Exploring Allelochemistry in Aquatic Systems - ACS Symposium

C h a p t e r 13

Exploring Allelochemistry in Aquatic Systems K. Irwin Keating

Downloaded by UNIV OF ROCHESTER on September 2, 2017 | http://pubs.acs.org Publication Date: January 8, 1987 | doi: 10.1021/bk-1987-0330.ch013

Rutgers University and New Jersey Agricultural Experiment Station, New Brunswick, NJ 08903 Allelochemistry in aquatic systems has been little studied. A secondary, opportunistic, form of activity resulting from the natural capacity of water as a carrier complicates studies in aquatic systems. Reflecting this complexity, experimental manipulations of apparent allelochemical relationships require unusual parameters of control, including control of inorganics to ppb levels and absolute partitioning of all organisms (including bacteria) in test systems. Comparisons of more than 200 pairs of algal species, all isolated from Linsley Lake, Connecticut, indicate that two-thirds of these pairs exhibit allelochemical activities. Since only autoclave-labile activity in cell-free filtrates was considered, and since much additional activity (not based solely on metabolites with these characteristics) was also noted, an ubiquitous presence and a pervasive influence of allelochemical activity on aquatic communities is postulated.

During the last several decades, as its potential for generating natural productbased pesticides emerged, the study of allelochemistry in terrestrial ecosystems has intensified. Yet, during the same period little effort has been invested in the pursuit of allelochemistry in aquatic ecosystems. In spite of this apparent neglect many field and laboratory examples have accumulated. A number of reviews are available; among the more extensive are those by Hartman, Lucas, Schwimmer and Schwimmer, Pourriot, Ruggieri, Keating, Maestrini and Bonin, and Provasoli and Carlucci (1-8). This lack of development in aquatic allelochemistry appears an anachronism. It is not. Specifically reflecting the pervasive and peculiar effects of water, analysis of allelochemical events in aquatic systems has presented unique problems. Among the most significant are (a) the widespread and unpredictable occurrence of secondary activity, and (b) the difficulties of distinguishing between ultra-trace nutrient requirements and allelochemical effects. Both generate a need for unusually rigid experimental control. The state of the art of aquatic organism culture has been inadequate to this challenge. Only recently (9) has the level of control been sufficient 0097-6156/87/0330-0136$06.00/0 © 1987 A m e r i c a n C h e m i c a l Society

Waller; Allelochemicals: Role in Agriculture and Forestry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


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to insure that organisms, other than relatively few selected algae, (a) could be isolated and kept, while isolated, in good health, and (b) kept on a long-term basis in this state in defined, controlled, and repeatable experimental circumstances. Thus, only recently has it been possible to partition the dimensions of an aquatic niche to suit the demands of analysis of allelochemical events. Primary allelochemical activity has essentially the same genesis and role in terrestrial and aquatic systems—offering an advantage to the producing organism which justifies the investment of resources in some active metabolite. In the aquatic environment, however, the water carrying the active substance circulates, randomly bathing the surfaces of many organisms other than the target. Some of these nontarget organisms respond to the active metabolite. This secondary response occurs only because the active substance happens to be there. It requires neither a competitive advantage for the producer, nor an association between the producer and target organisms. In fact a secondary target organism may benefit from an interaction that offers nothing to the producer. The absence of a special relationship that generates some competitive or symbiotic advantage for the producer makes the possibilities for such secondary actions much more numerous, and much less likely to be recognized, than would be the possibilities for primary reactions. Thus, to assure the needed partitioning of, and the correct identification of, producer and target organisms, exertion of exceptionally tight control over experimental details becomes imperative. Sometimes the active metabolic product of a planktonic organism is surprisingly familiar. One algal species, Chlorella vulgaris, manages to produce both hydrogen cyanide (10) and chlorellin (1^ 12). Chlorellin, a toxin peculiar to this species, is a mixture of chlorophyllide derivatives (13). Undoubtedly, such toxins offer Chlorella an advantage in its competition for a niche. They also affect many organisms which in no sense compete with Chlorella for a niche or for any single dimension (e.g. nutrients, space) of a niche. Thus, this single organism offers at least two distinct types of allelochemically active metabolic products and each, in turn, offers a capacity for both primary and secondary actions. This dichotomy of allelochemical activity is clearly evident among algal species that dominate the waters of Linsley Lake, North Branford, Connecticut. The natural programming of the series of algal species which dominate in the lake is greatly influenced by allelochemistry. In our laboratory tests the products of bloom-dominant blue-green algae (Cyanophyta) were either negative or neutral in their effects on predecessors and/or positive or neutral in their effects on successors (14). That is, they inhibited the algae they were replacing and left behind selective fertilizers when they, themselves, ceased to dominate. Comparisons of algal growth in waters collected before, during, and after blooms indicated that these waters produced positive, negative, and "neutral" effects in a pattern similar to those of the bloom-dominant blue-greens that had been prevalent. The "neutral" effects probably indicate that we were not testing enough parameters, or were not properly handling the metabolic materials. In Linsley Lake this pattern persisted for at least 5 years. In a sense dominants selected their successors. This, however, was a secondary action. No value to the producer is required to justify its existence. The producer would no longer be present when the allelochemistry is in action. A s a dominant weakened, it would make little difference to its survival as a species, or to its reoccur-



