinity fluctuations between 30%, and lo%, fell lower than when exposed to a 12 h wavelength cycle of the same amplitude. They also found that the time lag between external and internal osmotic and ionic changes was affected by the composition of the fresh water used as a dilutant in their experiments, presumably because of the influence of calcium on permeabilities.

    (b) Osmoconformers with behavioural reactions to salinity. The first, and so far the most comprehensive of osmotic/ionic studies upon osomoconformers which exhibit behavioural osmotic control, is that of Shumway (1977a). She worked upon six bivalve species; two sublittoral offshore forms, the queen scallop Chlamys opercularis L. and the horse mussel Modiolus modiolus L.; two were species with both littoral and estuarine distributions, the mussel Mytilus edulis and the oyster Crassostrea gigas while the last two species, Scrobicularia plana (da Costa) and the clam M y a arenaria were both characteristic of brackish water. Shumway collected a wealth of data, too numerous to present here in full, some of which are summarized in Figs 41, 42, 43 and 44. Throughout, her assumption was that these bivalves were osmoconformers. From a large body of work reviewed by Potts and Parry (1964) and Prosser (1973) this would seem entirely reasonable; the only marine bivalve known unequivocably to be an osmoregulator is the low salinity brackish water clam Rangia cuneata (Gray) (see Bedford and Anderson, 1972). The data for

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 215

    intertidal and estuarine bivalves demonstrate how effective be- havioural osmotic control can be in protecting osmoconformers. Whereas the sublittoral C. opercularis, which cannot exclude the external medium since it cannot sustain shell valve closure (Brand and Roberts, 1973), exhibits an almost perfect osmoconformer response (see Fig. 41), M . edulis closes its shell valves completely to restrict fluctuations in the concentration of the haemolymph to half the amplitude of those of the external environment (see Fig. 42). Mya arenaria, a clam particularly well adapted to brackish water habitats

    Hours H o u r s

    Hours Hours

    Hours Hours

    H o u r s H o u r s

    FIG. 41. Chlamys opercularis. Changes in haemolymph osmolality, and Na+, Mg' and Caz+ concentrations during exposure to (a) 50% seawater minimum sinusoidal salinity regime and (b) 50% seawater minimum square-wave salinity regime. Stippled areas represent changes in external medium. Each point is mean of three scallops. Error bars represent 95% confidence limits. From Shumway (1977a).

  • 216

    55 100

    L aJ 0 -

    65 v)

    .-"

    30

    12 24 Hours Hours

    10 100 100

    L W 0 L c

    gz

    d o s 65g 56 65

    .$ 30 30

    24 2

    12 24 0 Hours Hours

    Hours Hours

    Fu:. 42. Mytilus edulis. Changes in haemolymph (circles) and mantle fluid (squares) osmolality and Na', Mg2+ and Ca2+ concentrations during exposure to (a) 30% and (b) 0% seawater minimum sinusoidal salinity regime. Stippled areas represent changes in external medium. Arrows indicate points of shell-valve closure ( J. ) and opening ( f ) (from Bettison, unpublished). Each point is mean of three mussels. Error bars represent 95% confidence limits. From Shumway (1977a).

  • 217

    Hours Hours

    Hours Hours

    Hours Hours

    FIG. 43. Myu arenaria. Changes in haemolymph osmolality and Na', Mgz+ and Ca2+ concentrations of 1 0 0 ~ o seawater-acclimated burrowed (open circles) and non-burrowed (filled circles) clams exposed to (a) 30% seawater minimum sinusoidal salinity regime and (b) 30% seawater minimum square-wave salinity regime. Stippled area represents rhanges in external medium. Each point is mean ofthree clams. Error bars represent 95% confidence limits. From Shumway (1977a).

  • 218 .J. DAVENPORT

    Hours

    Hours

    5 E 0 V

    Hours

    Hours

    Hours

    Hours Hours

    FIG. 44. Modiolus modiolus. Changes in haemolymph (circles) and mantle fluid (squares) osmolality and Na', Mgz+ and Ca2+ concentrations during exposure to (a) 50% seawater minimum sinusoidal salinity regime and (b) 50% seawater minimum square-wave salinity regime. Stippled areas represent changes in external medium. Arrows indicate points of shell-valve closure ( 4 ) and opening ( t ). Each point is mean of three bivalves. Error ban represent 95'7' confidence limits. From Shumway (1977a).

    cannot close its shell valves nor withdraw its well developed siphons. However the edges of the mantle, like the siphons are covered by periostracum and are completely united except for a small pedal aperture. In conjunction with muscular sphincters at the tip of each siphon which control pumping (Chapman and Newell, 1956), these structural adaptations appear to minimize the influence of the external medium as effectively as shell valve closure does in M . edulis. M y a arenaria lives deep in the substratum, but, as Shumway's

  • ENVIKONMEKTAL SIMULATION EXPERIMENTS 219

    data show, the burrowing habit does not contribute to osmotic control (see Fig. 43) and presumably has more importance as a protection against predators. The results for one species studied by Shumway were rather unexpected. As may be seen from Fig. 44, the horse mussel Modiolus modiolus exhibited quite effective behavioural osmotic control despite its large byssal aperture, and the general acceptance that its distribution is sublittoral. Previously Coleman and Trueman (1971) had shown that M . modiolus could not retain water within the mantle cavity during aerial emersion and Pierce (1970) using steady-state salinity experiments had established that the species could not survive below 80% sea water. Shumway suggested that her results might have some environmental signifi- cance since Coleman and Trueman reported that horse mussel beds were occasionally exposed on very low spring tides when they might be affected by rain water. However it seems likely that aerial emersion would simply allow water to seep out of the mantle cavity and salinity effects would be negligible. The observations (Davenport, 197913) on propped open Mytilus edulis appear to be relevant here. It seems likely that as long as horse mussels keep their inhalent and exhalent apertures closed and do not actively pump water through the mantle cavity, exchange of fluid between the mantle cavity and exterior will be poor, even though the byssal aperture prevents effective valve closure, just as wedging the shell valves of M . edulis apart does not ensure exposure of their tissues to external salinites. Only during aerial emersion will the patent byssal opening of Modiolus modiolus be disadvantageous. In support of these comments the author offers some further personal observ- ations. In northern Norway, horse mussels do occur intertidally, but only in rock pools, not where they may be aerially exposed. Some M . modiolus were observed in very shallow pools so high on the shore that they were exposed to strong melt water influence at low tide during the subarctic spring; the water surrounding the bivalves was virtually fresh. This suggests that Shumways observations are perfectly valid in the field for M . modiohs in some parts of its geographical range. In subsequent studies three more species have been studied under simulated estuarine conditions, the American oyster Crassostrea virginica (see Hand and Stickle, 1977), the West African blood clam Anadara senilis L. (see Djangmah et al., 1979) and the Atlantic ribbed mussel Modiolus demissus (see Shumway and Youngson, 1979). All three species are euryhaline and can close their shell valves most effectively; they showed similar responses to those of Mytilus edulis and Crassostrea gigas.

  • 220 J. DAVENPORT

    As already discussed in Section VI.B, Shumway and Davenport (1977) showed that the lugworm Arenicola marina became quiescent and stopped irrigating its burrow when the salinity of the water overlaying its substrate was low. From Fig. 45 the effectiveness of this behaviour may be appreciated. Worms held naked in water fluctuating between 32 and 9%, experienced great changes in

    rn-osmole 2oo r

    Na+ 500 r

    K l 2 r

    0- 0 12 24

    Ca2+ l 2 c

    Mg2 6o r

    30 r s o p

    0- 0 1 2 24

    Time (h)

    FIG. 45. The changes in coelomic fluid osmolality and Na, K + , Mg, Ca2+ and SO, concentrations of burrowed and non-burrowed A . marina exposed to a 30% seawater minimum sinusoidal salinity regime. Each point is a mean of five animals. Error bars at the 95% confidence level are smaller than the actual points. From Shumway and Davenport (1977).

