Standardized aquatic microcosms By Frieah B. Toub
grazers are more sensitive and are re-
Ecotoxicology is a three-legged stool that, to date, has been balanced on two legs: chemical fate and single-species toxicology. These are necessary but insufficient components of the field. The third leg, that of organism interactions, has been slower to develop as a tool for assessing how an ecosystem is affected by and recovers from chemical stresses. All three aspects must be understood if we are to predict ecosystem
duced for a longer period of time. Such unexpected responses make understanding interspecies relationships essential for predicting ecosystem responses. If ecological interactions are to be studied in ecotoxicology, how large, complex, and site-specific must the study units be? Mesocosms, such as ponds or tanks. and enclosures of tural aquatic communities are being used as test units for new pesticides. In gen-
points. A test system is needed that includes important species interactions and allows clear, unambiguous demonstrations of the effects of a test chemical without being too complex to understand. Regulatory processes require experimental results that can be verified by subsequent studies at the same or other laboratories. Ow standardizedmicrocosm The Standardized Aquatic Microcosm (SAM) differs from other micro-
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The typical experiment consists of 24-30 microcosms, with six replicates in each of four o r five treatment groups, of which one group is a control. The microcosms, consisting of sterile medium and sediment, are inoculated with algae on Day 0 and with animals on Day 4, and are randomly assigmd to the treatment groups on Day 7. Organism abundances, pH, and oxygen dynamics are measured twice each week for the duration of the experiment, usually 63 days. Nutrients and the test chemical are measured twice a week for the first month and weekly thereafter. The exact procedures can be modified as required for the specific test, but some changes may affect the sensitivity with which the microcosm can detect an effect-for example, increasing the pH buffering or chelation potential of the medium changes the responses to toxic metals. The end points of the SAM involve changes in algal abundance, species dominance, magnitude and timing of grazer populations, respiration and photosynthesis patterns, and nutrient cycling. Data are handled by a computer program that converts data from the l a b
pleted, the zooplankton bloom, especially Dophnia m n g ~ is , increasing and consuming the algae. As the zooplankton overgraze the algal food sup ply, algal nutrients are recycled and thus become available for sustained algal growth. The effects of test chemicals are observed within the context of the processes described above. Malathion, an insecticide, killed all of the Daphnia present, and an algal bloom resulted. The malathion degraded in about a week, and reinoculated Duphniu-having a huge food supply-survived, ate, reproduced, and eliminated the algal bloom (IS). Streptomycin, an antibiotic and a selective algicide, temporarily reduced photosynthesis and eliminated some of the algal species. Grazer populations were reduced because of the reduced food supply, although some direct toxicity also might have been involved (IS. 16). Copper acted as both an algicide and zooplankton inhibitor: At low concentrations, it eliminated the Daphniu and allowed dense algal blooms of a few resistant algal species. Reinoculated Duphniu were able to become established, and the communities
Duphniu populations was delayed for a longer time. At low concentrations, the effects were temporary; at progressively higher concentrations, the effects were more severe and lasted longer (I 7-20). Three other laboratories performed the copper sulfate experiments and obtained similar results (18-20). The participating laboratories were DuluthEPA and the University of Minnesota at Duluth; Aberdeen Proving Grounds (U.S. Army, Maryland); and Marine Bioassays, a private environmental engineering company. Seven experiments were compared in which the nature of the effects was the same, and in each case the higher concentrations were associated with more severe effects and longer times before recovery. The timing and magnitude of the effects differed enough between experiments to be statistically distinct; therefore, each experiment must have its own simultaneous control. Other laboratories have performed SAM experiments successfully on other chemicals. For example. EPA at Corvallis, OR, has studied atrazine (21. 22). a widely used herbicide, and the
ratory units to standard units, performs statistical analyses (ANOVA), and provides tables and graphics to aid in reporting and interpretation. The statistical properties of the experimental design allow the graphical display of the test’s statistical power and differences between the treatment groups and controls (13, 14). Past work was performed on a mainframe, but programs using popular microcomputers are now being developed and tested.
moved along a path similar to the controls. At higher concentrations, more and more of the algal species were eliminated and the development of
Army at Aberdeen Proving Grounds has studied effects of brass and graphite (23-24) on various microcosms.
