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Phytotoxins: Environmental Micropollutants of Concern? Thomas D. Bucheli*,† †
Agroscope Institute for Sustainability Sciences ISS, CH-8046 Zurich, Switzerland mixture of (micro)-pollutants in the aquatic environment. To properly assess risks of (emerging) anthropogenic contaminants, the background stress/toxicity exerted by natural toxins should be known. Natural toxins comprise bioactive compounds from different kingdoms of life, and are usually separated in mycotoxins (produced by fungi), bacterial toxins, phycotoxins (from algae), phytotoxins (from plants) and zootoxins (from animals).2 While environmental exposure and effects of bacterial and phycotoxins have been relatively well investigated due to their threat to the pelagic food web (e.g., refs 4, and 5), similar studies for mycotoxins (e.g., refs 6−12) are few, and those for phytotoxins (e.g., refs 13−19) scattered. Here, the focus will be Natural toxins such as mycotoxins or phytotoxins (bioactive on phytotoxins, because they are systematically approachable compounds from fungi and plants, respectively) have been due to the knowledge in related disciplines such as medicine or widely studied in food and feed, where they are stated to outplant toxicology. Moreover, circumstantial evidence from other compete synthetic chemicals in their overall human and animal fields of research such as agronomy, plant protection, and toxicological risk. A similar perception and awareness is yet ecology points to their potential relevance for environmental largely missing for environmental safety. This article attempts health, as will be illustrated in the following. to raise concern in this regard, by providing (circumstantial)
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evidence that phytotoxins in particular can be emitted into the environment, where they may contribute to the complex mixture of organic micropollutants. Exposures can be orders-ofmagnitude higher in anthropogenically managed/affected (agro-)ecosystems than in the pristine environment.
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TOXIC PLANTS: AN OVERVIEW The most comprehensive compilation of toxic plants is probably provided by Quattrocchi,20 who lists thousands of plant species on close to 4000 pages. However, they contain no further information about the geographical distribution or the actual phytotoxins of individual plants. In this respect, Burrows and Tyrl21 provide an excellent compendium about toxic plants of North America. Several homepages with databases of poisonous plants are available as well. One of the largest may be the one by the U.S. Food and Drug Administration.22 This database contains references to studies describing the toxic properties and effects of plants and plant parts, and includes some 4624 vascular plants.23 The European Food Safety Authority compiles about 700 botanicals containing toxic substances of concern.24 From this literature it appears that most toxic plant species identified by today belong to the division of the magnoliophyta (angiosperms; flowering plants). While their relative number may currently be rather low (i.e., several thousand out of about 250 000−400 000 species25), they spread widely over at least 75 different families21 (out of about 415 families25).
INTRODUCTION
In the early 1990s, Ames and colleagues evaluated the toxicological significance of human dietary exposures to synthetic chemicals in light of those to naturally occurring chemicals.1 They concluded that 99.99% of dietary pesticides are chemicals that plants produce to defend themselves. Consequently, natural toxins in food and feed are regulated by law in many countries.2 Ames et al. further stated that “Toxicological examination of synthetic chemicals such as pesticides and industrial pollutants, without similar examination of the chemicals in the natural world to use for comparison, has generated an imbalance in both data and perception about potential hazards to humans...”.1 This article hypothesizes that the very same holds true for environmental exposure, and that consequently, the quoted sentence would need to expanded by “... and the environment”. There are many reasons why we should care for natural toxins in the environment,3 and this article is going to address some of them in more detail. The most relevant ones are probably the following: (1) Natural toxins come in legions, and with an incredible diversity; (2) Exposures can be orders-ofmagnitude higher in anthropogenically managed/affected (agro-)ecosystems than in the pristine environment; (3) Natural toxins are toxic by definition, although their mode of action is often unknown; (4) Effects (on nontarget organisms) have been shown in case of allelochemicals, invasive plant species, and biopesticides; (5) They contribute to the complex © 2014 American Chemical Society
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PHYTOTOXINS ARE SECONDARY PLANT METABOLITES Secondary plant metabolites serve a wide range of physiological purposes.26−28 They support the primary plant metabolism (e.g., photosynthesis, ATP production) and plant development (e.g., germination, organ development), facilitate symbiosis with rhizobia and mycorrhiza, and act as signaling compounds to attract pollinators or seed dispersers. Furthermore, they are Published: October 17, 2014 13027
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Environmental Science & Technology produced to defend the organism against abiotic (e.g., UV radiation, temperature, drought) or biotic (e.g., herbivores such as insects or vertebrates, microbes such as viruses, bacteria or fungi, or plant parasites) stress. Finally, such compounds may help the plant to compete with others in a process termed allelopathy (see below). Secondary plant metabolites are chemically very diverse. Important compound classes and some approximate numbers encompass tannins, coumarins, quinones, flavonoids (4000), polyketides (750), terpenes (incl. mono-, sesqui-, di-, triterpenes, saponins, and steroids; all together around 15 000), alkaloids (12 000), cyanogenic glucosides (60), and glucosinolates (100).26−28 Effects of secondary plant metabolites, for example, on human (and animal) health can be positive or negative, and depend among other things on dietary composition and exposure concentrations,26 as we know since Paracelsus.
