ECOTOXICOLOGY: A Comprehensive Treatment - Chapter 32 potx

Although microcosms are designed to simulate specific portions of a natural ecosystem, themost valuable experiments investigating effects of contaminants on ecosystem processes have been

32 The Use of Microcosms,

Mesocosms, and Field

(Fisher 1960)

32.1 INTRODUCTION

Results of field surveys and other descriptive approaches have provided a solid foundation by which

to evaluate the effects of contaminants on ecosystem processes These studies have shown that certainfunctional characteristics of ecosystems, especially productivity, nutrient flux, and decomposition,are quite sensitive to anthropogenic disturbance However, as we noted in the previous chapter,descriptive studies are limited because of the inability to demonstrate cause-and-effect relationshipsand because of difficulties identifying underlying mechanisms responsible for changes in these eco-system processes Complex interactions and indirect effects of chemicals are likely to be the rulerather than the exception in many ecosystems In addition, community inertia, defined as the tend-ency for communities to persist under unfavorable conditions following disturbance (Milchunas andLauenroth 1995), complicates evaluation of ecosystem responses to perturbation Isolating causalmechanisms is particularly important in ecosystem studies because these processes are often complexand controlled by an assortment of direct and indirect effects For example, litter decomposition inaquatic and terrestrial ecosystems is regulated by microbial processes and activity of invertebrates.Because effects of contaminants on decomposition rate are dependent on the relative sensitivity

of microbial and macroinvertebrate communities, experimental approaches that isolate these ferent mechanisms are necessary to predict effects This is an ideal application of microcosm andmesocosm experiments, which are often designed to manipulate single or multiple environmentalvariables, providing an opportunity to isolate specific factors and identify underlying mechanisms

dif-It is the ability to isolate and manipulate individual factors that makes application of microcosm andmesocosm experiments particularly powerful in ecotoxicological research

In this chapter we turn our attention to experimental approaches that have been employed todemonstrate effects of contaminants and other stressors on ecosystem processes We will examine theuse of both small-scale approaches such as microcosms, as well as larger, more ecologically realistic

687

and field-based approaches such as mesocosms and whole ecosystem manipulations Because of thelimited spatiotemporal scale, measuring responses of ecosystem processes to contaminants in micro-cosms presents significant challenges The duration of microcosm and mesocosm experiments is ofcritical importance when assessing effects of contaminants For example, a common phenomenon insoil microcosms is the natural reduction in microbial biomass or activity over time As a consequence,effects of stressors on soil microbial processes become more difficult to quantify as experiments pro-gress Although microcosms are designed to simulate specific portions of a natural ecosystem, themost valuable experiments investigating effects of contaminants on ecosystem processes have beenconducted in larger systems.

32.2 MICROCOSM AND MESOCOSM EXPERIMENTS

There is an increasing belief amongst risk assessors that model ecosystems do not possess ecologicaladvantages that were originally assumed, and that an instrumentalist approach to the prediction of toxiceffects in ecosystems will yield the most cost-effective results

use-of anthropogenic disturbance, model ecosystems can be used to characterize ecological resistanceand resilience However, in his critique of model ecosystems used in ecotoxicological research,Crane (1997) argues that research priorities should shift from understanding these ecological com-plexities to questions regarding repeatability, precision, and the relationship between experimental

Year

1989 1990 1991 1992 1993 1994 1995 1996 1997

0 10 20 30 40 50

60

Limnology Toxicology Microbiology Terrestrial

FIGURE 32.1 The number of studies published between 1990 and 1996 that included the words microcosm

or mesocosm in the title or abstract (Data from Table 1 in Fraser and Keddy (1997).)

and natural systems He notes the “dangers of allowing model ecosystem studies to be driven byecological theory” and argues that model ecosystems are most appropriate as a tool to provide envir-onmentally realistic exposure conditions Because microcosm and mesocosm experiments oftenprovide complex responses across levels of biological organization, interpreting results can be chal-lenging Recall that problems with data interpretation were provided as a primary justification for thedecision by the U.S EPA to drop mesocosm testing for pesticide registration (Touart 1988, Touartand Maciorowski 1997,Chapter 23) Stay et al (1988) argued that a lack of correspondence betweenpopulation, community, and ecosystem-level responses observed in their experiments indicated thatmeasurements at one hierarchical level may not be useful for predicting effects at other levels.Sorting out these complex responses and relating alterations in community structure to ecologicalprocesses should be a priority for microcosm and mesocosm research We also believe that a criticalarea of research is to determine the extent to which these experimental systems reflect natural con-ditions One of the challenges associated with the use of microcosms and mesocosms is the change