Accomplishments to date About 30 SAM experiments have been performed successfully on a variety of chemicals in various laborate ries. The microcosm has a predictable sequence of nutrient depletion as algal biomass is accumulated by the IO algal species; as algal nutrients are being de-
Frieda Toub
Comparison of results Treatment with low concentrations of copper sulfate led to algal blooms because grazers were eliminated for a longer period than it took some algae to recover (25). Similar results have been found in freshwater studies (26)and some marine studies (27). Atrazine also demonstrated effects in microcosms similar to those observed in ponds (2122). Comparison of microcosm results with those in natural communities suffers from the same problems as comparisons between different natural communities, o r even in the same community over time. For example, Envimn. Sci. Technol.. WI. 23. No. 9. 1989 1W5
the large marine enclosures (CEPEX) were tested in June and again in September (27). Treatment with copper in September showed responses like those observed in the SAM tests: Most zooplankton were eliminated and primary production increased. However, when the CEPEX communities had been treated with the same copper concentration in June, both zooplankton and primary production decreased. The development of an algal bloom in the September experiment when zooplankton were more abundant suggests that grazing was controlling algal abundance. Thus the microcosms simulate the effects on a community in which algal abundance is controlled by grazing.
Simulation modeling MICMOD (MICrocosm MODel) is a simulation model that includes the algal nutrients (N and P), eight phytoplankton groups, and five zooplankton groups (28-29). Toxicant effects can be modeled by changes in physiological rate parameters such as nutrient uptake, nutrient assimilation, photosynthesis, and respiration for phytoplankton; and rates of grazing, assimilation, respiration, growth, reproduction, natural mortality, and chemical-induced mortality for animals. Nutrient cycling is modeled by phytoplankton uptake and release, and animal grazing, egestion, and excretion. For example, in the case of streptomycin, toxicant effects were modeled as mortality of zooplankton and growth reduction of phytoplankton, with parameter values based on single species experiments (30, 31). The model output suggests some testable hypotheses; for example, the streptomycin’s effects on Daphnia may act through delayed mortalities or sublethal effects of reduced feeding or fecundity, or the mortality may have been caused by excess ammonia concentrations associated with the damaged algae. Genetically engineered microorganisms Although the microcosms reported here were developed for testing chemical effects, they are being used with some modification for studying the effects of microbes on ecological processes. The SAM is currently being used to study the degradation of a sensory irritant by naturally occurring bacteria at Aberdeen Proving Ground (32). The Mixed Flask Culture Microcosm has been used to test the naturally occurring Bacillus thuringiensis israelensis for its effects on microcosms that included the mosquito (Aedes aegypti) and midges (Tanytarsus) in addition to the other organisms (33. 34). For genetically engi1066
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neered organisms some methods will require modification to minimize risks of contamination.
Future uses Potential industrial uses include the testing of new chemicals when they are available in small quantities and when too little may be known to consider field testing. Determining fate and ecological effects in the same test is more cost-effective because both types of information are needed. If environmental testing were performed early in the development of new chemicals, companies would be spared the cost of developing and scaling up procedures for chemicals that are not likely to pass regulatory review. Early testing would help define alternative structures or formulations that might be more effective and less ecologically damaging in natural ecosystems. The use of microcosm data for regulatory decisions has been discussed (9, 35). They could be considered at several points, ranging from a high-level tier prior to field testing to a low-level, screening tier. As the credibility, relevance, and applicability of microcosm protocol are demonstrated, agencies will be better able to fit such data into their regulatory decision-making process. Acknowledgments Considerable thanks are owed Michael C. Harrass, Buzz L. Hoffmann, John C. Matheson 111, Wayne G. Landis, and William van der Schalie, the laboratories that took part in the interlaboratory tests (mentioned above), and my colleagues and coauthors cited in the references. Funding has been received from the Food and Drug Administration, EPA, the U . S . Army, and the Holland Fund. References (1) Cairns, J . , Jr. Community Toxicity Testing; ASTM Spec. Tech. Pub/. 920; 1986, 165-85. (2) Giesy. J. I?, Jr. Microcosms in Ecological Research; U.S. Department of Energy: U.S.Government Printing Office: Washington, DC, 1980; CONF-781101. (3) Franco, I? J . et al. Eni.iron. Toxicol. Chem. 1984,3, 447-63. (4) Giddings, J. M. Hazard Assess. Chem.: Cum Dev. 1983. 2. 45-94. ( 5 ) Giddings, J. M~.’ASTMSpec. Tech. Pub/. N o . 920. 1986 121-34. (6) Shannon, L. J: et al. ASTM Spec. Tech. Publ. No. 920; 1986, 135-57. (7) Sheehan, P. J . ; Axler, R. E ; Newhook, R. C. ASTM Spec. Tech. Publ. No. 920; 1986, 158-79. (8) Taub, E B.; Kindig, A. C . Standardized Aquatic Microcosm Prorocol. Final Report. Section I1 of V, FDA Contract 22383-7000, modified 1988: Society of Environmental Toxico~ogy and Chemistry Short Course (available from the author). 1986. (9) Fed. Regist. 1987,52(187), 36344-352. ‘10) Taub. E B. In Algae as Ecological Indi(.arors: Shubert, L, E , , ~ d , Academic : Press: New York, 1984: pp. 363-94.