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 22 1

    coelomic fluid osmotic and ionic composition as indeed is to be expected from the work of Schlieper (1929) who showed that the lugworm was an osmoconformer. In contrast, worms allowed to burrow in seawater-laden sand beneath the fluctuating salinity water showed no significant changes in coelomic fluid composition whatsoever.

    Finally Shumway (197th) monitored changes in haemolymph osmolarity in hermit crabs exposed to salinity fluctuations. Naked hermit crabs manifest an osmoconformer response similar to the queen scallop, and become isosmotic with low salinities during 12 h wavelength square wave 32 + IS%, salinity regimes. Specimens of Pagurus which had been allowed to keep shells, into which they retreated at low salinities, maintained significantly higher haemo- lymph concentrations than naked animals, thus demonstrating that some degree of protection is afforded by the shell. However, as Davenport et al. (1980) showed, this protection is somewhat marginal and cannot compete in effectiveness with the efficient structural/ behavioural mechanisms of more euryhaline species such as M . edulis and A . marina.

    (c) Osmoregulators. Few laboratory salinity fluct.uation simulation studies have been performed upon osmoregulators, although Spaargaren (1974) predicted from his equations that a combination of damping and osmoregulation would restrict internal osmotic changes in the shore crab Carcinus maenas (L.) to one third of the external fluctuations during a sinusoidal tidal salinity cycle. Findley and Stickle (1978) studied an even more euryhaline crab, Callinectes sapidus which was exposed to laboratory 24.8 h wavelength salinity cycles. These cycles were either of 20 + 10 + 20%,, 30 + 10 -+ 30X0 or 10 + 30 -+ lo%,. The results for haemolymph osmolarities are displayed in Fig. 46 and demonstrate that internal osmotic conditions are almost independent of external fluctuations in the blue crab. In none of the three regimes did the haemolymph osmolarity alter by as much as 100mOsmoles/kg despite external changes five to six times greater. Generally speaking ionic concentrations were equally stable as may be seen from the values displayed in Table XVII. Of particular interest are the haemolymph magnesium concentrations. It has been known for some time that there is a close relationship between activity and blood magnesium concentration in decapod crustacea. This was discovered by Robertson (1953, 1960) who attributed the effects to the interference of magnesium with neuromuscular transmission. The phenomenon has been further

  • 222

    900 800-

    700

    400

    300 200

    900

    =:: 2 800- 0 700- E 600- 8 = - 500- 5 400-

    .- - 0

    .-

    300 200

    900

    50 0 400

    2 0 0 300

    J. DAVENPORT

    20-10-20 Hemolymphw

    - L A , ; : : : w -

    &Ambient Water

    - 30-10-30

    v -

    - -

    10-30-10 -

    !!!;R, 0 2 4 6 8 10 12 14 16 18 202224 26 discussed by Lockwood (1962) and Potts and Parry (1964). High blood magnesium levels are associated with slow moving crustacea (e.g. Maia squinado Latreille) while low haemolymph magnesium concentrations characterize active crabs and prawns. In the nominal 30 -+ 10 -+ 30%, regime mean magnesium levels in the haemolymph of C. sapidus varied from 41.9 -+ 32.2 -+ 45.1 m eq./litre while those of the medium ranged from 92 + 32.3 -+ 91 m ey./litre. Thus, when the external magnesium levels fell by 65% the internal concent- rations only dropped by 23%. However, despite this damping, one would still predict from Robertsons observations that activity would be increased at low salinity; certainly Findley et al. (1978)

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 223

    T A B L E XVII. LEVELS OF IONS ( M EQ / LITRE ) IN S E A WATER (S.W.) AND HAEMULYMPH (HL) OF BLUE CRABS (Cullinectes supidus) EXPOSED TO 30 -+ 10 + 30% DIUHNAL

    SALINLTY REGIME (MODIFIED FROM FINDLEY AND STIC'KLE, 1978)

    Time (hours) Ion 0 4.17 8.33 12.42 16.58 20.75 24.83

    Na' S.W. 321 191 137 101 213 273 314

    C1- S.W. 360 234 163 106 246 308 355

    K + S.W. 108 5.4 3.4 3.2 6.4 9.0 10.6

    Mg2+ S.W. 92.0 603 37.5 32.3 722 87.1 91.0

    HL387514 361f8 34659 343+8 351f8 363f9 365516

    HL 339+12 338511 335510 330+8 323f8 314+12 304+18

    HL 11.2k0.5 1 0 0 f 3 8 . 2 5 0 4 7.450.4 81k0.4 9.9f0.4 l0.8f0.7

    HL 11.9f2.7 37.5k1.7 34.0k1.4 32.2f1.7 32.9f1.7 37.151.6 45.1f3.1

    Haemolymph values are means predicted from regression analysis with 95% confidence intervals.

    found that oxygen uptake was enhanced at the low salinity extreme of such tidal cycles, but no measurements of activity were performed and it should be pointed out that Robertson's work was performed at high constant salinity.

    The only other study performed upon an osmoregulator so far is that of Davenport and Vahl (1979) who worked upon the intertidal benthic teleost Blennius pholis. The blenny often lives in gullies on the shore fed by freshwater streams which produce marked salinity decreases associated with low tides. In a square wave tidal salinity regime fluctuating between 34%, and fresh water the plasma osmolality did not change significantly, but remained at about 380 mOsmoles/kg. House (1963) had earlier shown that the blenny's blood concentration did fall significantly when the fish was left in a salinity of lo%, for 48 h; however House also showed that Blennius differed from other euryhaline teleosts such as the eel Anguitla anguilla L. (see Keys, 1933) and the trout Salmo gairdnerii Rich (see Houston, 1957) in that it exhibits a very rapid physiological response to salinity changes, switching in less that 5min from pumping salts out across the gills in concentrated media to taking up actively ions from hypoosmotic solutions. This rapidity of response, which operates in both directions, means that passive loss of salts caused by exposure to fresh water will be partially offset by active salt uptake so that a decline in blood concentration will occur relatively slowly. In Davenport and Vahl's experiments i t was clear

  • 224 d DAVEKPORT

    that 6 h exposure to fresh water was insufficient to cause a significant decline in plasma osmoconcentration.

    More data for osmoregulators would obviously be desirable. However, part of the reason for the lack of study follows from the generally greater mobility of osmoregulators by comparison with osmoconformers; they do not need to take refuge during periods of low external salinity and their very mobility makes it difficult to be certain of the precise short term salinity fluctuations that they encounter in nature.

    2. Volume regulation

    A detailed discussion of all of the differences and relationships between ionic regulation, osmoregulation and volume regulation would be out of place in this review since these topics have been dealt with elsewhere (e.g. Florkin, 1962; Potts and Parry, 1964; Gilles, 1979). There are two types of volume regulation. First there is regulation of the volume of extracellular fluid; this is sometimes referred to as whole animal volume regulation since a great proportion of the total volume of many animals consists of extracellular fluid. Because it is usually difficult to measure the volume of extracellular fluid or the volume of a whole animal, i t is a normal experimental procedure to weigh animals repeatedly while they are exposed to salinity changes to assess volume regulatory capacity. In forms such as the euryhaline osmoregulating crab Carcinus maenus, changes in urine output in response to salinity stress are so rapid that weight changes are almost undetectable (Schwabe, 1933). In volume regulating osmoconformers such as Pagurus bernhardus initial volume ( = weight) changes of con- siderable magnitude may occur which can take many hours or even days to reverse (Davenport, 1972b). In osmoconformers which have weak or negligible volume regulatory capacity, volume changes induced by salinity stress are never fully reversed except by further salinity changes in the opposite direction. In salinity simulation studies certain problems have arisen. First, it is tempting to assume that an animal which does not change weight in a fluctuating salinity regime is a volume regulator, whereas an animal whose weight fluctuates is not. However, it must be remembered that the original definitions of volume regulators stemmed from direct transfer experiments. Thus if one takes an osmoconformer such as a starfish from full strength sea water and places i t immediately in a dilute medium, water will be taken up rapidly by osmosis while solutes