in functional measures over time, independent of the effects of contaminants These changes, whichare often a result of container artifacts, compromise our ability to make comparisons among treat-ments Williams et al (2002) compared structural characteristics of microcosms and natural ponds,and recommended refinements to the design of model systems to improve their ecological realism.Unfortunately, few studies have made this comparison based on functional measures or ecosystemprocesses Kurtz et al (1998) measured reproducibility and stability of structural and functionalprocesses in estuarine microcosms Both structural (relative abundance and density of sulfate redu-cing bacteria) and functional (CO2assimilation, sulfate reduction) measures in microcosms weresimilar to conditions in natural sediments after 7 days However, the authors cautioned against longerterm experiments without modifying the system Suderman and Thistle (2003) examined changes instructural and functional measures in sediment microcosms derived from a shallow estuary Chloro-

phyll a, primary production and most measures of meiofauna community composition remained

relatively stable over the 3-month period

Another potential criticism of microcosm and mesocosm experiments is their relatively limitedtemporal scale Because long-term mesocosm experiments (e.g.,>1 year) are rare, our understand-

ing of prolonged exposure to stressors is incomplete This is an especially important issue whenconsidering ecosystem processes that often show delayed responses compared to structural altera-tions Bokn et al (2003) exposed rocky intertidal communities to long-term nutrient enrichment inmarine mesocosms Despite large inputs of N and P (maximum target concentrations were 32 and2.0µM, respectively) and significant increases in periphyton biomass, there were essentially no

effects on NPP, GPP, or respiration These unexpected results were attributed to competition amongmacroalgal species, grazing by herbivores, and physical disturbance Although this study was con-ducted for 2.5 years, a relatively long time period when compared with most mesocosm studies,this was not a sufficient amount of time for opportunistic algal species to become established andrespond to nutrient enrichment (Bokn et al 2003)

In addition to comparing processes in microcosms and mesocosms with those in natural systems and assessing changes in controls over time, additional research is necessary to optimizeexperimental designs and to evaluate the statistical power of these systems (Kennedy et al 1999) InChapter 23, we discussed strengths and weaknesses of different experimental designs (e.g., ANOVAversus regression; assignment of replicates to experimental units) for community-level assessments.Consideration of statistical power is especially critical for assessments of ecosystem processesbecause variability of these measures is often greater than for structural measures Kraufvelin(1998) estimated the number of replicates necessary to detect significant differences for 50 differentvariables derived from land-based, brackish water mesocosms Although calculations were based

eco-on populatieco-on and community-level variables, the results have important implicatieco-ons for cosm experiments designed to assess functional endpoints Relatively few of the structural variablesexamined had coefficients of variation (CV) less than 20% Using an endpoint with a modest CV

meso-of approximately 30%, 24 replicates were necessary to detect a statistically significant difference

(α = 0.05) of 25% between control and treatment mesocosms Kraufvelin (1998) also noted large

differences in the amount of variation among response variables Assuming that ecosystem processeswill show a similar or greater level of variation, statistical power will obviously be an importantconsideration when selecting functional endpoints

32.2.1 MICROCOSMS ANDMESOCOSMS INAQUATICRESEARCH

Microcosms and mesocosms have been employed extensively to assess the effects of contaminants

on processes in aquatic ecosystems Although the majority of these investigations have focused

on changes in primary production, respiration and other aspects of ecosystem metabolism, points related to nutrient processing and decomposition rates have also been considered Mostexperiments conducted in stream microcosms and mesocosms have focused on the response ofperiphyton Because of their small size, rapid rate of development, and diverse taxonomic composi-tion, periphyton are sensitive indicators of water quality in natural and experimental streams (Lowe

end-et al 1996) Changes in the structure and function of epilithic assemblages exposed to ants can occur very rapidly Colwell et al (1989) attributed increased respiration in outdoor streammicrocosms treated with Zn to the establishment of Zn-tolerant bacteria and algae We should alsoremember that structural characteristics of ecosystems may directly or indirectly influence ecologicalprocesses For example, physicochemical characteristics in macrophyte-dominated systems are oftencontrolled by biological processes that are directly related to ecosystem metabolism (Brock et al.1993) Kersting and van den Brink (1997) describe a dissolved oxygen–pH–alkalinity–conductivitysyndrome, in which each of these variables is expected to respond to toxic substances in parallel.These interrelated responses may result in feedback between contaminants and ecosystem processes.For example, it is likely that alterations in community metabolism resulting from exposure to contam-inants may affect pH and thereby modify contaminant bioavailability To improve our understanding

contamin-of the complex responses frequently observed in microcosm and mesocosm experiments, samplingprotocols should be designed to quantify relationships between these physicochemical and biologicalvariables

