Part 2: Experimental design of perturbation experiments
7
Laboratory experiments and benthic mesocosm studies
Steve Widdicombe1, Sam Dupont2 and Mike Thorndyke2,3
Plymouth Marine Laboratory, UK
Department of Marine Ecology - Kristineberg, University of Gothenburg, Sweden
3
Royal Swedish Academy of Sciences, University of Gothenburg, Sweden
1
2
7.1
Introduction
There is an expectation from society and policymakers that the scientific community will be able to provide
the knowledge that will guide them as they address the big environmental questions of the day. In the field
of climate change, which includes both global warming and ocean acidification, the overriding political
and societal needs are for predictions of the environmental consequences associated with continued
atmospheric emissions of CO2. In particular, what will happen to species, populations, communities and
ecosystems in the future and how will any changes affect the function of marine ecosystems? To fully
understand the biological consequences of ocean acidification it is essential that the scientific community
generates a holistic understanding of climate change impacts. However, to develop a comprehensive
understanding takes time and this strategy can only be achieved too late to inform the political decisions
needed to underpin strategies of carbon mitigation and societal adaptation. So, whilst the overall aim
of any ocean acidification experiment should be to provide new data and understanding that will help
the scientific community advance towards a holistic understanding, we must be realistic in what we
can achieve with limited resources so as to provide clear answers or messages that help politicians,
environmental managers and the public take action. For example, can we identify critical thresholds (or
tipping points) of change? Which species, communities and ecosystems are most at risk? Will organisms
be able to adapt to the predicted changes? The use of perturbation experiments is an extremely effective
way of addressing these questions.
Field observations are usually confounded by the presence of many potentially important variables in addition
to the one that interests us, and the relationships observed can only be described as correlative. Therefore, in
order to demonstrate a direct “cause and effect” relationship it is necessary to conduct controlled, manipulative
experiments. These experiments allow a single, or a limited suite of variables, to be manipulated and the effect
on particular end-points compared against a control condition. It is important at this stage to recognise that all
experiments are in fact abstractions from reality and all approaches have significant strengths and weaknesses
(these will be discussed later). No single approach or experiment can explain all the potential impacts of
ocean acidification on marine organisms, populations, communities and processes so instead we should see
each individual experiment as a means of answering a specific question or providing specific knowledge that
will advance our holistic understanding of the issue concerned. By considering how and by whom the results
of your experiment will be used (see Figure 7.1) it is easier to appreciate what makes a “good and useful”
experiment. In all cases, the methodologies must be appropriate for the questions posed, the results must be
clear and statistically robust, and the study must be adequately reported.
The issue of ocean acidification is a young and maturing question of immense and increasing scientific concern.
Unfortunately there are relatively few published papers (<50) that describe laboratory-based experiments
specifically designed to examine the impacts of ocean acidification on benthic organisms and processes.
More experiments are therefore vital across a range of groups, species, populations, functional groups, and
physiological traits. The aim of this section is not to recommend particular experimental approaches or subjects
for study over others, but to ensure that any experiments that are conducted are of maximum benefit to the
whole ocean acidification research community and its stakeholders.
Guide to best practices for ocean acidification research and data reporting
Edited by U. Riebesell, V. J. Fabry, L. Hansson and J.-P. Gattuso. 2010, Luxembourg: Publications Office of the European Union.
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Figure 7.1 A framework to visualise the role of experiments in identifying the consequences of ocean acidification.
7.2
Approaches and methodologies
The oceans harbour incredible biological diversity (May, 1994; Reaka-Kudla, 1997) and the majority of this
biodiversity is made up of invertebrates either residing in (infauna) or on (epifauna) the seafloor (Snelgrove,
1999). The benthos contains 98% of all marine species and harbours living representatives of all but one of the
29 non-symbiont animal phyla so far described. Given the importance of the benthic environment as a reservoir
for biodiversity, there has been much speculation as to whether ocean acidification has the potential to reduce
benthic biodiversity by impacting on key physiological (e.g. growth, respiration, calcification, metabolic rate)
and ecological (e.g. competition, predation) processes in marine organisms (Widdicombe & Spicer, 2008).