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 225

    diffuse outwards less quickly; consequently the animal will swell. If the animals integument is especially permeable to solutes, however, and the salinity changes of the environment relatively slow, then outward solute diffusion may be rapid enough to prevent the build up of substantial osmotic gradients and therefore prevent excessive swelling. This would appear to be the case for the three echinoderm species studied by Stickle and Ahokas (1974). Conversely, Shumway (1978~) noted substantial fluctuations in the volume of individual hermit crabs exposed to simulated tidal salinity regimes. Volume regulation in this species is a relatively slow process accomplished largely by changes in urine output which are not triggered until substantial weight changes have occurred. A second problem is experimental in origin. In several cases i t is technically difficult to weigh single animals repeatedly during exposure to a salinity regime. An alternative procedure is to expose many animals to the regime and withdraw some at intervals which are then weighed, freeze dried and reweighed to allow calculation of the proportion of the animals weights which consists of water. If the latter rises during the course of the experiment the animals overall volumes are assumed to have risen and vice versa. Particular difficulty arises with bivalve molluscs since they have to be cut out of their heavy non-living shells to be wet weighed and it is difficult to avoid significant haemolymph loss. If haemolymph is lost then the investigator will tend to be measuring tissue hydration rather than whole body hydration and will inadvertently be studying cellular volume regulation (see below)! To further complicate this picture some workers have unfortunately and inaccurately used the terms Yo body water and Yo tissue water interchangeably.

    Because of these problems it is difficult to do more than divide the animals so far studied into those which do exhibit significant total volume changes in fluctuating salinity regimes and those which do not (see Table XVIII). Obviously changing total volumes are generally undesirable, since they will interfere with various biological processes, primarily locomotion and respiration, but also diffusion generally.

    The second type of volume regulation occurs at the cellular level; it is commonly known as intracellular fluid isosmotic regulation (Florkin, 1962). The cells of multicellular animals, whether osmoconformers or osmoregulators, are isosmotic with the surround- ing body fluids (Conway and McCormack, 1953). If the body fluid concentration changes as a result of external influences, the cells will tend to swell or shrink because of osmotic gain or loss of water.

  • 226 J . DAVENPORT

    TABLE XVIII. SPECTIES SHOWING CONSTANT VOLUMES (A) OR V.4RIAKI.E VO1,I'MEh (B) IN FL~K~TUATIN(: SALINITY REGIMES (DATA FROM VAKIOL~S SOIWES)

    (A) Constant volume species Source of information

    1 . Echinoderms Pisaster ochraceus Cucumaria rniniata Strongyloeentrotus drobachiensis

    Arenicoh marina (burrowed)

    Thais larnellosa

    Stickle and Ahokas (1974) Stickle and Ahokas (1974) Stickle and Ahokas ( 1974)

    Shumway and Davenport (1977)

    Stickle and Ahokas (1975)

    2. Polychaetes

    3. Molluscs

    (B) Variable volume species

    1 . Echinoderms Asterias rubens Solaster papposus Henricia sanguinolenta Astropecten irregularis

    2. Polychaetes Arenicola marina (non-burrowed)

    3. Molluscs (gastropods) Scutus breviculus Thais haemastoma

    4. Molluscs (amphineurans) Mopalia mucosa Katherina tunicata

    5. Molluscs (bivalves) Crassostrea virginica

    6. Crustaceans Pagurus bernhardus

    Shumway (1977b) Shumway (197713) Shumway (1977b) Shumway (1977b)

    Shumway and Davenport (1977)

    Tucker (1970a) Stickle and Howey (1975)

    Stickle and Ahokas (1975) Stickle and Ahokas (1975)

    Hand and Stickle (1977)

    Shumwav (197%)

    Experiments with isolated cells have shown that both swelling and shrinking do occur, but are reversed in cells from euryhaline species by regulatory processes which are much more rapid in action when reducing swelling than in resisting shrinking (Gainer and Grundfest, 1968; Gerard and Gilles, 1971; Pierce, 1971; Gilles, 1975); such processes are not exhibited by cells of stenohaline species. Over the past twenty years it has been demonstrated that a variety of intracellular free amino acids and other amino compounds (e.g. glycine-betaine) are involved as osmotically active compounds in cellular volume regulation in both osmoconformers and osmoregu-

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 227

    lators (for reviews see Florkin and Schoffeniels, 1969; Schoffeniels and Gilles, 1970, 1972; Schoffeniels, 1976). In response to hypo- osmotic stress, which tends to cause tissue swelling, intracellular amino compound concentrations fall (whether by extrusion or incorporation into proteins) in order to reduce intracellular osmotic pressure; hyperosmotic stress has the reverse effect. In bivalve molluscs for example an almost linear relationship between tissue free amino acid levels (measured as ninhydrin positive substances- N.P.S.-levels) and environmental salinities has been recorded for the osmoconformers Mytilus edulis and Crassostrea virginica (Lange, 1963; Lynch and Wood, 1966). Many studies have been devoted to confirming the role of the intracellular free amino acid pool in intracellular isosmotic regulation in a variety of animals; equal effort has gone into evaluating the importance of individual amino acids and the mechanisms for regulating the size of the pool (for a brief review of the relevant literature see Livingstone et al., 1979).

    All of this work featured either direct transfer experiments or was performed on animals taken from a variety of natural habitats where salinity was constant (e.g. Lange, 1963); not until the work of Shumway (1977~) and Shumway et al. (1977) were measurements made of cellular volume regulation in animals exposed to simulated estuarine conditions. Shumway ( 1977c) measured tissue water levels in eight bivalve species (Chlamys opercularis, Modiolus modiolus, M y a arenaria, Scrobicularia plana, Mytilus edulis, Cerastoderrna edule L., Mercenaria mercenaria L. and Crassostrea gigas). Unfortunately many of her data were from propped open animals and perhaps ought to be reinterpreted given the results of the subsequent study by Davenport (1979b). However, if normal animals alone are referred to i t is clear that all species except the quahog Mercenaria mercenaria showed significant changes in tissue hydration during square wave or sinusoidal salinity cycles with hydration levels rising at low salinity and falling when salinities rose again (see Fig. 47). Propped open quahogs exhibited similar tissue water changes, as did normal specimens of the ribbed mussel Modiolus demissus Dillwyn in a later study (Shumway and Youngson, 1979). In none of these species did the hydration level return to a higher level than the initial full seawater control level when the animals were returned to full sea water after a period of exposure to low salinities; taken with the observed fluctuating in tissue water content this indicated that the bivalves cells were behaving like simple osmometers with no volume control, little solute loss and passive ebb and flow of water. However, for Mytilus edulis alone, Shumway performed further measurements

  • 228 J . DAVENPORT

    HOURS

    FIG. 47. Changes in tissue water content of normal ( 0 ) and wedged open ( 0 ) M. edulis exposed to gradual and abrupt salinity fluctuations. Each point is a mean of three animals. Error bars represent 95% confidence limits. From Shumway (1977~) .

    upon mussels which had been held in a fluctuating salinity regime for 1 week. After this period she found that the tissue water content of the mussels remained almost constant throughout the salinity cycles and showed none of the changes exhibited by animals during the first 24 h of exposure. The corresponding N.P.S. determinations per- formed by Shumway etal. (1977) and Shumway and Youngson (1979) were most interesting. With nine species in all investigated, and both N.P.S. (ninhydrin positive substances) and individual amino acid determinations performed upon all of them in several salinity regimes, a great deal of information was collected, but a few generalizations may be made. It is clear that tissue N.P.S. levels do not simply fall and rise with salinity as might be predicted from the hypothesis of Florkin and Schoffeniels (1965, 1969). In several cases, including M . edulis, falling salinity was associated with rising N.P.S. levels; Shumway et al. speculated that amino acid accumulation might result from anaerobic processes during shell valve closure, but unfortunately glycine, which formed a major part of the enlarged N.P.S. pool, is not produced during anaerobiosis in the mussel (De Zwaan et al., 1976). Specimens of M . edulis held for 1 week in a fluctuating salinity regime showed no significant N.P.S. concen-

  • ENVIRONMENTAL SIMlTLATION EXPERIMEXTS 229

    tration changes during the tidal cycle; N.P.S. values simply remained steady at a value roughly midway between the extremes of N.P.S. changes shown by mussels during the first 24 h of exposure to the same regime. Shumway et al. interpreted all of this information to mean that mussels and other bivalves living in estuaries did not continually build up and lose intracellular free amino acids, but eventually assumed a constant tissue hydration and N.P.S. level which ensured a minimum expenditure of energy and material by the animals. Livingstone et aE. (1979) extended this approach with specimens of M . edulis exposed to salinity regimes which did not stress the animals enough to induce shell valve closure. The N.P.S. response during the first few tidal cycles was again equivocal, but over 48 cycles the mean N.P.S. level averaged over each cycle fell significantly indicating that adaptation in the form of isosmotic intracellular regulation had taken place.