32.2.1.1 Separating Directand IndirectEffects

One of the important applications of microcosm and mesocosm research has been to separate thedirect effects of contaminants from the indirect or secondary effects on ecosystem processes Select-ive application of contaminants that have specific effects on one group of organisms but relativelylimited effects on another group is a useful approach for quantifying these direct and indirect effects(Pratt et al 1997, Slijkerman et al 2004) Pearson and Crossland (1996) measured photosynthesisand respiration in outdoor experimental streams exposed to the herbicide atrazine and the insect-icide lindane Atrazine had a direct inhibitory effect on photosynthesis at 100µg/L In contrast,

photosynthesis increased in lindane-treated streams due to the elimination of grazing rates Because the direct toxicological effect on invertebrates is limited, atrazine has been used

inverteb-to identify botinverteb-tom-up responses in model systems (Pratt et al 1997) (Figure 32.2) In this example,reduced food availability to higher trophic levels would be considered an indirect effect of herb-icide exposure Conversely, exposure of model systems to insecticides can have a direct effect

on invertebrate grazers, resulting in increased algal biomass and production Predicting effects

of contaminants on intermediate trophic levels where elimination of one group may impact bothlower and higher trophic levels presents special challenges Boyle et al (1996) quantified indir-ect effects of the insecticide diflubenzuron, a chitin inhibitor, on ecosystem processes in 0.1 hamesocosms Significant declines in abundance of grazing insects and zooplankton following difluben-

zuron treatment resulted in increased chlorophyll a biomass and GPP (a top-down response due to

reduced grazing) and reduced biomass of juvenile bluegill (a bottom-up response due to reducedfood supply) Brock et al (1993) reported similar increases in periphyton and phytoplankton when

20 30 40 50 60

70 Dissolved oxygen Species richness

FIGURE 32.2 Changes in structural (species richness) and functional (dissolved oxygen concentration)

variables in microcosms treated with the herbicide atrazine (Data from Table 1 in Pratt et al (1997).)

mesocosms were treated with the insecticide chlorpyrifos, which reduced abundance of grazinginvertebrates

The relationships between structural and functional components of model ecosystems are oftencomplex and may be dependent on contaminant concentration Slijkerman et al (2004) observedthat at intermediate concentrations of the fungicide carbendazim (17µg/L), structural changes were

observed but there were no corresponding effects on ecosystem function Functional impairmentoccurred at higher exposure concentrations (219µg/L), indicating that functional redundancy could

not compensate for changes in community structure Similar results were reported by Carman et al.(1995) for meiofauna exposed to PAHs in sediment microcosms Despite significant changes inmeiofaunal community composition in high PAH treatments, there were no effects on bacterial ormicroalgal activity

Elimination of invertebrates by chlorpyrifos in experimental ditches had modest effects on system metabolism by decreasing respiration and increasing oxygen concentration; however, effects

eco-on community structure and decompositieco-on rates were much more dramatic (Kersting and van denBrink 1997) Exposure of macroinvertebrates to chlorpyrifos reduced abundance of shredders andresulted in decreased litter decomposition (Cuppen et al 1995) These researchers also speculatedthat elimination of grazing invertebrates by insecticides may enhance effects of eutrophication byreducing top-down control of primary producers Detenbeck et al (1996) measured biomass, GPP,and respiration in mesocosms treated with the herbicide atrazine in wetland mesocosms GPP wasreduced at the lowest exposure level (15µg/L), but respiration was either reduced (25 µg/L) or

enhanced (75µg/L) An increase in ammonium, dissolved N, and dissolved P in treated mesocosms

was attributed to reduced nutrient uptake by periphyton Bester et al (1995) observed significantreductions in primary production at low levels of atrazine exposure (0.12µg/L) in marine mesocosms

An increase in concentrations of dissolved organic N and P in treated microcosms was attributed torelease from damaged cell walls

Mesocosm experiments also allow investigators to compare responses across levels of biologicalorganization, thereby providing opportunities to examine underlying mechanisms and relate struc-tural changes to functional alterations As noted inChapter 31, reduced decomposition rate observed

in contaminated streams may result from either lower abundance of macroinvertebrate shredders orchanges in microbial activity Stream mesocosm experiments have been used to assess the relativeimportance of these two explanations Newman et al (1987) measured litter processing rates, shred-der abundance, and microbial colonization in outdoor experimental streams dosed with chlorine.Although no effects were measured at intermediate concentrations (64µg/L total residual chlorine),

lower rates of decomposition in streams receiving 230µg/L were attributed primarily to reduced

abundance of amphipod shredders The bacterial insecticide Bacillus thuringiensis significantly

increased microbial respiration and decreased decomposition in laboratory microcosms weiser et al 1996) Although a similar trend was observed in outdoor stream channels, this trendwas not significant because of high variation among replicates