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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

rence a year later, during the next annual bloom sequence, what organism was favored sufficiently to dominate immediately after it in the current year. If some dominant left behind a product damaging to the organism that dominated next, that "next" organism would cease to be next. Some other of the hundreds of candidates would replace it in the annual bloom sequence. Thus, it would be most unlikely that a producer would negatively affect its successor. On the other hand, promoting its own replacement is also not a reasonable evolutionary basis in an organism for the selection of a trait that results in production of metabolically active extracellular materials. When a producer releases metabolites that selectively favor successor-dominants, this benefit results from a successor's opportunistic use of some metabolite produced for another purpose. It is likely that the general, justifying, basis for the production of such active metabolites is in the widespread metabolite-based interference with those organisms with that producers do actively compete for dominance. Once this variety of metabolic material is present in the water, any possible successordominant which could use the material to its advantage would be favored. Methods and Materials Complete methodology for algal experiments (15) and for all zooplankton/algal experiments (9) are available elsewhere. Results and Discussion Several hundred algal pairs were tested for allelochemical activity and fully two-thirds (Table I) showed activity. In most tests algae were isolates from Linsley Lake. Except as noted in Table I tests were limited to autoclave-labile allelopathic materials carried in the cell-free filtrates of bacteria-free cultures of producer-algae. This usually translated into lability to a sudden increase in temperature, pH, or both. Most of our algae produced effects on most of our algae. Yet, no effects were inappropriate to the natural sequence of bloom dominance in the lake. In addition to the effects tallied in Table I, a variety of additional effects of the same cell-free filtrates was observed. Because so many instances of allelopathy were readily demonstrated, detailed study was restricted. Heat/pH-labile materials were selected for in-depth study including concentration, isolation, and partial identification of substances produced by dominant algal species (15). Study of materials related to remaining allelochemical events was limited to confirmation that the activity in the filtrate was tied to the presence of dissolved organic material that could be removed by activated charcoal. Since algal .cultures were bacteria-free and unialgal, and were always in inorganic media, all organics in filtrates of those cultures could be identified as having been produced by the algae. It is, specifically, the sheer number of instances of allelochemical activity in these studies which militates against drawing too close a parallel between the roles this phenomenon plays in terrestrial and aquatic systems. In contrasting the significance of allelochemistry to aquatic systems with its significance to terrestrial systems, it is especially constructive to take into account the absence of the need for protection against desiccation. This permits an exposure of action sites on the cell surfaces of aquatic organisms that would be destructive to organisms exposed to the drying effects of even excep-


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Table I. Allelopathic Effects of Cell-Free Filtrates of Axeoic Cultures of Dominant Algae on Nondominant Algae (Number of species with +, -, ο responses)

DIATOMS

BLUE-GRNS

GREENS

MOTILE

TOTAL SPECIES

+

-

+

ο

-

Ο

TESTED


PRODUCER Oscillatoria aghardi*

0

5

3

5

0

3

2 0 1

6

1

3

29

Oscillatoria rubescens*

0

6

0

3

3

2

2

3

1

6

27

Anabaena sp.