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(1 1) Taub, E B. In Aquatic Ecotoxicology Vol. 11; Boudou, A , , Ribeyre, E , Eds.; CRC Press: Boca Raton, FL, in press. (12) Taub, E B.; Crow, M. E. In Microcosms in Ecological Research; Giesy, J. E , Ed.; DOE Symposium Ser. 52, U . S . Government Printing Office: Washington, DC, 1980, pp. 69-104; CONF-781101. (13) Conquest, L. L. Int. J . Environ. Stud. 1983,20, 209-2 1. (14) Conquest, L. L.; Taub, E B. ASTMSpec. Tech. Publ. NO. 1027: 1989.12. 159-77. (15) Taub, E B. et al. ASTM Spec. Tech. Publ. N O . 802; 1983, 5-25. (16) Harrass, M. C.; Kindig, A. C.; Taub, E B. Aauar. Toxicol. 1985. 6. 1- 11. (17) Taub, !I B. In Chapter 13, Mulrispecies Toxicio Testing; Cairns, J., Jr., Ed.; Pergamon: Oxford, U.K., 1985, pp. 165-86. (18) Taub, E B.; Kindig, A. C.; Conquest, L. L. ASTM Spec. Tech. Publ. 920: 1986, 93-120. (19) Taub, E B.; Kindig, A. C.; Conquest, L. L. ASTM Spec. Tech. Publ. No. 971; 1988, 10, 384-405. (20) Taub, E B. et al. ASTM Spec. Tech. Publ. NO. 1007; 1989,11, 368-94. (21) Larsen, D. I?, DeNoyelles, E , Jr.; Stay, E Environ. Toxicol. Chem. 1986,5, 17990. (22) Stay, E S. et al. ASTM Spec. Tech. Publ. NO. 865; 1985,75-90. (23) Haley, M. V. et al. ASTM Spec. Tech. Publ. NO. 971; 1988, 10, 468-79. (24) Landis, W. G. et al. U.S. Department of Defense: Aberdeen Proving Ground, MD, 1988; CRDEC-TR-88133. (25) Harrass, M. C.; Taub, E B. ASTM Spec. Tech. Pub/. NO. 865; 1985, 57-74. (26) McKnight, D. Limnol. Oceanogr. 1981, 26. 518-31. (27) Thomas, W. H. et al. Bull. Mar. Sci. 1971,27, 34-43. (28) Swartzman, G . L.; Rose, K. A. Ecol. Model. 1984, 22, 1231-34. (29) Rose, K. A. et al. Ecol. Model. 1988, 42, 1-32. (30) Swartzman, G . L.; Kaluzny, S . E Ecological Simulation Primer. Macmillan: New York, 1987. (31) Swartzman, G. et al. Aqua?. Toxicol. 1989,14, 109-30. (32) Haley, M. V. et al. “Naturally Occurring Organisms Resistant to Dibenz-l,4Oxazepine”; Abstract, Program of the Society of Environmental Toxicology and Chemistry; SETAC: Washington, DC, 1988, p. 150. (33) Shannon, L. J. et al. ASTM Spec. Tech. Publ. No. 1027; 12, in press. (34) Shannon, L. J . ; Flum, T. E. “Survival and Effects of Bacillus thuringiensis var. lsraelensis Introduced into Aquatic Microcosm Communities”; U . S . Environmental Protection Agency. U . S . Government Printing Office: Duluth, MN, 1987; DU E104, PPA 12, Milestone 7254. (35) Harrass, M. C.; Sayre, E G. ASTM Spec. Tech. Pub/. No. 1027; 12, in press.
Frieda B’ received her B’A‘ degree in biology and chemistry at Newark College of Arts and Sciences of R~~~~~~university in 1955, and her M.s*degree and ph.D. in ZoOlogY at Rutgers University in 1957 and 1959. She- is currently professor of Jisheries and adjunct professor Of the lnStimte Of Environmental Sciences at the University of Washington,Seattle. Much of her researchfocuses on the organization of ecological communities and their responses to stress.