(Kreutz-32.2.1.2 Stressor Interactions

The key strengths of microcosm and mesocosm experiments are the opportunity to assess effects ofchemical mixtures and to quantify interactions among stressors under controlled experimental condi-tions Cuppen et al (2002) observed significant effects of a mixture of insecticides (chlorpyrifos andlindane) on decomposition rates of particulate organic matter in litterbags Despite rapid dissipation

of both insecticides (t1/2= 9–22 days), elimination of shredders and reduced microbial activity

res-ulted in lower decomposition rates Results of experiments measuring interactions between nutrientsand agricultural contaminants (e.g., herbicides, insecticides, sediments) are especially enlighten-ing because these stressors frequently co-occur More importantly, bioavailability of contaminantsmay vary depending on the nutrient status and the amount of organic material in an ecosystem Forexample, sorption of contaminants is likely to be greater in more productive ecosystems Barreiroand Pratt (1994) used a factorial experimental design to measure the interactive effects of nutrientenrichment and the herbicide diquat on primary productivity in microcosms Although structural

variables responded to nutrients, there was no effect of diquat on algal biovolume, chlorophyll a, or

protein levels In contrast, GPP was significantly reduced in treated microcosms These researchersalso reported that recovery was greater in systems with higher nutrient levels, most likely due tofaster contaminant dissipation (Pratt and Barreiro 1998) The influence of nutrient concentration oncommunity resistance and resilience was also reported by Steinman et al (1992), indicating thatfunctional responses to chemical perturbations are often context-dependent

Comparatively few studies have examined effects of contaminants on N cycling and flux inaquatic microcosms Petersen et al (2004) compared the effects of two antifouling biocides (zinc pyr-ithione, ZPT; and copper pyrithione, CPT) on nitrification and denitrification processes in sediments.Flux of nitrate from sediment increased significantly after additions of ZPT and CPT (Figure 32.3).This increase was a result of increased nitrification (NH4 → NO2 → NO3) and/or a decrease indenitrification (NO3→ N2) The greater sensitivity of nitrification observed in this experiment was

0 100 200 300 400 500 (b)

likely a result of greater functional redundancy of denitrification processes (Petersen et al 2004).Nitrification is a process performed by a limited number of bacteria, whereas denitrification is ageneral process performed by many species.

Microcosm and mesocosm experiments can also be used to compare functional responses ofcommunities derived from different sources, thereby providing an opportunity to understand howintrinsic features of an ecosystem may influence susceptibility to contaminants Stay et al (1988)reported that effects of fluorene on respiration and rates of recovery differed among communitiesdepending on the source of these organisms Fate of the insecticide chlorpyrifos in microcosms andits effects on community metabolism, decomposition, and nutrient cycling was influenced by thepresence of macrophytes (Kersting and van den Brink 1997) Balczon and Pratt (1994) comparedeffects of Cu on littoral and open water communities Effects of Cu on oxygen production andrespiration were reduced in microcosms with an established littoral zone, most likely because ofgreater adsorption and complexation by macrophytes and sediments Although these results showingvariable responses in different ecosystems complicate our ability to make broad generalizations,understanding the underlying mechanisms responsible for this variation may ultimately improve ourpredictive ability

Interactions between biotic and abiotic factors may also influence the response of primary ducers to contaminants Steinman et al (1992) observed that the physical structure and integrity

pro-of periphyton mats influenced resistance and resilience pro-of carbon fixation rates (a measure pro-ofprimary productivity) to chlorine exposure Hill et al (2000) measured bioaccumulation of Cd

by periphyton and subsequent effects on photosynthesis Effects of Cd on photosynthesis wereregulated by periphyton biomass, with greater effects observed in treatments with less bio-mass Although there were differences in community composition among biomass treatments,reduced effects in high biomass treatments were attributed to contaminant dilution and lower

Cd bioavailability

32.2.1.3 Ecosystem Recovery

Although the short duration of many microcosm and mesocosm experiments precludes assessment

of recovery, some researchers have used these experimental systems to evaluate improvements inecosystem processes when contaminants are reduced or eliminated Oviatt et al (1984) measuredrecovery of benthic respiration and nutrient flux for 21 months in mesocosms containing sedimentscollected along a pollution gradient Within 5 months, water quality characteristics (nutrients, chloro-

phyll a, and dissolved oxygen) and net system production were similar among treatments, indicating

that recovery may occur rapidly after pollutants are eliminated Rapid recovery (4 weeks) of tosynthesis following exposure of marsh plants to crude oil was also reported by Pezeshki andDeluane (1993) Similarly, periphyton productivity in outdoor experimental stream channels dosedwith the herbicide, hexazinone, was reduced by 80%, but recovered within 24 h following treatment(Schneider et al 1995) The estimated LC50 of hexazinone for periphyton production (3.6µg/L)

pho-was reported to be less than published values based on single species tests, demonstrating the greatersensitivity of this functional measure

32.2.1.4 Comparisons of Ecosystem Structure and Function

The majority of published microcosm and mesocosm experiments measure either structural or tional characteristics Because of concerns over sensitivity, variability, and the rate of response ofsome functional indicators, we suggest that a practical application of these experimental systems

func-is to compare the efficacy of structural and functional endpoints Questions such as the number ofreplicates required to detect statistical differences between reference and treated microcosms andthe rate at which structural and functional variables respond to chemical stressors are of particularimportance Rigorous control over exposure conditions and the ability to manipulate several variables

simultaneously in microcosm and mesocosm experiments provide a unique opportunity to compareeffects of stressors on structural and functional characteristics (Culp et al 2003).