Most benthic ocean acidification studies published to date have used highly, or fully, controlled single species
experiments to look at specific responses and improve understanding of physiological mechanisms. Whilst
these experiments are extremely important it should not be forgotten that they represent the most artificial of
experimental situations, being isolated from many of the biological interactions (e.g. competition, predation,
facilitation) that occur in the natural environment. In support of such controlled experiments it is important
also to consider experiments that use not only single species but simple assemblages or even complex subsets
of whole ecosystems. These microcosm or mesocosm experiments have been used with great success by the
pelagic acidification research community (see chapter 6) but have yet to be fully exploited by researchers
studying the effects of seawater acidification on benthic systems.
Studies to date have examined a wide range of physiological and ecological responses to ocean acidification;
calcification (e.g. Gazeau et al., 2007; Findlay et al., sbm), acid-base regulation (e.g. Miles et al., 2007; Spicer
et al., 2007), respiration (e.g. Wood et al., 2008), fertilisation (e.g. Kurihara & Shirayama, 2004; Havenhand
et al., 2008; Byrne et al., 2009), larval development (Dupont & Thorndyke, sbm), tissue damage (e.g. Wood
et al., 2008), immune response (e.g. Bibby et al., 2008), assays of general health (e.g. Neutral Red Retention
assay - Beesley et al., 2008), feeding (e.g. Dupont & Thorndyke, 2008), burrowing (Widdicombe & Needham,
2007), survival (e.g. Dupont et al., 2008), abundance (Dashfield et al., 2008), diversity / community structure
(e.g. Widdicombe et al., 2009), all of which have been important in their own right. However, what is now
evident is that changes in physiological processes can occur simultaneously within an organism. Consequently,
in order to determine the effects of acidification on an individual’s performance and survival, it is essential
that data are collected that can be used to identify the physiological and ecological trade-offs which occur
and therefore we consider impacts at a “whole organism” level (see Wood et al., 2008 for a full description).
This does not necessarily mean that experiments have to measure all of the parameters listed above but it does
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highlight the necessity that studies are reported with sufficient detail as to allow accurate comparisons between
experimental results.
Most published studies have so far only focused on limited aspects of natural life histories such as a single
life stage, environment, etc. However, an individual may experience very different environmental conditions
during its life. Consequently, an organism’s performance and adaptation potential to a new stress can be
extremely variable in space and time, e.g. many benthic organisms, particularly in coastal areas, spend a
part of their life cycle as pelagic larvae, exposing them to potentially greater variability in seawater pH and
carbonate saturation than when they are buried within the sediment as adults. Many organisms are mobile
and can change their local environment, for example via daily/seasonal migrations or avoidance of stressful
conditions by moving. A review of published studies indicates that many of the experiments so far conducted
have been carried out over relatively short time scales; ranging from less than 24 hours exposure to a maximum
of 30 weeks. While such comparatively short exposure times may be relevant to studies designed to assess
the potential of ocean acidification to affect certain physiological and ecological processes, they do not truly
reflect the rate or scale by which changes in seawater chemistry will occur and thus affect organisms over a
longer timescale. Moreover, very few analyses have been attempted over several generations. Consequently,
there is currently a severe lack of studies that adequately assess the potential for individuals, populations and
communities to adapt to ocean acidification in the longer term.
Apart from previous studies that have explicitly investigated the impacts of acidification on calcification in
corals and calcareous algae (see chapter 13) the majority of benthic experiments have used the addition of
CO2 gas to manipulate seawater chemistry; either as pure CO2 gas (e.g. Widdicombe & Needham, 2007), as
a specific air/CO2 mixture (e.g. Kurihara et al., 2007) or, in one case, the addition of flue gas generated by a
power plant furnace (Palacios & Zimmerman, 2007). These approaches are described in detail in chapter 2.
7.3
Strengths and weaknesses
As previously noted, all experiments are an abstraction from reality and therefore represent a series of
compromises (Figure 7.2). This does not mean that any one approach is better than another but we should be
conscious of what each approach can provide and what it cannot.
Figure 7.2 A summary of the strengths and weaknesses of single species cultures and multiple species
mesocosm experiments.
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7.3.1
Experiments on single species
As these studies are conducted on individuals or
species in isolation they are relatively easy to interpret
(Figure 7.3). In general these experiments can be highly
controlled with only a single variable being manipulated.