0

29

3

3

2

Pseudanabaena galea ta

0 7 1

1

4

3

7

Oscillatoria sp.

0 5 1

1

4

Synechecoccus sp.

0

0

9

4

Aphanizomenon flos-aquae

0

4

3

3

Anabaena sp.

1

Nostoc muscorum**

0

Nostoc sp.**

0

3

5

TOTALS

1

63

28

0

0

4

2

4

1

0

3

2

2

0

0 2 2

29

3

0 5 1

1 2 3

26

4

0

3

2

2

3

0

29

2

3

3

0

3

1 0 4

26

3

4

27

5

1

26

50

8

2

11

0

3

0

19

20

1

13

2

0 1 2

17

14

9

22

20

*Not axenic **Not isolated from Linsley Lake


257


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tionally humid ambient air. The capacitj of water as a solute/carrier and the common cell surface characteristics of aquatic organisms offer far more opportunity for a form of allelochemical activity which, due to its arbitrary pairing of producer and target organisms, is more appropriately characterized as secondary than as primary. There are reports of algae releasing as much as 90S of their total photosynthetic product into the water (3-8). A great deal of energy is invested in those complex molecules. If these were of no value, such incredibly inefficient organisms could not dominate a community. Since they do, there is a value and the trait is fixed in evolutionary terms. This is the basis for primary allelochemical activity. While direct competitions with other algae for niche allocation must be included as explanations for some primary allelochemical activity, there are other benefits to be considered. Algae are the prey, the food, of zooplankters of the second trophic level. If a particular algal species could produce a metabolite that made the zooplankters sick (indications: sluggish movement of feeding and/or swimming appendages; gut passage of nondigested algal material; loss of fecundity; loss of color; assumption of irregular swimming position), it would certainly provide a survival advantage—primary allelochemistry. This would also assist other phytoplankters, direct competitors. Thus, it would carry a price, but the net result would be beneficial to the producer. The potential interactive pattern is enormously complex. Coevolution, planktonic forms (both phytoplankton and sooplankton) constantly responding to the metabolites of other planktonic forms, must be considered as one of the basic themes of aquatic systems. The ties that impose that coevolution would be allelochemical. After observing how intricately the pattern of allelochemical actions was woven, and taking into account the amount and variety of bioactive material dissolved in ambient waters, we decided to develop an in vitro demonstration of the action of phytoplankton metabolites on zooplankters. Some activities were obvious. Since the algae are the basic food of the animals, there were positive effects. Any trace organic-based nutritional value could be an example—vitamins, coenzymes, etc. Even the provision of ordinary calories would be a legitimate "positive activity" although in no sense an allelochemical one. Also, several algae have been shown to be extremely toxic to just about any animal on which they were tested (4). The most thoroughly studied of these are the toxins of blue-greens (16), some of which (17) can kill a cow in minutes. We sought something less dramatic. The algal producer need not be a usual prey of the zooplankter. Some algal species always seem to prosper when the zooplankters are in trouble. The blue-greens, in particular, have often been observed to dominate waters in which there are few, or no, zooplankters. They have been consistently shown to be poor foods not only for zooplankters, but also for a variety of other eukaryotic organisms (1-8). Algal extracellular products, present in great quantities during blooms, are of interest. To study such interactions highly controlled sooplankton cultures in which all the possible producers could be identified would be essential. That is, random microbial infections in cultures would be unacceptable.



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Complex exogenous organics could also not be allowed. These requiremenis almost ended our studies. This was because, although there are hundreds, perhaps thousands, of published papers concerning zooplankton culture, there were no prior examples of controlled looplankton culture from which both all bacteria and all exogenous complex organics were excluded. The latter guaranteed that there were none in which inorganic trace inclusions were controlled, because even a diet comprised of carefully selected pure proteins and lipids suffers from the inevitable contamination that accompanies commercial proteins and lipids. All that is needed is a few sulfhydryl groups and substitutions will occur. In fact, with the single exception of some very special work by Provasoli and several students (18—21) in extremely high-organic media, the only successful long-term cultures of zooplankters were in lake, well, or tap water with mixed microbes. A l l were useless for our studies of allelochemistry. It took five years to develop the cultures we needed. We refer to the final product of our efforts as the "MS" Cladoceran maintenance system (9). All media, both food and animal versions, are essentially the same, Table II. The inevitable contaminants carried into animal cultures by organic nutritional components (plant, animal, protein, lipid, peptone, etc.), which have plagued nutritionists for years, were by-passed by producing food algae in 100% inorganic media. The algae produce everything organic that the animals need, excepting vitamin B . Since the organics made by those algae cannot be J2