The conventional wisdom is that, because of functional redundancy and greater variability offunctional measures, changes in community composition are likely to occur before alterations in eco-system processes are observed (Schindler 1987, Schindler et al 1985) However, like many examples

of conventional wisdom, there are exceptions to these generalizations in the literature Some studieshave reported that functional measures are equally sensitive or even more sensitive than measures

of abundance, biomass, or community composition Functional measures (periphyton productivity)were considerably more sensitive than structural measures (periphyton biomass; macroinvertebrateabundance and drift) to the herbicide hexazinone in outdoor stream mesocosms (Schneider et al.1995) Concentration–response relationships between copper and several functional endpoints wereestablished by Hedtke (1984) in laboratory microcosms GPP and respiration were reduced at 9.3µg

Cu/L, but changes in community composition were observed only at higher concentrations of Cu(30µg/L), suggesting that ecosystem processes were more sensitive than structure in these experi-

ments Clements (2004) reported that EC10values for heavy metals based on community respirationand abundance of metal-sensitive species were similar Jorgensen et al (2000) calculated no effectconcentrations (NECs) for a variety of structural and functional measures in large pelagic meso-

cosms exposed to anionic surfactants (linear alkylbenzene sulfonates) Biomass (as chlorophyll a)

and biovolume of the dominant taxonomic groups were affected only at the highest concentrationstested, whereas phytosynthetic activity was the most sensitive parameter for phytoplankton After4.5-day exposure, NECs for photosynthetic activity were similar to values for structural characterist-ics (abundance of protozoans, crustaceans, and diatoms) Detenbeck et al (1996) reported that grossproductivity of periphyton was significantly reduced in microcosms exposed to 15µg/L of atrazine,

a concentration that significantly reduced survival of Daphnia but had no effect on other response

variables measured (biomass of cattails; growth of tadpoles and fathead minnows) Fairchild et al.(1987) compared community composition, nutrient dynamics, leaf decomposition, and primary pro-duction in experimental streams exposed to clean and contaminated (triphenyl phosphate) sediments.Sediment exposures altered patterns of macroinvertebrate drift and increased nutrient retention, buthad no effects on leaf decomposition

Some stream microcosm experiments have been conducted specifically to validate results oflaboratory toxicity tests and provide an opportunity to compare ecosystem functional measureswith more traditional toxicological endpoints Exposure of stream mesocosms to relatively highlevels of Cd (143µg/L) resulted in reduced abundance of grazing snails and increased periphyton

biomass, but had no effects on gross or net primary productivity (Brooks et al 2004) Concurrent

single species toxicity tests with Ceriodaphnia dubia and Pimephales promelas showed that survival

was significantly reduced at this concentration The lack of a response at lower Cd concentrations(15µg/L) was attributed to high concentrations of dissolved organic materials in these effluent-

dominated streams, which likely reduced metal bioavailability Richardson and Kiffney (2000)compared structural and functional measures in outdoor experimental streams dosed with mixtures

of metals Significant concentration–response relationships were developed for several measuresrelated to mayfly abundance and drift, but no effects of metals on algal biomass or bacterial respirationwere observed These researchers recommended that regulatory agencies should include estimates

of mayfly abundance and richness as indicators of metal impacts in streams

Balczon and Pratt (1994) derived maximum allowable toxicant concentrations (MATCs) forlittoral and aquatic microbial microcosms exposed to Cu The MATCs were generally greater forprocess (photosynthesis, respiration) as compared to measures of community composition (species

richness, chlorophyll a biomass), indicating greater sensitivity of structural responses Similar

res-ults were reported by Melendez et al (1993) in which microbial communities were exposed to theherbicide diquat Changes in productivity and respiration were observed only at the two highest con-centrations (10 and 30 mg/L), and these ecosystem-level responses recovered after 2 weeks exposure

In contrast, the MATC for protozoan species richness and bacterial cell density was 0.32 mg/L, and

these responses showed little evidence of recovery Barreiro and Pratt (1994) observed that gross

community productivity of periphyton was considerably more sensitive than chlorophyll a to diquat.