Therefore the responses observed are not confounded by
additional variables or by biological or environmental
interactions. In cases where multiple variables are
manipulated, the experiment can be designed with
suitable replication to disentangle main effects and
interactions (see chapter 4). In fact, the opportunity to
fully replicate provides considerable statistical power
and is a key strength of these kinds of laboratory
experiments. In addition, sampling such experiments
Figure 7.3 Larval cultures at the Sven Lovén ocean
acidification facilities (University of Gothenburg).
is usually relatively straightforward and easy access
pH is dynamically controlled using pH-computers
to the experimental subject can allow the observation
(Aquamedic) by injection of pure CO2 in 5 l closedof impacts over time. A major drawback, however, is
circuit aquaria allowing an accuracy of 0.04 pH
the relevance of the data collected to the real world.
units (photo credit: S. Dupont).
In removing an organism from its natural habitat it is
difficult to know whether it is in any way stressed and
therefore whether it is behaving or functioning normally.
Also, whilst isolation from the highly variable natural environment can make identification of causality much
easier, it should be remembered that an organism is normally exposed to a variety of stressors and the response
identified in the artificial environment created in the laboratory may not actually manifest itself in the natural
world. This means care in interpretation is critical.
7.3.2
Mesocosm experiments on natural assemblages and communities
This approach aims to enclose an intact sample of a natural community or ecosystem and expose it to
experimental perturbation (see also chapters 6 and 13). These experiments are biologically complex which
brings with it both major advantages and problems. The incorporation of natural, biological interactions means
that results obtained from mesocosm studies are considered more relevant to natural situations than those
from laboratory experiments. This additional realism aids in the scaling up of experimental results to field
situations. In addition, keeping organisms in a more natural environment means they are less likely to become
stressed than those in smaller scale laboratory experiments. However, although more “realistic” than singlespecies approaches, mesocosms are still not a precise replication of a natural system and this should be borne
in mind when interpreting results and scaling up. In natural coastal environments, benthic organisms may be
strongly linked to the pelagic environment. In addition to the coupling via pelagic larvae of benthic species,
there are other interactions between pelagic and benthic species. These include feeding, predation, parasitism,
propagation and seeding with dead organisms or products (e.g. house of appendicularians). For example, the
quality of the food naturally coming from a complex pelagos may not be reproduced in mesocosm experiments,
or there could be a reduction in competition because of an absence of natural recruitment.
A weakness of mesocosm experiments is that they are often large and it can be difficult and costly to achieve
an ideal level of statistical replication. Whilst this should not be seen as a reason not to conduct mesocosm
experiments, it is important to acknowledge statistical deficiencies and ensure interpretation of the results does
not exceed the limits imposed by statistical constraints. For example, replication within a single mesocosm
could be considered as pseudoreplication and therefore applicability of observations to situations outside of
that mesocosm are limited (for a full explanation of pseudoreplication see chapter 4).
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7.3.3
Mesocosm experiments on “artificial” assemblages and communities
Another approach can be employed to combine the strengths of experiments on single species (control of
the variables manipulated, real replicates, decrease of experimental variability, etc.) with those of mesocosm
experiments on natural assemblages and communities (biological complexity). The principle is to create
artificial assemblages that mimic natural communities but with a controlled species composition, density,
habitats etc. (Figure 7.4). This approach integrates biological interactions (feeding, predation, parasitism etc.)
but decreases the variability due to the sampling effect of natural assemblages and communities, a factor
particularly important in natural mesocosm experiments where replication is often limited.
In summary, benthic perturbation experiments can adopt a range of approaches that increase in complexity and
reality from tightly controlled single species experiments through artificially assemblages to natural communities
in mesocosms. All of which have different, yet complimentary strengths and weaknesses. Consequently, to
fully appreciate the impacts of ocean acidification on benthic organisms and ecosystems we must utilise all of
these approaches in addition to the field experiments and observations described in chapter 8.
7.4
Potential pitfalls
Many of the pitfalls associated with ocean acidification experiments will be the same as those encountered for
all experiments involving marine organisms. For a good review of experimental practice see “Recommended
texts for further reading” in chapter 4. Here we briefly summarise the major pitfalls.
7.4.1
Experimental stress
In any laboratory experiment organisms are being removed from their natural surroundings to some extent.