contaminated with inorganics that are not present in algal growth media, inorganic contamination can be limited to the level of the purest available inorganic salts. The MS system supports an extensive variety not only of Cladocera, but also of other aquatic animals from several trophic levels. It is also, literally, the only extant system for maintaining permanent, healthy, cultures of zooplankters in defined circumstances. Unfortunately for its ultimate purpose (the study of allelochemistry between the first and second trophic levels), the system has two less than ideal characteristics. The first concerns the concentrations of several inorganics, especially copper, molybdenum, and calcium, which we consider higher than desirable. The second is the possibility that the algae which serve as food for our animals might also produce allelochemicals, not only directly, but also indirectly by stimulating animals or other algae into production. Our recent efforts have been directed at refining the system and, as these problems are addressed, allelochemical interference repeatedly imposes itself. Initially, our extreme control of inorganics produced a disaster. Animals were falling apart, losing their major swimming appendages. In some ways they were quite healthy, producing larger broods than had been reported in the literature; however, they showed the deteriorated and ill-formed cuticle (thus the appearance) of old age in quite young adults. The problem could have been an infection—bacteria or fungus feeding on components of the cuticle, weakening its structure. It could have been a deficiency that interfered with the biosynthesis of critical structural components of the cuticle. It could have been allelochemicals generating either damage to the finished structure of the cuticle or interference with its formation in the first place. The more diatom (a food alga) added, the



Table II. Composition of MS Media Α-MS Algal Medium ·Μ· COMPONENTS* Disodium EDTA Β

(H BO ) s

s

Fe (FeCls)

5 ppm

5

1000 ppb

1000

ppm ppb

400 ppb

400

ppb

Mn(MnCl MH 0)

200 ppb

200

ppb

Li (LiCl)

100 ppb

100

ppb

Rb (RbCl)

100 ppb

100

ppb

100 ppb

100

ppb

50 ppb

50

ppb

Mo(Na Mo0 *2H 0)

50 ppb

50

ppb

Cu (CuCl ' 2H 0)

25 ppb

25

ppb

Zn (ZnCl )

25 ppb

25

ppb

5 ppb

5

ppb

5 ppb

5

ppb

2

ppb

2


MS Animal Medium

2

Sr (SrCl -6H 0) 2

2

Br (NaBr) 2

4

2

2

2

2

Co (CoCl -6H 0) 2

2

(KI)

I

2 ppb

Se (Se0 ) 2

(NH VO )

0.5 ppb

0.5

ppb

COMPONENTS ** Glycylglycine

250 ppm

250

ppm

NaNO

V

4

s

120 ppm

50

PPm

CaCi '2H 0

38 PPm

38

PPm

MgS0 '7H 0

20 ppm

20

PPm

145 ppm

10

PPm

10 ppm

10

PPm

Κ ΗΡ0 · 3H 0

10 ppm

10

PPm

KH P0

25 ppm

10

PPm

1

ppb

s

2

2

4

2

NaîSiOa' 9H 0 2

KCl 2

4

2

2

4

ÎTAMINS*** Thiamine (HC1) Biotin B 12

75 ppb 0.75 ppb 0.75 ppb

*Target i o n or element i s l i s t e d f i r s t . Compound employed i n s o l u t i o n i s i n parentheses. C o n c e n t r a t i o n i s f o r i o n or element. **Compound employed i n s o l u t i o n i s l i s t e d . Concentration i s f o r whole compound. ***Vitamin B 1 2 should not be i n c l u d e d i n Chlamydomonas r e i n h a r d t i cultures. A l l vitamins can be omitted from such c u l t u r e s .