    In the only other simulation study involving tissue N.P.S. measurements Shumway and Davenport ( 1977) demonstrated that tissue N.P.S. concentrations in lugworms buried in sand did not change when the salinity of the overlying water fluctuated. On the other hand, naked specimens of Arenicola did show tissue N.P.S. changes in both sinusoidal and square wave salinity regimes; the patterns were not especially coherent although there was a general tendency for a fall in N.P.S. level as time elapsed. In any case these data probably have no relevance for lugworms in nature.

    Stickle and his co-workers (Stickle and Howey, 1975; Hand and Stickle, 1977; Findley and Stickle, 1978) have also carried out work on invertebrates which is relevant to cellular osmoregulation but have monitored only N.P.S. levels in haemolymph? not tissue concentrations. This approach seems to stem from the observations of Pierce and Greenberg (1973) who showed that the isolated hearts of Modiolus extruded free amino acids during exposure to low salinity. Thus a rise in haemolymph N.P.S. would indicate a loss of free amino acids from the intracellular pool in response to reduced extracellular fluid concentrations. Stickle and Howey (1975) found that haemo- lymph N.P.S. rose in oyster drills as surrounding salinities fell, and Hand and Stickle (1977) reported similar data for the oyster Crassostrea virginica. These results support the cellular volume regulation hypotheses of Florkin and Schoffeniels. However, Findley and Stickle (1978) also reported some haemolymph N.P.S. fluctu- ations in the blue crab Callinectes sapidus. Although these fluctuations were described as minor by Findley and Stickle, changes of as much as 100% occurred during a 2 0 4 10+20%,

  • 230 J. DAVENPORT

    salinity cycle, which seems very strange in a crab with such pronounced control over extracellular fluid concentration. However Findley and Stickles haemolymph N.P.S. values were far below those reported for the crab by other workers, possibly due to starvation, so the results may not have been applicable to normal animals. It should also be pointed out that relying solely upon haemolymph N.P.S. concentrations for information about cellular volume regulation is somewhat unsafe. It is by no means clear that amino acids are always extruded intact from the cells, particularly in crustaceans in which Florkin and Schoffeniels (1965, 1969) proposed that intracellular free amino acids were degraded into keto acids and ammonia.

    In summary it would appear that regulation of the intracellular amino acid pool is not effective in preventing cellular volume changes induced by tidal salinity fluctuations. However, in animals exposed to such fluctuations over long periods of time the size of the pool is altered to minimize volume changes.

    G. Oxygen consumption

    The cost in energy terms of existence in variable as opposed to stable habitats is obviously of interest to both physiologists and ecologists. As far as salinity fluctuations are concerned, the available evidence is confusing. Dehnel (1962) suggested that oxygen consumption would be enhanced at low salinity because of osmoregulatory (and volume regulatory?) work. However calcu- lations based on thermodynamic criteria by Potts and Parry (1964) suggest that osmoregulation of the extracellular fluids should cost very little. In any case the majority of intertidal and estuarine species are osmoconformers. No data appear to be available about the minimal cost of isosmotic intracellular osmoregulation. On the other hand, as discussed in earlier sections, fluctuating external salinities have profound effects on the behaviour and physiology of intertidal and estuarine organisms which are likely to alter respiration rates in a variety of ways independently of the cost of osmoregulatory work.

    Before the development of simulation equipment, studies of the effects of salinity on oxygen uptake were technically unsatisfactory because animals were physically disturbed whenever they were transferred from one salinity to another-a procedure bound to alter respiration at least temporarily. Independently Davenport (see Shurnway, 1978a) and Findley et al. (1978) developed through-flow respirometers which allowed continuous measurement of oxygen

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 23 1

    uptake during salinity fluctuations without otherwise disturbing the animals under investigation. A further refinement of such apparatus described by Vahl and Davenport (1979) allows feeding during exposure to experimental regimes.

    The first worker to use such equipment was Bettison (un- published data) who monitored oxygen uptake in several estuarine bivalve species. All showed zero oxygen uptake when isolated from deleterious external salinities by shell valve closure or siphon retraction. Usually oxygen uptake was apparently enhanced after such a period of isolation but some of this increase was probably an artifact caused by expulsion of deoxygenated water from the bivalves mantle cavity. Similar data was later collected by Shumway and Youngson (1979) for the Atlantic ribbed mussel Modiolus demissus.

    Shumway (1978a, b, c, 1979) also monitored oxygen consumption in a number of other animals which respond behaviourally to salinity fluctuations. These were the sea anemone Metridium senile, the sea squirt Ciona intestinalis, the hermit crab Pagurus bernhardus and a number (1 1 species) of gastropod molluscs. Except for P. bernhardus all behaved in similar fashion with low ( M . senile) or negligible (all other species) oxygen uptake occurring at low salinities when the animals reduced contact with the environment and ceased producing respiratory currents. Oxygen consumption results for P. bernhardus (see Shumway, 197th) are difficult to understand since no alterations in oxygen uptake were observed in salinity regimes (32 --+ 16 --+ 32%,) except for brief increases which occurred at 24%, and appeared to be associated with transient bursts of activity. In a later study Davenport et al. (1980) showed quite clearly that there was no oxygen uptake by hermit crabs which had withdrawn into their shells in response to low salinity. Obviously if the scaphognathites continue to direct a respiratory current through the branchial chambers water would necessarily be taken up as well as oxygen and the animal would gain no osmotic benefit from withdrawal into the shell. Since Shumway demonstrated osmotic benefit the continuation of oxygen uptake seems inconsistent.

    Oxygen uptake measurements have been performed by Findley et al. (1978) and by Davenport and Vahl(l979) on animals which do not isolate themselves at low salinity. Findley et al. (1978) investigated the osmoconforming predatory whelk Thais haemastoma and the osmoregulating crab Callinectes sapidus collected from the same habitat and exposed to both steady and fluctuating salinities. They showed dissimilar respiratory patterns; T . haemastoma acclimated to

  • 232 J . DAVENPORT

    steady salinities showed higher respiration rates at 30%, than at lo%, and a reduction in oxygen consumption during salinity fluctuations which deviated in either direction away from an acclimation salinity. In contrast, oxygen uptake by the blue crab was higher at constant, lo%, or 20%, than at 30%,, and during salinity fluctuations was always greatest at the lowest salinities. The results for C. sapidus can be interpreted in terms of heightened oxygen uptake which could be caused by increased activity at low salinity resulting from lowered haemolymph magnesium concentrations or from the cost of active inward transport of ions. Findley et al. suggested that the results for T . haemastoma followed from its incomplete volume regulation capacity. Presumably a general reduction in physiological efficiency resulted from either osmotic swelling or shrinkage, causing oxygen uptake to fall in response to salinity changes in either direction.