This artificially imposed stress (termed here as experimental stress) could have a significant impact on an
organism’s response and attempts should be made to reduce it as much as possible. Additional experimental
Figure 7.4 Community based experiments being conducted in the seawater acidification facility housed at the
Plymouth Marine Laboratory (photo credit: S. Widdicombe).
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stress can occur in recalculating seawater systems if levels of metabolic waste are allowed to build up or if
levels of oxygen are allowed to fall. However, it should also be recognised that organisms will be exposed to
environmental stresses in the field as part of their natural existence (e.g. predation and competition). Bringing
organisms into the laboratory or mesocosm can therefore liberate them from these natural stresses, which in
turn could affect the results of any laboratory-based experiment.
7.4.2
Natural cycles
All organisms go through natural cycles; reproductive, growth, feeding, temperature; which can occur on a
variety of time scales; diurnal, monthly, annually. For example, in the reproductive season there will be a different
cycle of energy allocation with more resources going to the gonads and less going to support body maintenance
or growth. At what point an organism is in a particular cycle can have a considerable effect on its physiological
condition and how it will respond to specific perturbations. An appreciation of an organism’s physiological
condition at the time of the experiment is therefore vital for accurate interpretation of the results.
7.4.3
Influence of populations
An individual’s response will be affected by its environmental history; intertidal vs. subtidal; range edge vs. mid
range; naturally high or variable CO2 areas. Thus, it is vital to take account of the ecophysiological parameters of the
collection site as well as exploring the responses of samples taken from a variety of sites and populations.
7.4.4
Variability between individuals
Even within populations there will also be variability in response between individuals. This variability is part
of the natural “noise” of experimental data and should be accounted for by ensuring adequate replication at the
appropriate experimental level (see chapter 4).
7.4.5
Plasticity
Many organisms have the ability to alter their behaviour and/or physiology in response to an environmental
change. However, this plasticity or acclimation depends on the speed at which the change occurs. Consequently,
in perturbation experiments the speed at which the experimental treatment levels are imposed could have a
significant impact on the results obtained. Presently there is no consensus as to whether acclimation should
be used prior to exposure experiments or how long any acclimation period should be. Consequently, to assist
future interpretation of results, papers should clearly report whether or not organisms were acclimated to the
experimental treatment levels prior to exposure, and for how long.
7.4.6
Loss of natural processes
As previously discussed, experiments are an abstraction of reality and responses could be different when
confounded by natural biological interactions.
7.4.7
Exposure time vs. organism longevity
In all experiments there is a need to consider the exposure time in relation to an organism’s life span or the
length of a particular life stage (life history and generation time). Long-lived species may have physiological
mechanisms which enable them to cope with relatively short-term perturbations.
7.4.8
Synergistic stressors
So far there has been little attention on multiple environmental stressors. For example, the effects of climate
change will include a number of stressors, such as temperature, pH, anoxia, salinity and physical disturbance,
all occurring simultaneously.
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7.5
Suggestions for improvements
The level of understanding currently available with which to make the required predictions of ecosystem
responses to the environmental changes associated with ocean acidification is low. Consequently, all new
studies generating observations and data are extremely valuable and all efforts should be made to ensure these
studies are conducted and reported in the most appropriate and comprehensive manner.
As reported in section 7.4, two major pitfalls of perturbation experiments are associated with experimental
stress and natural cycles. To ensure that the results of future experiments are as applicable to natural systems
as possible, every attempt should be made to reduce experimental stress and understand natural cycles. To do
this there should be an attempt to mimic, as closely as possible, the organism’s natural conditions and cycles,
for example temperature, salinity, light, food availability, tidal cycle and water flow. This means that before
designing an experiment, a researcher should understand the underlying biological and chemical conditions
of the area from which a study organism is to be taken. For the chemistry there is a need to assess natural
variations (daily, seasonal, etc.) taking into account parameters such as the impact of biological activities on
seawater chemistry, for example respiration, plus an organism’s natural response to changing conditions, for
example temporary migration to more favourable areas. These conditions will be life stage dependent and
observations of field conditions should be mindful of this. If the intention is to conduct long-term studies, it is
necessary to also take into account the natural behaviour of the species being studied as they may migrate to
different habitats depending on season or age. In long-term experiments it may also be necessary to provide
food. This should be supplied in relevant concentrations and in an appropriate form (e.g. algal species or prey
item), which can vary according to season and life stage. When interpreting experimental results it would be
useful to provide a measure of experimental stress. This could be done by comparing the experimental control
group with individuals from the natural field population. Assessments of stress could include measures of
metabolic activity (e.g. respiration, ventilation or heart rate) or assays of general health (e.g. Neutral Red
Retention assay).