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more trouble introduced. This was prima facie indication of allelochemistry. If a metabolite were involved, it should have been possible to locate it in either the cells or the cell-free filtrates of food cultures. Damage appeared proportional to the amount of diatom present in cultures. Yet, when the diatom was withdrawn from the animal's diet, the damage, though less pronounced, persisted. Thus, it could not have been the diatom alone that generated the problem. It was clearly demonstrable (22) that the diatom did cause part of the trouble. This was initially interpreted as indicating that more than one of the food algae was producing an undesirable material. We sought to isolate that material, but were unsuccessful. The basis for this problem proved to be a selenium deficiency (23). The addition of 1 χ 10~ mg/L eliminated overt cuticle deterioration in the first generation. Ultimately, it was determined that a minimum of 2 χ 10~ mg/L of selenium in culture media (9) is necessary to avoid selenium deficiency in the animals tested in the MS system with exogenous organics excluded. Prior to this time no certain information had been published concerning specific requirements for inorganic materials essential to zooplankters. Therefore, when media were originally formulated, reasonable suppositions concerning nutritional require ments, based on nonspecific nutritional information relating directly to Cladocera, or on specific information relating to other organisms, including mammals, were made. Since a requirement for selenium had never been suggested for organ isms even remotely related to the Cladocera; it had not been included in the ori ginal formulations. It was, therefore, available at critically low concentrations. That damage observed to be proportional to the amount of diatom present in cul tures was the result of the food diatom taking up the last traces of selenium that had been carried into the culture system as contamination. More diatom simply meant a greater depletion of the already deficient trace element. There was no allelochemistry involved. The selenium was apparently being sequestered in a part of the alga that was not digested. In fact, its presence or absence did not initially affect diatom reproduction as measured by cell number although color and longevity of algal cultures were both diminished. It is important that this problem be recognized as an ultra-trace inorganic nutritional problem masquerading as an allelochemical one because this reinforces the demand for exceptional control of both inorganic and organic incorporations into test systems. There are natural waters, for instance those of Lake Superior, which are sufficiently low in selenium to restrict zooplankton reproduction (24). Thus, the absence of secure information concerning minimum requirements for inorganic trace nutrients can interfere with accurate interpretation not only of laboratory results, but also of events in natural settings. In short, a good food, one that is a source of desirable organics and useful calories (25), limits growth and shortens life span in proportion to its presence—this presents the appearance of allelochemistry; however, it is the exaggeration of a deficiency which proportionally increases. It is not the production of some allelochemical substance that proportionally increases. Like it or not, in aquatic systems the interplay of allelochemistry and nutri tion can not be ignored. Diatoms also have their nutritional problems. Many blue-greens interfere with diatom growth simply by interfering with their uptake of silica, which 4


S



144


happens to be singularly critical to diatoms (26). Since blue-greens give no indication of a nutritional requirement for silica, this does not represent direct competition for a nutrient. However, sequestering so critical a nutrient in a manner that makes it difficult for diatoms to satisfy their needs is a useful way to inhibit diatom populations (14, 27)—with which blue-greens do compete for just about every other nutrient. This is a relatively uncomplicated example of primary allelochemistry. When the blue-greens produce a bloom population, diatom numbers are greatly reduced. This does not help the zooplankton of the second trophic level. Although they might benefit by eliminating diatoms in that uncommon natural system in which selenium were somewhat deficient, generally, the animals lose the food value of the diatoms, and do very poorly on the bluegreens as food. Blue-greens have also long been suspected to produce allelochemical substances that interfere with zooplankton physiology (4,5,7,8,15). When blue-greens bloom, they release a variety of allelochemical substances into the water. Some sequester nutrients (25), some act as toxins (4,15,16). All must serve some purpose for their producers. These metabolic products of blue-greens interfere with the normal growth and development of many other species. It is not surprising that once entrenched, blue-greens are difficult to eliminate. Diatoms and blue-greens are not the only algae that cause trouble under the right circumstances. Even our most desirable food algae produce negative effects. We usually feed three algae (Nitzschia frustulum, Chlamydomonas reinhardti, and Ankistrodesmus convolutus) to our zooplankters, being careful to feed from the log phase which offers high protein (28) and the likelihood of low allelochemical complications. Chlamydomonas reinhardti has been included in our standard diet for several years because it is generally regarded as a desirable food (18,29-32). Two years ago, when the supply of food algae was less than adequate, cells were harvested from cultures that were about a week and a half older than usual. It was soon clear that, as the Ç. reinhardti cultures got older, the animals fed from these cultures got sicker. Animals fared better if they went hungry. In some cases, the Ç. reinhardti actually killed them. The same "desirable" food species, C. reinhardti, introduces another problem in animal cultures. When C. reinhardti is reared in algal Α - M S medium containing vitamin B , a diet containing relatively young such cells produces a mild negative effect on animals. Progeny of older mothers fed this diet show a pronounced Lansing effect—a loss of fecundity of progeny which is directly proportional to the mother's age at the time progeny are born (33, 34). Since there is no Lansing effect whatsoever when animals are fed a diet that includes the same clone of C. reinhardti reared in the same Α - M S medium excepting that the Α - M S contains no B , it appears to be the algal response to the presence of 12