The lowest observable effect concentration (LOEL) for P/R in planktonic communities exposed tofluorene, a polycyclic aromatic hydrocarbon (0.12 mg/L), was comparable to chronic toxicity valuesbased on single species tests with cladocerans, chironomids, and bluegill (Stay et al 1988) How-ever, the magnitude of change in ecosystem processes did not reflect the near complete elimination ofmost zooplankton at concentrations exceeding 2 mg/L Exposure of microbial communities derivedfrom natural sediments to a fungicide, herbicide, or insecticide reduced microbial biomass but had

no significant effects on respiration or denitrification (Widenfalk et al 2004) These differencesamong experiments suggest that not only is the relative sensitivity of structural and functional meas-ures contaminant-specific, it may also vary with level of contamination and characteristics of theexposure system

In a comprehensive analysis of structural and functional responses of outdoor aquatic mesocosms

to the insecticide diflubenzuron, Boyle et al (1996) observed relatively little effects on GPP, but

a significant increase in chlorophyll a, and reduced abundance and species richness of secondary

consumers (zooplankton, insects, and bluegill) in treated mesocosms (Figure 32.4) Although munity metabolism and decomposition rates were affected in microcosms treated with chlorpyrifos,these processes were generally less sensitive and occurred only after changes in structural measures,suggesting functional redundancy of these systems (Brock et al 1993) Cuppen et al (2002) repor-

com-ted that no observable effects concentrations (NOECs) for decomposition rate of Populus leaves

and abundance of several macroinvertebrate shredders were similar Interestingly, the structural and

0 20 40 60 80 100

120

Bluegill (adults) Bluegill (recriuts)

Zooplankton Insects

FIGURE 32.4 Effects of the insecticide, diflubenzuron, on structural (abundance, biomass, richness) and

functional (primary production) measures in lentic mesocosms Increased chlorophyll a biomass in mesocosms

treated monthly and biweekly compared with controls was attributed to reduced grazing pressure (Data fromBoyle et al (1996).)

functional NOECs derived from this microcosm experiment were considerably less than the LC50value derived from standard toxicity tests using known sensitive organisms Because of the com-plex and often unpredictable relationship between structural and functional measures observed insome studies, we suggest that an appropriate strategy will be to include endpoints reflecting bothpattern and process when designing microcosm and mesocosm experiments We agree with Brock

et al (1993) that an understanding of contaminant effects on ecosystem function cannot be fullyappreciated without an understanding of community structure

32.2.1.5 Effects of Contaminants on Other Functional

Measures

Although we traditionally consider changes in community composition to be a structural measure,some researchers consider alteration in the abundance of groups that play an important functional role(e.g., abundance of shredders in streams; abundance of grazing zooplankton in lakes) to be intimatelyrelated to ecosystem processes and therefore an appropriate surrogate functional measure (Gruessnerand Watzin 1996, Wallace et al 1996) Field (Wallace et al 1982) and stream microcosm experiments(Carlisle and Clements 1999) have assessed the effects of contaminants on functional feeding groupcomposition The export or loss of materials from an ecosystem is an important functional processthat has received relatively little attention in the ecotoxicological literature Similarly, emergence ofadult insects represents a net transfer of energy from aquatic to terrestrial habitats and therefore could

be considered a functional response Gruessner and Watzin (1996) reported increased emergence ofinsects in stream microcosms treated with atrazine Culp et al (2003) measured increased algalbiomass and changes in taxonomic composition in stream mesocosms dosed with 5% or 10% pulpmill effluents Although most measures of benthic macroinvertebrate community composition weresimilar between treatments, emergence of mayflies was significantly reduced in treated streams.Increased nutrient loading from nonpoint sources is expected to have significant impacts on aquaticecosystem structure and function Elevated levels of nutrients are likely to produce excess organicmatter, which will result in greater biomass or increased export An understanding of the ability

of an ecosystem to assimilate this excess production is necessary to predict the potential negativeeffects of nutrient enrichment Barron et al (2003) observed no change in GPP, NPP, respiration,

or biomass following 27 months of nutrient addition in marine rocky intertidal mesocosms Carbonbudgets calculated in this system showed that the lack of a response to nutrient enrichment resultedfrom increased export of dissolved organic carbon The ability of an ecosystem to export relativelylarge amounts of excess carbon may offer some protection from nutrient enrichment in coastal areas