    Davenport and Vahl (1979) investigated the intertidal teleost Blennius pholis in square wave and sinusoidal salinity regimes fluctuating between 0 and 33-5%,. The results indicated heightened oxygen consumption at low and rising salinities with minimum uptake rates at high salinity. This again suggests a correlation between oxygen consumption and osmotic work, but the changes in oxygen consumption during salinity cycles were relatively small ( c . 25%). Kinne (196410) and Holliday (1971) have stressed the well known fact that the oxygen content of water depends upon salinity and Holliday stated that since oxygen uptake of teleost fish is to a large extent determined by the oxygen concentration of the surrounding water it is difficult to assess whether a change in respiration rate is related to salinity or oxygen concentration. True, in Davenport and Vahls experiments, as with those of Findley et al., salinity fluctuations were accompanied by changes in oxygen content (but not oxygen tension which remained constant) between 6.4 ml 02/1 at 33Ym and 8.0ml 02/1 in fresh water. The change in oxygen content (c. 25%) is comparable with the changes in oxygen uptake displayed by the blenny. However, it should be pointed out that Hollidays statement that uptake is a function of concentration is dubious since oxygen tension-the equivalent of its thermodynamic po ten tial-and not its concentration, controls oxygen ex change across the gills. Also, the uptake changes in the blenny were asymmetrical whereas the oxygen content of their environment fluctuated symmetrically with salinity. Whatever the underlying cause the energy cost to the blenny is higher in a fluctuating salinity regime than at constant high salinity. Subsequently Vahl and Davenport (1979) measured the increase in metabolic rate of the

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 233

    blenny, expressed as enhanced oxygen consumption, following a meal. This increase, known as specific dynamic action (S.D.A.) of the food consumed (Kleiber, 1961) may be as great as 60% and reaches a peak 2 4 h after a meal. Vahl and Davenport calculated that S.D.A. and the energetic cost of living in fluctuating salinity environments could interact to reduce the blennies scope for activity seriously if large meals were eaten at the wrong time during a tidal cycle (see Fig. 48). Of course the blenny tends to be a browser (Qasim, 1957), thus spreading the influence of S.D.A., but it does take large meals in the wild on occasion (Grove, personal communication).

    VII. OXYGEN TENSION STUDIES

    In the open sea oxygen tensions at the surface are usually reasonably high and apparently do not limit animals in any way. Abnormally low or high oxygen tensions only occur in a few specialized areas, and are usually associated with equally unusual temperatures or pH, carbon dioxide or sulphide levels. Some habitats are usually anaerobic, for example beneath the surface of mud flats or some deeper areas of enclosed seas (e.g. Black Sea). On the other hand, turbulent open coast areas or waters characterized by dense kelp beds may feature intermittent oxygen supersaturation (Shelford and Powers, 1915). Some specialized habitats feature pronounced oxygen tension fluctuations; such occurrences in rockpools have already been discussed in Section I1 (2) but similar changes between zero and 2-300% air saturation have also been reported in Zostera beds (Powers, 1920; Broekhuyser, 1935). Organic pollution, usually by sewage, together with resultant eutrophication can result in low or fluctuating oxygen tensions in estuaries or enclosed areas of the sea.

    Rather less research has been directed at determining oxygen tension limits to survival and reproduction in marine animals than for either temperature or salinity; the most recent review of the field appears to be that of Vernberg (1972). Many physiological and biochemical studies have been performed on the dependence of oxygen consumption on external oxygen tension, the importance of blood pigments as oxygen carriers and the workings of anaerobic metabolism. The literature devoted to these topics is too large and diffuse to be reviewed here and in any case, as for the tolerance studies, no work has been performed under conditions of fluctuating oxygen tension.

    Cycles of oxygen tensions were first used in an experimental study

  • 234 J . DAVENPORT

    40

    30

    20

    10

    0

    40

    - J 30 v

    x z

    m 0

    .r 20 -

    10

    0

    40

    30

    20

    10

    0

    A feeding - scope for activity salinity

    eeding

    I feeTng

    C

    feerng

    60

    40

    20

    0

    x .5 .= .- 1

    0

    60 c

    a, a

    40 2

    E 5

    20 E" .- X

    L

    8 0

    60

    40

    20

    0

    Hours

    FIG. 48. Blennius pholis. Combined effects of salinity and S.D.A. upon scope for activity; models for various feeding times. From Vahl and Davenport (1979).

    by Davenport and Fletcher (1978) on the mussel Mytilus edulis. Shumway (1977a) showed that, in a sinusoidal salinity regime, the osmolarity of the mantle fluid of mussels closely followed that of the external environment until shell valve closure occurred; the mantle fluid concentration then remained virtually constant at the

  • ENVIRONMENTAL SIMULATION EXPERIMENTS 235

    equivalent of about SO%, until the shell valves reopened in response to rising salinities (see Fig. 42). In a separate study Bettison and Davenport (in preparation) showed that the oxygen content of the mantle fluid of Nyt i lus fell dramatically when the she11 valves closed in response to an abrupt salinity change, from a mean value of 5-6 ml O,/litre just before closure to 0.9 ml O,/litre 15 min later. After 6 h of valve closure the oxygen content was still 0.6 ml O,/litre: evidently the mantle fluid does not become com- pletely anoxic. To summarize, the mantle fluid of Mytilus varies substantially in both salinity and oxygen tension during an estuarine salinity cycle. Davenport and Fletcher were interested in how these conditions affected the ciliary activity of the gills of M . edulis. In their studies they used gill preparations and methods of the type developed by Ajana (1975) from the earlier techniques of Gray (1923, 1924). Salinity and oxygen tension regimes of the type shown in Fig. 49 were applied separately or in combination to the gill preparations. The results are summarized in Figs 50, 51 and 52. Reduced oxygen tensions or salinities acting alone reduced ciliary activity (expressed as particle transport rates) by about 40%. When both were combined (see Fig. 52) ciliary activity fell by about 40y0 during the period of gently falling salinity and normal oxygenation; when low oxygen tension was added to the salinity stress the ciliary activity fell by a further 25%. On a return to high salinities and oxygen tensions the cilia completely recovered and it seems certain that such regular fluctuations in ciliary activity occur under natural conditions.

    The only other study which has involved laboratory simulations of oxygen tension fluctuations is that of Ritz (1980) who investigated the effects of fluctuating conditions of salinity, oxygen tension and copper concentration on the intertidal amphipods Gammarus duebeni Liljeborg and Marinogammarus marinus (Leach). Both species survived tidal salinity fluctuations between full sea water and pure fresh water for 4 days, but when a tidal oxygen tension fluctuation between 100% and 4% air saturation was superimposed upon this salinity regime (with low salinity and low oxygen tension being simultaneous), 50% of M . marinus were dead in 2 days in square wave regimes; no mortality occurred in G. duebeni or in either species exposed to sinusoidal regimes. Unfortunately no data for oxygen tension acting alone were available but this study reinforces the conclusions derived from many multivariate steady-state investig- ations that separate stresses may interact and reinforce each other.

  • J. DAVENPORT 236

    100

    - - 1 50 g v)

    0 I , I I , I

    0 5

    0

    5 50 - - c 0 1 - a

    0 0 - 1 1 I

    0 - 0 6 12 I I Hours 0 6 12

    H0E

    FIG. 49 (left). Simulated mantle cavity conditions in estuarine salinity cycle. Upper graph indicates salinity levels with the solid line representing the mantle cavity concentrations and the circles representing the hypothetical environmental salinities. Lower graph shows the oxygen tension conditions: 100~o air saturation is equivalent to about 150 mm Hg. Arrows indicate the onset and cessation of the stimulated shell valve closure period. From Davenport and Fletcher (1978).

    FIG. 50 (right). Effects of salinity upon frontal ciliary activity. Upper graph represents the salinity regime. Lower graph shows ciliary activity; the symbols represent mean values for five gill preparations, with 95% confidence intervals. From Davenport and Fletcher (1978).

    VIII. POLLUTANT STUDIES

    The literature devoted to the effects of pollutants upon marine organisms is vast and unwieldy, but the reviews by Bryan (197 1) and Phillips (1977a), together with the report of the symposium organized by Cole (1979) are relevant here.