12

ambient

B

12

that

changes

this desirable

food

organism

into a

self-protec

ting, allelochemical-producing, "prey" of the animals. A t this time we speculate that C. reinhardti (a facultative producer/user of B ), when reared in the presence of exogenous B , would produce a B -binder (35). To date effort has not been invested in isolation of the binder; however, in controlled cultures in which both C. reinhardti and any of several other food algal species are reared, C. reinhardti produces the same growth 12

12

12


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curve while the second species is grossly inhibited. Direct competition for avail able nutrients can not satisfactorily explain the absence of growth (reproduc tion) for the second alga. For example, simultaneous inoculation of C. reinhardti and the diatom, Nitzschia frustulum, into a dual-algal culture results in C. reinhardti growth quite similar to that of an uni-algal culture, but little perceivable growth for the diatom initially and elimination (death) of the diatom within two to three weeks. When these algae are transferred into animal cultures (as food), it is suggested that the binder would interfere with the animal's utilization of ambient B in a manner that inhibits the development of reproductively viable progeny. If correct, this could be interpreted in natural circumstances as either primary or secondary allelochemical activity (or both). It is primary in that the production of a B -binder would offer a variety of advantages in terms of


12

12

C. reinhardti's competition with other algae for occupancy of its niche and second ary in that the alga's capacity to thwart predators is coincidentally enhanced. It is, however, important to consider that the speculated interference with the predator would also offer protection to other, competing, algal species. To emphasize the complexity of this phenomenon, it must be noted that while additional exogenous B might help some of C. reinhardti's algal compe titors, and it appears in preliminary tests to improve the animal's condition, as little as 0.5 μξ of additional ambient B (150% of "optimum") introduces 12

12

a consistent drop in animal reproduction in some circumstances (35). This com plexity of allelochemical interferences that desirable food algae introduce under writes our belief that exceptional levels of control must be established prior to study of allelochemistry at the interface of the first and second trophic levels. What it all means is hard to say. It is certain that, without exception, a community in a freshwater system offers an incredible number of interacting allelochemical phenomena all at once, all of the time. Every organism is affected, not just by predators and foods, but by every other organism which releases metabolites in some form into the water in which they all dwell. When we take the concepts of terrestrial ecology and impose them on aquatic systems, we lose a lot of this allelochemical local color. Algae release just about any substance they make, whether useful to the producer or not, into the water. They have a storage problem, so they dump. With that array of plausibly active metabolic material in the water, allelochemical events of the primary sort are easy to find simply by looking for useful effects and seeking out the metabolites involved. Examples of secondary allelochemistry, while plentiful, are a bit more difficult to recognize since they need not tie the producer and target organism together by a logical association. Allelochemistry is so pervasive in aquatic systems that in our laboratory, even when we specifically try to avoid it, we find it wherever we look. Our greatest problem is sorting it out. Acknowledgments This work was supported by NSF Ecology Program Research Grant N S F - D E B 7823258, by the New Jersey Agricultural Experiment Station, and by state funds. NJAES Publication No. D-07496285.



146

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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

32. 33. 34. 35. 36.

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RECEIVED January 3, 1986


Exploring Allelochemistry in Aquatic Systems - ACS Symposium

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