Another pitfall was concerned with the natural plasticity of organism response. Compared to the time
available in which to conduct experiments, ocean acidification is actually occurring very slowly. Whilst
rapid, “shock” experiments are very useful to identify the potential physiological and ecological processes
that are vulnerable to high CO2 levels, more effort needs to be made in mimicking the slow steady increase
in ocean acidity. This includes long-term exposure experiments that superimpose a gradual decrease
in seawater pH onto the natural pH cycle. In addition, experiments that deal with developmental and
transgenerational issues should be conducted. For example, most experiments on larvae to date have
taken eggs and sperm from specimens collected in the wild or from control aquaria and then exposed the
resultant fertilised eggs and larvae to the lowered pH conditions. Here it is vital that adults too should be
acclimated to the same pH ranges as those to which the larvae will be exposed. This is a truer reflection
of the natural situation.
By using a range of treatment levels (>5) it is possible to describe the relationship observed between a perturbation
and a response. When trying to identify critical thresholds or parameterise models, such relationships are more
valuable than observations of impact based on one or two treatment levels. Experimentalists should not be
reluctant to publish experiments that show “no response”. Providing it can be shown that the lack of a response
was genuine and not due to a lack of statistical power or inappropriate methodology, such observations are
extremely valuable in determining the structure and function of future ecosystems. To ensure any study
maximises its value to other users (scientists, policymakers, environmental managers etc.) it is essential that
all relevant information be reported. These requirements are outlined below.
7.6
Data reporting
To ensure that the data and understanding generated from experiments provides the maximum benefit and
value to all relevant end-users, particularly fellow scientists and policymakers, it is essential that all relevant
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data be accurately reported in scientific publications. For ocean acidification perturbation experiments on
benthic organisms and processes we suggest the following data should be collected and reported.
7.6.1
−
Where (location, habitat, place) and when (date and time) the specimens were collected.
−
The organisms mean body size plus the maximum and minimum sized animal used.
−
The sex and reproductive state of the organisms.
−
Organism’s age or developmental stage.
7.6.2
Relevance of experimental treatments to natural field conditions
−
The environmental conditions at the specimen collection site.
−
The natural values observed in field populations for each of the response variables measured in the study.
7.6.3
7.7
Specimen state and quality
Experimental conditions
−
The mean (± a measure of error), maximum and minimum temperature and salinity in each treatment.
−
The mean (± a measure of error), maximum and minimum for at least two measures of carbonate
chemistry from each treatment (see chapter 1).
−
Accurate description of the methods used to measure carbonate parameters including pH scales and
buffers where appropriate.
−
State whether the experimental subjects were given a period of gradual acclimation (state the size of
the incremental changes and the length of time between steps or total acclimation period) or whether
they were immediately exposed to the full treatment levels.
−
Report any indications or measures of stress in the experimental organisms. Compare these data to
field specimens where possible.
−
State the length of time organisms were exposed to the treatments.
Recommendations for standards and guidelines
1. Optimise the limited resources (time, space, money, people) to provide answers that help politicians,
environmental managers and society to adapt to and mitigate change.
2. Methodologies and experimental design must be appropriate for the question posed (Figure 7.1).
3. To have a realistic picture of the real impact of ocean acidification on a species, it is important to take
into account the “whole organism” level and to integrate environmental variability in space and time
experienced by an organism.
4. When the goal is to study the ecological impact, an experiment needs to be realistic and mimic the
real conditions experienced by the organisms both for biotic and abiotic conditions.
5. The stresses experienced by an organism during an experiment, other than that caused by the perturbation
of interest, should be kept at its natural level (reduce experimental stress and keep “normal” stress).
6. Experiments should be designed to assess natural plasticity within species and populations. These
will necessitate the use of long-term exposures that cover multiple generations.
7. Ocean acidification should be investigated in synergy with other stressors.
8. All data should be reported with all the information to allow comparison between studies, even data
showing “no effects”.
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7.8
References
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