    As described in earlier sections the mussel reacts to fluctuating external salinities by shell closure during periods of low salinity. Clearly in an estuarine mussel bed the tissues of M . edulis are not exposed to the full influence of the freshwater input. In polluted estuaries pollutants are often freshwater borne and hence both pollutant and freshwater influences are closely linked.

  • ENVIRONMENTAL SIMI'LATIOS EXPERIMENTS

    O J I , I

    2137

    100

    is 0 -

    50 P .. 4

    0

    00 J O O J I I I 0 6 1 2 0 6 12

    Hours Hours

    FIG. 51 (left). Effects of oxygen tension upon frontal ciliary activity. Upper graph represents the oxygen tension regime. Lower graph shows ciliary activity; the symbols represent mean values for five gill preparations, with 95% confidence intervals. Prom Davenport and Fletcher (1978).

    FIG. 52 (right). Combined effects of salinity and oxygen tension upon frontal ciliary activity. Upper graph indicates the regimes used; solid line indicates salinity levels, dashed line represents oxygen tensions. Lower graph shows ciliary activity: the symbols represent mean values for five gill preparations, with 95% confidence intervals. From Davenport and Fletcher (1978).

    Given the closure response to low salinities exhibited by Mytilus the closure mechanism may protect the bivalve against some of the effects of freshwater-borne pollutants; this possibility was in- dependently suggested by Phillips (1977b) and Davenport (1977).

    Davenport (1 977) decided to use copper to test this hypothesis. The metal was chosen because there was considerable data concerned with copper toxicity in the antifouling literature while Bryan and Hummerstone (1971) showed that i t was common in estuaries associated with dumped mining wastes. First a control experiment was performed with continuous levels of either 0.5 ppm or 0.25 ppm copper being added to sea water (3333 which was delivered to mussels. In the former Concentration all animals were dead in 3 4 days, the median lethal time (M.L.T.) being about 2 days. In 025ppm copper the M.L.T. was 4-5 days. However if copper was

  • 238 J. DAVENPORT

    delivered intermittently to mussels during a square wave tidal salinity cycle with copper being associated with low salinity and its absence occurring at high salinity, no mortality occurred in a 5-day experiment (see Fig. 53). On the other hand, if copper was added during the phase of high salinity, mortality was rapid (see Fig. 54). So far everything agreed with Davenports and Phillips hypotheses that shell valve closure induced by low salinity acted as a protection against pollutants as well as fresh water. However, a further experiment was performed with intermittent (6 h on, 6 h off) 0-5 ppm copper being delivered to mussels maintained in full strength sea water. As may be seen from Fig. 55 mortality was again zero. It rapidly became clear that mussels could actually detect heightened external copper concentrations and close their shell valves to avoid

    6

    trppp; 0 1 2 3 4 5 D a v r O 1 2 3 4 5

    12 24 12 24 Time (h) Time (h)

    FIG 53 (left). Myt i lus edulis. The effects of discontinuous 0 5 ppm added copper delivered under fluctuating salinity conditions. Copper on at low salinities. A, Percentage of animals able to form byssus during previous 24 h. B, Cumulative percentage of animals unable to maintain valve closure. C, Cumulative percentage of animals moribund. D, Cumulative percentage mortality. Squares = first experimental run. Triangles = second experimental run.

    FIG 54 (right). M y t i l w edulis. The effects of discontinuous 05 ppm added copper delivered under fluctuating salinity conditions. Copper on a t high salinities. A-D, as in Fig. 53 Squares = first experimental run. Triangles = second experimental run. From Davenport (1977).

  • 0 1 2 3 4 5 D a y r O 1 2 3 4 5

    0 - 12 24

    Time (h)

    FIG. 55. Mytalus edulis. The effects of discontinuous 0.5ppm added copper delivered under constant lOOyo S.W. conditions. A, percentage of animals able to form byssus during previous 24 h. B, Cumulative percentage of animals unable to maintain valve closure. C , cumulative percentage of animals moribund. D, cumulative percentage mortality. Squares = first experimental run. Triangles = second experimental run. From Davenport (1977).

    Valves open

    FIG. 56. Strain-gauge traces of four successive periods of shell valve opening in a mussel exposed to a discontinuous added copper regime in lOOyo S.W. From Davenport (1977).

  • 240 .J. DAVENPORT

    damage; some stress gauge traces which confirm this are shown in Fig. 56. In subsequent studies by Davenport and Manley (1978). Manley and Davenport (1978) and Manley (in preparation) the concentration threshold which initiates changes in behaviour in Mytilus (about 0.02 ppm total copper in 33%, Menai Strait sea water) has been determined. The phenomenon has been demonstrated in several other bivalves (Crassostrea gigas, Modiolus demissus, Modiolus modiolus and Anadara senilis), and is now known to be more clear cut for copper than for other heavy metals. It is still debateable whether the response to copper is ever of significance in nature, since fluctuations in copper content of estuarine waters with mussel populations have yet to be demonstrated. However, the results do cast a measure of doubt upon the value of mussels as biological indicators of pollution. The common bivalves meet many of the criteria for biological monitors proposed by Butler et al. (197 l ) , but the usefulness of a biological monitoring system depends upon its providing an accurate integration of all changes in environment pollutant levels. If it fails to register transient or recurrent short term slugs of highly polluted water because of its ability to isolate its tissues from them, the integration could be misleading.

    Ritz (1980), employing the same equipment and techniques, imposed simultaneous salinity, oxygen tension and copper fluctu- ations upon Marinogammarus marinus and Gammarus duebeni; the multiple variables make it difficult to abstract conclusions about single factors. However it is clear that G. duebeni survives intermittent exposure to 1 ppm added copper in sea water whereas continuous exposure is rapidly lethal. Presumably periodic access to clean sea water allows removal of the accumulated pollutant in the urine (Bryan, 1971). In fresh water the situation is quite different, both intermittent and continuous 1 pprn copper regimes are equally lethal. However, in low salinities copper is known to be especially toxic (Jones, 1975; Jones et al., 1976). Moreover, G. duebeni is faced with maintaining active ion uptake and producing hypo-osmotic urine both of which can reduce resistance to pollutants (Inman and Lockwood, 1977 ).

    IX. CONCLUSIONS

    The value of environmental simulation experiments lies in their ability to reveal responses and mechanisms not predictable from steady state experiments. Many of the studies reviewed here meet

  • EKVIHONMESTAL SIMULATIOX EXPERIMENTS 24 1

    this criterion at least to some extent, though in some cases, of course, the results obtained were not entirely unexpected. For example, given the excellent field work of Milne (1940), the data obtained by Shumway (1977a) concerning mantle fluid and haemolymph concentrations in Mytilus exposed to fluctuating salinities were predictable except in detail. On the other hand many of the thermal and salinity tolerance limits found for species exposed to cyclic regimes were dramatically different from those previously accepted.

    Similarly exciting were, for example, the observation by Diaz (1975) that damage to oyster larvae by brief exposure to high temperature may not be expressed for several days, a feature of great significance to oyster fisheries operating near power stations. The remarkable behavioural osmotic control exerted by burrowed lugworms (Shumway and Davenport, 1977), and the subtle isolation behaviour of Mytilus by closing the exhalant aperture (Davenport, 1979a) could not have been foreseen.

    Throughout the simulation study literature, are scattered criticisms of steady-state experiments. In most cases these comments are valid though perhaps expressed with the overstatement of the enthusiast! However it seems clear that simulation studies with varying factors must represent a supplementary approach to the conventional methods rather than a replacement.

    Some potential pitfalls are evident in fluctuating factor experiments. Once simulation equipment is available it is tempting to use it indiscriminately. There is a fine dividing line between using say a sublittoral species for comparative purposes in a study primarily aimed at understanding the biology of its intertidal and estuarine relatives, and using the species simply because it is available. Unless there are good scientific reasons which indicate otherwise (e.g. to elucidate basic mechanisms), it seems logical that only animals which have been proved to encounter physico-chemical fluctuations in nature should be exposed to idealized simulations in the laboratory; here we need much more information. In this respect especial care should be taken with highly mobile marine animals (e.g. fish, crustacea) which may live in variable habitats but avoid fluctuations by swimming, crawling or making appropriate vertical migrations.