32.2.2 MICROCOSMS ANDMESOCOSMS INTERRESTRIALRESEARCH

While aquatic ecotoxicologists have long recognized the value of microcosms and mesocosms asresearch tools for investigating effects of contaminants on ecosystem processes, these systems havereceived considerably less attention in terrestrial ecotoxicology (Figure 32.1) Fraser and Keddy(1997) reported that despite a general increase in the use of microcosms and mesocosms to addressbasic and applied research questions during the mid-1990s,<5% of the studies were conducted in

terrestrial ecosystems For practical reasons, much of the research using terrestrial microcosms andmesocosms has focused on soil microbial systems As described in previous chapters, alterations

in abundance and activity of soil microbes can have significant effects on decomposition rates andnutrient processing By examining both structure and function of soil communities, it is possible

to link direct and indirect effects of contaminants, and identify important regulating mechanisms(Bogomolov et al 1996) Although the vast majority (>95%) of soil respiration in terrestrial ecosys-

tems is a result of microbial activity, nematodes, arthropods, annelids, and other organisms contributesignificantly to decomposition Experiments have been conducted to determine the relative contri-butions of microbes and invertebrates to detrital food webs Salminen et al (2001) measured the

effects of heavy metals and detritivores (enchytraeid oligochaetes) on respiration in soil microcosms.Invertebrate detritivores were eliminated at the highest Zn concentrations (>2500 mg/kg) and effects

of Zn on microbial respiration were dependent on detritivore density Clear effects of Zn were onlyobserved in treatments with the greatest density of detritivores These researchers reported someevidence of functional redundancy, but noted that elimination of species that play an important role

in regulating soil microbial processes will have disproportionate impacts on ecosystems

Unique properties of the soil environment may complicate our ability to assess bioavailabilityand contaminant effects For example, in aquatic ecosystems, the assumption that contaminants areevenly distributed within the water column is generally valid Although most experiments conductedwith soil microcosms attempt to achieve a relatively homogeneous distribution of contaminants,chemicals in natural soils are often patchily distributed In addition, small-scale spatial variation

in the physicochemical characteristics of soils may alter chemical bioavailability (Salminen andSulkava 1997) Some of the characteristics of soils that modify chemical bioavailability, such asparticle size and amount of organic material, are analogous to properties of aquatic sediments Wewill see that experimental designs that account for soil type and modify soil characteristics are acommon feature of many terrestrial microcosm experiments In the following sections, we willreview some of the experiments conducted to assess the effects of heavy metals, organics, and otherstressors on soil processes

32.2.2.1 Heavy Metals

Effects of heavy metals on ecosystem processes have been measured in soil microcosms containingboth natural and synthetic assemblages of microbes In addition to measuring bacterial, fungal,nematode, and arthropod abundance and biomass, typical functional endpoints reported in thesestudies include soil respiration, nitrification, and N mineralization Although some experimentalstudies have directly measured leaf litter decay rates (Cotrufo et al 1995, Kohler et al 1995),most of the research has focused on underlying microbial processes that regulate decomposition.Bogomolov et al (1996) measured a suite of structural and functional characteristics in microcosmsexposed to Cu Increased pools of dissolved organic N and ammonium, reduced soil respiration, andreduced litter decay were observed in Cu-treated microcosms Soil respiration was the most sensitiveprocess examined, with effects observed at 50 mg Cu/L These changes in ecosystem processes werethe result of direct toxic effects on structural measures (reduced microbial biomass and abundance

of nematodes)

As in aquatic ecosystems, one major advantage of microcosm and mesocosm experiments isthe ability to manipulate several independent variables or site characteristics to quantify factorsthat determine contaminant effects and bioavailability Khan and Scullion (2000) examined effects

of heavy metals on microbial biomass, respiration, and mineralization in soils with varying clayand organic content Metal bioavailability and effects were generally greater in sandy loams ascompared to soils with higher organic content Ammonification and nitrification were found to bemore sensitive to Cd in calcareous soils than noncalcareous soils (Dusek 1995) Nitrate accumulated

in Cd-treated calcareous soils primarily as a result of the greater sensitivity of nitrite oxidizers.Niklinska et al (1998) established concentration–response relationships between heavy metals andrespiration in litter collected from beech-pine and oak forests Despite differences in physical andchemical characteristics of the two litter types, storage time was more important in controlling effects

of metals than litter type Decreases in litter respiration rates with storage time were most likely aresult of rapid reduction in the amount of easily degraded material and/or increased respiration rateimmediately following litter collection (Niklinska et al 1998) For litter respiration to be a usefulindicator of ecosystem responses to contaminants, a better understanding of the effects of storagetime is required On the basis of the estimated EC50 values for respiration (Figure 32.5), Niklinska

et al (1998) reported the following range of toxicity: Cu> Zn ≥ Cd  Pb It is interesting to note

Beech-pine Oak

FIGURE 32.5 Average EC50 values for soil respiration rates measured in microcosms exposed to heavy

metals Forest litter was collected from beech-pine and oak-hornbeam forests (Data from Niklinska et al.(1998).)