    Despite these various problems, simulation studies appear to have a bright future. Likely trends in equipment trends and techniques have been referred to in Section 111. Experimental possibilities are probably endless and certainly unpredictable, but a few tentative suggestions may be made. First, it seems probable that

  • 242 J. DAVENPORT

    fluctuations in environmental factors such as pressure, pH or food concentration (for filter feeders) may be added to the present repertoire. Pollutants other than heavy metals might also be investigated (e.g. oil, dispersants). Secondly, it appears likely that studies involving fluctuations in salinity or temperature may tackle more fundamental questions of ion transport or enzyme behaviour. Finally, and of particular interest, is the idea that animals may live and grow better if they are exposed to variable rather than constant conditions. Most evidence for this derives from behavioural or psychological studies on terrestrial vertebrates. However, 30 years ago Allee et al. (1949) demonstrated that aquatic organisms might survive variable thermal conditions better than they do constant temperatures. Most of the factor fluctuation studies performed so far have concentrated upon the effects of extreme stresses, but what consequences would low amplitude factor fluctuations have for development in marine organisms? The equipment and methods reviewed here could provide the answers.

    X. ACKNOWLEDGEMENTS

    I am grateful to Professor D. J. Crisp, C.B.E., F.R.S. for critically reading the manuscript. I also wish to thank Mrs D. Clapton and Mrs E. Hughes who patiently typed it.

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    Ganning, B. (1971). Studies on chemical, physical and biological conditions in Swedish rockpool ecosystems. Ophelia 2 ( l ) , 51-105.

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    Gruffydd, L1. D. and Beaumont, A. R. (1970). Determination of the optimum concentration of eggs and spermatozoa for the production of normal larvae in Pecten maximus (Mollusca, Lamellibranchia). Helgolander wissenschaftliche Meeresuntersuchungen 20, 486497.

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    127-137.

    125-1 30.

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    Hand, A. C. and Stickle, W. B. (1977). Effects of tidal fluctuations of salinity or pericardial fluid composition of the American oyster Crassostrea virginica. Marine Biology 42, 25S27 1 .

    Haskin, H. H. (1964). The distribution of oyster larvae. Proceedings of the Symposium of Experimental Marine Ecology. Occasional Publications No. 2, pp. 76-80. Graduate School of Oceanography, University of Rhode Island.

    Heath, W. G. (1963). Thermoperiodism in sea-run cutthroat trout (Salmo clarki clarki). Science 142, 486488.

    Heinle, D. R. (1969). Temperature and Zooplankton. Chesapeake Science 10, 18G209.

    Hidu, H. and Haskin, H. H . (1978). Swimming speeds of oyster larvae Crassostrea virginica in different salinities and temperatures. Estuaries 1, 252-255.

    Hillman, N. S. (1964). Studies on the distribution and abundance of decapod larvae in Narragansett Bay, Rhode Island with a consideration of morphology and mortality. MAC. Thesis, University of Rhode Island.

    Hochachka, P. W. and Lewis, J. K. (1970). Enzyme variants in thermal acclimation: trout liver synthetases. Journal of Biological Chemistry 245, 6567-6573.

    Hochachka, P. W. and Somero, G. N. (1968). The adaptation of enzymes to temperature. Comparative Biochemistry and Physiology 27, 659-668.

    Hokanson, K. E. F., Kleiner, C. F. and Thorslund, T. W. (1977). Effects of constant temperatures and die1 temperature fluctuations on specific growth and mortality rates and yield of juvenile rainbow trout, Salmo gairdneri. Journal of the Fisheries Research Board of Canada 34, 63-48,

    Holliday, F. G. T. (1971). I n Marine Ecology (0. Kinne, ed.), Vol. I, Part 2. pp. 997-1083. Wiley Interscience, New York.

    Hoss, D. E., Coston, L. C., Baptist, J. P. and Engle, D. W. (1975). Effects of temperature, copper and chlorine during simulated entrainment in powerplant condensor cooling systems, pp. 519-527. Vienna, International Atomic Energy Agency (Ref. IAEA-SM-189/19).

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    Houston, A. H. (1957). Responses of juvenile chum, pink and coho salmon to sharp seawater gradients Canadian Journal of Zoology 35, 37 1-383.

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    Hubbs, C. (1964). Effects of thermal fluctuations on the relative survival of greenthroat darter young from stenothermal and eurythermal waters. Ecology 45 (2), 37G379.

    Inman, C. B. E. and Lockwood, A. P. M. (1977). Some effects of methylmercury and lindane on sodium regulation in the amphipod Gammarus duebeni during changes in the salinity of its medium. Comparative Biochemistry and Physiology 58C,

    Jansson, B. 0. (1962). Salinity resistance and salinity preference of two oligochaetes Akerdrilus monospermatecus Knoller and Marionina preclittochaeta N.sp. from the intertidal fauna of marine sandy beaches. Oikos 13, 293-305.

    Jones, L. H., Jones, N. V. and Radlett, A. J . (1976). Some effects of salinity on the toxicity of copper to the polychaete Nereis diversicolor. Estuarine and Coastal Marine Science 4, 107-111.

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    Keys, A. B. (1933). The mechanism of adaptation to varying salinity in the common eel and the general problem of osmotic regulation. Proceedings of the Royal Aociety (Series B ) 112, 184-199.

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    Khlebovich, V. V. (1969). Aspects of animal evolution related to critical salinity and internal state. Marine Biology 2, 338-345.

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    Korringa, P. (1952). Recent advances in oyster biology. Quarterly Review of Biology 27, 266-308, 339-365.

    Krogh, A. (1939). Osmotic Regulation in Aquatic Animals, 242 pp. Cambridge Univemity Press, London.

    Lange, R. (1963). The osmotic function of amino acids and taurine in the mussel, Mytilus edulis. Comparative Biochemistry and Physiology 10, 173-179.

    Lange, R. (1970). Isosmotic intracellular regulation and euryhalinity in marine bivalves. Journal of Experimental Marine Biology and Ecology 5 , 17(r179.

    Lange, R. and Mostad, A. (1967). Cell volume regulation in osmotically adjusting marine animals. Journal of Experimental Marine Biology and Ecology 1, 209-2 19.

    Lewis, J. B. (1960). The fauna of rocky shores of Barbados, West Indies. Canadian Journal of ZooZogy 38, 391435.

    Lewis, J . B. (1963). Environmental and tissue temperatures of some tropical intertidal marine animals. Biological Bulletin, Marine Biological Laboratory, Woods Hole, Mass. 124, 277-284.

    Ling, S. W. (1969). Methods of rearing andcylturing Macrobrachium rosenbergii (de Man). F A 0 Fisheries Reports No. 57, Vol. 3, pp. 607-619.

    Livingstone, D. R., Widdows, J. and-Fieth, P. (1979). Aspects of nitrogen metabolism of the common mussel MytiZus edulis: adaptation to abrupt and fluctuating changes in salinity. Marine Biology 53, 41-55.

  • ENVIRONMENTAL SIMlLAlYOK EXPERIMENTS 25 1

    Lockwood, A. P . M. (1962). The osmoregulation of Crustacea. Biological Reviews 37,

    Loosanoff, V. L. (1945a). Precocious gonad development in oysters induced in midwinter by higher temperatures. Science 102, 124-125.

    Loosanoff, V. L. (1945b). Effect of sea water of reduced salinities upon the starfish Asterias forbesi of Long Island Sound. Transactions of the Connecticut Academy o j Arts and Sciences 36, 813-833.