0.30

Control litter

Polluted litter

FIGURE 32.6 Litter decay rates of oak leaves (Quercus ilex) collected from control and metal polluted sites

after 2 months exposure to clean and metal contaminated soil CC= soil and litter from control site; PC =polluted soil and clean litter; PP polluted soil and polluted litter; CP= clean soil and polluted litter (Data fromTable 3 in Cotrufo et al (1995).)

that this order of toxicity is quite different from most aquatic studies in which Cd is significantlymore toxic to primary producers than either Cu or Zn

Information concerning the relative toxicity of heavy metals in soil versus leaf litter is necessaryfor remediation of metal contaminated sites In microcosm experiments Cotrufo et al (1995) com-pared soil respiration, microbial and fungal biomass, and litter decomposition rates of oak leavescollected from clean and metal-polluted sites Lower respiration rates and reduced fungal abundancewere observed in litter contaminated by metals Decomposition rates were significantly reduced formetal-polluted litter, regardless of the soil source (Figure 32.6) These data indicate that heavy metals

in litter were directly responsible for reduced decomposition rates The addition of organic materialthrough natural decomposition processes can reduce the effects of heavy metals in terrestrial eco-systems Boon et al (1998) observed that the combined effects of low pH and Cu contamination onsoil ecosystem processes were significantly reduced in microcosms planted with Cu-tolerant grass.The positive influence of Cu-tolerant plants on soil processes, which provided organic material and

reduced metal bioavailability, has important implications for restoring habitats impacted by heavymetals.

32.2.2.2 Organic Contaminants and Other Stressors

Microcosms and mesocosms have been used to examine the effects of organic contaminants interrestrial ecosystems, with considerable effort devoted to assessing responses of soil communities.Some papers have adopted a comparative approach and examined responses to a large number

of organic chemicals on a few ecosystem-level endpoints (Pell et al 1998) Others have examinedeffects of a single chemical or class of chemicals on several ecosystem processes An excellent series

of papers describing the use of terrestrial microcosms in ecological risk assessment was published

in the journal Ecotoxicology in 2004 (Volume 13, Issue 4) This series of publications was the result

of a joint effort by university, private, and governmental partners to develop a standardized methodfor conducting microcosm experiments in terrestrial ecosystems The terrestrial model ecosystems(TME) experiments were conducted with intact soil cores collected from several different field sites.Initial experiments focused on ecosystem responses to carbendazim, a fungicide used extensivelyfor agricultural applications in Europe Experiments conducted at sites in the United Kingdom,Germany, Portugal, and the Netherlands examined effects of carbendazim on nutrient cycling andorganic matter processing In general, nutrient dynamics were not affected by contaminant exposureduring the 16-week experiment, a result that was also supported by field experiments conductedsimultaneously (Van Gestel et al 2004) In contrast to these results, Burrows and Edwards (2004)reported significant effects of carbendazim on nutrient dynamics, soil dehydrogenase activity, andseveral structural measures in soil microcosms treated with similar carbendazim concentrations.Alteration in decomposition rate of organic matter is likely to affect nutrient dynamics and istherefore considered an integrative functional endpoint in microcosm tests Because decompositionand nutrient dynamics in soils are closely coupled and regulated by microbial processes, structuralchanges in microbial communities are likely to have important effects on ecosystem function Thefungicide dithianon had relatively little influence on decomposition rates but significantly inhibitedmicrobial activity in soil microcosms (Liebich et al 2003) These alterations in functional processescorresponded to changes in microbial community composition and fungal biomass Forster et al.(2004) used cellulose paper as a standardized material to measure decomposition and assess inver-tebrate feeding activity A significant concentration–response relationship between carbendazimconcentration and decomposition rate was observed, with treated microcosms having 40%–80%lower decomposition and showing a significant reduction in invertebrate feeding activity SimilarLC50 values were calculated for both microcosm and field experiments (7.1–9.5 kg/Ha), lendingadditional support for the application of TMEs to assess contaminant effects on soil processes.Soil respiration, measured as evolution of CO2, is also a sensitive endpoint in microcosm experi-ments Salminen et al (2002) compared the effects of several organic chemicals and heavy metals onmicrobial respiration rate in soil microcosms This functional measure was correlated with microbialbiomass and estimates of microarthropod and nematode abundance Soil respiration was reduced inall contaminant treatments, and effects increased with chemical concentration Changes in respira-tion were accompanied by decreases in microbial biomass and abundance of soil organisms resultingfrom direct toxicity in most treatments Because some of these responses were not observed untillate in the study, Salminen et al (2002) recommend that soil microcosm experiments should beconducted for a sufficient period of time to avoid false conclusions regarding responses

Few investigators have conducted comparative studies of organic chemicals under differentphysicochemical conditions in soil microcosm experiments Chen and colleagues (Chen and Edwards

2001, Chen et al 2001) employed soil microcosms to examine the effects of fungicides on severalecosystem processes Soil properties, especially soil texture, regulated the effects of fungicides onsoil microbial activity and nutrient dynamics (Chen and Edwards 2001) Soil microcosms amendedwith either alfalfa leaves or wheat straw, materials with very different C:N ratios (alfalfa leaves,