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    Loosanoff, V. L. and Davis, H. C. (1950). Conditioning V . mercenaria for spawning in winter and breeding its larvae in the laboratory. Biological Bulletin, Marine Biological Laboratory, Woods Hole, Mass. 98, 6(f65.

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    Biology 51, 11 1-1 17.

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    Morgan, R. P. and Stross, R. G. (1969). Destruction ofphytoplankton in t,he cooling water supply of a steam electric station. Chesapeake Science 10, 165-171.

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    Pollution 13, 281-317.

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    Pierce, S. K . Jr (1971). Volume regulation and valve movements by marine mussels. Comparative Biochemistry and Physiology 39A, 103-1 17.

    Pierce, S. K. and Greenberg, M. J. (1972). The nature of cellular volume regulation in marine bivalves. Journal of Experimental Biology 57, 681-692.

    Pierce, 8. K . and Greenberg, M. J. (1973). The initiation and control of free amino acid regulation of cell volume in salinity stressed marine bivalves. Journal of Experimental Biology 59, 435-446.

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    Pritchard, D. W. (1953). Distribution of oyster larvae in relation to hydrographic conditions. Proceedings of the Guyand Caribbean Fisheries Institute, 1952, pp. 123- 132.

    Prosser, C. L. (1973). Comparative Animal Physiology, 966 pp. W. B. Saunders Co., Philadelphia.

    Pyefinch, K. A. (1943). The intertidal ecology of Bardsey Island, North Wales, with special reference to the recolonization of rock surfaces, and the rock pool environment. Journal of Animal Ecology 12, 82-108.

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    Read, K. R. H. (1969). Thermal tolerance of the bivalve mollusc Modio1.u.s modiolns L. American Zoologist 9, 279-282.

    Regnault, M. and Costlow, J. D. (1970). Influence de la temperature et de la salinitG sur le developpement larvaire de Crangon septemspinosa Say (Decapoda, Caridea), Vie et Milieu (Sir A . , Biol. mar.) 21, 453-466.

    Ritz, D. A. (1980). Tolerance of intertidal amphipods to fluctuating conditions of salinity, oxygen and copper. Journal of the Marine Biological Association of the United Kingdom 60, 489-498.

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    Rosenberg, R. and Costlow, J. D. Jr (1976). Synergistic effects of cadmium and salinity combined with constant and cycling temperatures on the larval development of two estuarine crab species. Marine Biology 38, 291-303.

    Sanders, H. L., Mangelsdord, P. C., J r and Hampson, G. R. (1965). Salinity and faunal distribution in the Pocasset River, Massachusetts. Limnoloyy and Oceanography 10, Suppl. R216-R229.

    Sandoz, M. and Rogers, R. (1944). The effect of environmental factors on hatching. moulting and survival of zoea larvae of the blue crab, Callinectes sapidus Rathbun. Ecology 25, 216-228.

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    Sastry, A. N. (1978). Physiological adaptation of Cancer irr0ratw.s larvae to cyclic temperatures. Zn Physiology and Behaviour of Marine Organisms, Proceedings of the 12th European Symposium on Marine Biology, Stirling, Scotland. September 1977 (D. S. McLusky and A. J. Berry, eds), pp. 57-65. Pergamon Press, Oxford.

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    Schoffeniels, E. and Gilles, R. (1970). Osmoregulation in aquatic arthropods. I n Chemical Zoology (M. Florkin and B. T. Scheer, eds), Vol. V, Part A , pp. 255-286. Academic Press, London and New York.

    Schoffeniels, E. and Gilles, R. (1972). Ion regulation and osmoregulation in Mollusca. I n Chemical Zoology (M. Florkin and B. T. Scheer, eds), Vol. VII , pp. 393-420. Academic Press, London and New York.

    Schwabe, E. (1933). Uber die Osmoregulation verschiedener Krebse (Maloco- stracen). Zeitschrift f u r vergleichende Physiologie 19, 183-236.

    Segal, E. and Dehnel, P . A. (1962). Osmotic behaviour in an intertidal limpet. Acmaea limatula. Biological Bulletin, Marine Biological Laboratory, Woods Hole, Mass. 122, 417430.

    Shelford, V. E. and Powers, E. B. (1915). An experimental study of the movements of herring and other marine fishes. Biological Bulletin, Marine Biological Laboratory, Woods Hole, Mass. 28, 315-334.

    Sherberger, F. F., Benfield, E. F., Dickson, K . L. and Cairns, J . Jr (1977). Effects of thermal shocks on drifting aquatic insects: a laboratory simulation. Journal of the Fisheries Research Board of Canada 34, 52S536.

    Shumway, S. E . (1977a). Effect of salinity fluctuation on the osmotic pressure and Na+ , Ca2+ and Mgz+ ion concentrations in the haemolymph of bivalve molluscs. Marine Biology 41, 153-177.

    Shumway, 6. E. (1977b). The effects of fluctuating salinities on four species of asteroid echinoderms. Comparative Biochemistry and Physiology, 58A,

    Shumway, S. E . (1977~). The effect offluctuatingsalinity on the tissue water content of eight species of bivalve molluscs. Journal of Comparative Physiology 116, 26S285.

    Shumway, S. E. (1978a). Activity and respiration in the anemone, Metridium senile (L.) exposed to salinity fluctuations. Journal of Experimental Marine Biology and

    Shumway, 8. E. (1978b). Respiration, pumping activity and heart rate in Ciona intestinalis exposed to fluctuating salinities. Marine Biology 48, 235-242.

    Shumway, S. E. (1978~). Osmotic balance and respiration in the hermit crab, Pagurus bernhardus, exposed to fluctuating salinities. Journal of the Marine Biological Association of the United Kingdom 58, 869-876.

    Shumway, S. E. (1979). The effects of fluctuating salinity on respiration in gastropod molluscs. Comparative Biochemistry and Physiology MA, 274-283.

    Shumway, S. E. and Davenport, J. (1977). Some aspects of the physiology of Arenicola marina (Polychaeta) exposed to fluctuating salinities. Jownal of the Marine Biological Association of United Kingdom 57, 907-924.

    Shumway, S. E. and Youngson, A. (1979). The effects of fluctuating salinity on the physiology of Modiolus demissus (Dillwyn). Journal of Experimental Marine Biology and Ecology 40, 167-181.

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    Ecology 33, 85-92.

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    Shumway, S. E., Gabbott, P . A. and Youngson, A. (1977). The effect of fluctuating salinity on the concentrations of free amino acids and ninhydrin positive substances in the adductor muscles ofeight species of bivalve molluscs. Journal of Experimental Marine Biology and Ecology 29, 131-150.

    Sleigh, M. A. (1962). The Biology of Cilia and Flagella. 242 pp. Pergamon Press, Oxford.

    Somero, G. N. (1969). Enzymatic mechanisms of temperature compensation: immediate and evolutionary effects of temperature on enzymes of aquatic poikilotherms. American Naturalist 103, 517-530,

    Southward, A. J. (1958). Note on the temperature tolerance of some intertidal animals in relation t o environmental temperatures and geographical distri- bution. Journal of the Marine Biological Association of the United Kingdom 37, 49-66.

    Spaargaren, D. H. (1974). A study on the adaptation of marine organisms t o changing salinities with special reference t o the shore crab Carcinus muenas ( L . ). Comparative Biochemistry and Physiology 47A, 499-512.

    Spaargaren, D. H. (1978). A comparison of the blood osmotic composition of various marine and brackish water animals. Comparative Biochemistry and Physiology

    Stephenson, T. A , , Zoond, A. and Eyre, J. (1934). The liberation and utilization of oxygen by the population of rock pools. Journal of Experimental Biology 11,

    Stickle, W. B. and Ahokas, R. (1974). The effects of tidal fluctuation of salinity on the perivisceral fluid composition of several echinoderms. Comparative Biochemistry and Physiology 47A, 469-476.

    Stickle, W. B. and Ahokas, R. (1975). The effects of tidal fluctuation of salinity on the haemolymph composition of several molluscs. Comparative Biochemistry and Physiology 50A, 291-296.

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