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ALLELOPATHY AND EXOTIC PLANT INVASION
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PHYTOTOXINS AS BIOPESTICIDES
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Allelopathy as a scientific discipline has been perceived with some ambiguity over the last decades. On the one hand, it got repeatedly into the spotlight of a broader scientific audience after reports about it being the reason for the success of invasive species. On the other hand, it came under a lot of critics for being scientifically dubious. Two incidences are noteworthy in this respect. First, zones of inhibition around Salvia leucophylla and Artemisia californica invading an annual grassland near Santa Monica, Ca, were reported in 1964.34 The second example is Centaurea maculosa, an invasive species in the western U.S. which displaced “native plant species by exuding the phytotoxin (−)-catechin from its roots”.35 The authors stated that their “results support a “novel weapons hypothesis” for invasive success”, a theory expressed by the group of Callaway at that time.36,37 However, both examples had later to be revised or at least corrected. In the first case, it was shown that plants could grow in the bare soil around the invasive species if herbivores were excluded.38 In the second example, the authors had to add an erratum, stating that they “have had difficulties replicating the high and consistent levels of catechin found in soils”, and that “in vitro exudation of catechin by C. maculosa roots has not been reproducible”.35 Consequently, leading scientists in the field critically reflected about allelopathy, and the necessity for more scientific rigor and quality in this discipline.39,40 While it is beyond the scope of this article to elaborate on this any further, the sheer possibility of “allelochemical weapons” motivates further research. After all, more environmental organic chemistry in the field of allelopathy seems urgently needed.
ALLELOPATHY: THE EFFECT OF ONE PLANT ON ANOTHER VIA PHYTOTOXINS
The maybe earliest written report about plants influencing each other via chemicals dates back to Solon (about 638−559 BC). Plutarch writes in his book “Lives”: “He [Solon] showed skill in his orders about planting, for any one that would plant another tree was not to set it within five feet of his neighbour’s field; but if a fig or an olive not within nine; for their roots spread farther, nor can they be planted near all sorts of trees without damage, for they draw away the nourishment, and in some cases are noxious by their eff luvia”. For a systematic review on the history of allelopathy, see Willis.29 In modern times, Molisch30 provided the first definition, which is (author’s translation from German): “The described strange observation of the influence of one plant on another... deserves to be also noted with a brief expression. For this, I suggest the word “allelopathy”, deduced from the two greek words “allelon”, “mutual, among each other” and “pathos”, “suffering” or “what happens to oneself””. While this original definition does not explicitly mention chemical compounds, they are introduced in more recent ones. For instance, the Oxford English Dictionary describes allelopathy as “the deleterious process by which one organism (esp. a plant) influences others nearby through the escape or release of toxic or inhibitory substances into the environment”.31 What strikes us as environmental scientists in this definition is that bioactive compounds are emitted “into the environment”, which would make them−in case of adverse effects−environmental micropollutants! In fact, allelochemicals are released by plants into the environment in various ways.32,33 They can volatilize or leach from roots, stems and leaves, can emit from pollen, flowers, fruits or seeds. Allelochemicals may also be released during plant injury or decay, or may form as a result of (a)biotic transformation of parent compounds. While their actual number is unknown, a literature review about allelopathic weeds listed 240, 64, and 25 species with allelopathic inhibitory activity on crops, other weeds, and themselves, respectively.33 In the following, we expand allelopathy to disciplines of relevance for environmental exposure to such chemicals (i.e., invasive species and biopesticides), before presenting studies that investigated the environmental fate and behavior of phytotoxins directly.
In light of allelochemicals acting against weeds, they have repeatedly been considered as “Nature’s own pesticides”.41 In fact, a number of natural toxins have led to the production of very successful commercial pesticides. For example, triketone herbicides were developed from leptospermone from Leptospermum scoparium. Cinmethylin herbicides are based on 1,4cineole, present in essential oils of a number of plant species. Pyrethroid insecticides have pyrethrins 1 and 2 from Chrysanthemum spp. as a natural analogue.39,40,42 While pesticides from allelochemicals may have some properties that render them less hazardous to the environment than synthetic ones (e.g., possibly a shorter half-life), there might be others that lead to the opposite. Vyvyan40 lists reasons why they actually might not be that environmental benign, which are (1) “Of the hundreds of identified allelochemicals, modes of action have been determined for a few only”, (2) “Many allelochemicals exert their influence through mechanisms not possessed by commercial herbicides”, (3) “Most biologically active natural products are at least partially watersoluble”, (4) “As a result of natural selection” active natural products are “more likely to exhibit some bioactivity at low concentrations”, and (5) “Many plant and microbial compounds are potent mammalian toxins”. Such bioactive natural compounds are often classified as “biopesticides”, defined by the U.S. Environmental Protection Agency (EPA) as “certain types of pesticides derived from... animals, plants, bacteria, and certain minerals”.43 Currently, the U.S. EPA regulates more than 400 active ingredients, and the European Commission (EC) approved on about 110 biological 13028
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Environmental Science & Technology
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pesticides.44 About one-fourth of these compounds or formulations are plant derived.44,45 Regulators in both the U.S. and the European Union (EU) consider biopesticides to be “usually inherently less toxic than conventional pesticides”, “effective in small quantity”, and to affect “only the target pest and closely related organisms”.43 For these reasons, they encourage their development and increased use, and facilitate their registration.43,46 However, quite a number of biopesticides registered in the US and/or the EU include natural toxins that may not a priori be considered nontoxic. This is illustrated with a few examples about nontarget effects of plant derived biopesticides. Rotenone is produced by plants such as Derris spp., Lonchocarpus spp., and Tephrosia spp. Roots of T. virginiana were used by some American Indian tribes as a fish poison and a vermifuge.21 It had been registered in the U.S. for use in food crop, ornamental plants, and pets to control flying and crawling insects.47 In 2007, it was reregistered as a piscicide.48 This is interesting to note, because above, biopesticides were considered to affect “only the target pest and closely related organisms”. The questions would then be what is the target pest in the first place (insects vs fish), and how closely related are these organisms really? More importantly, however, is the fact that nontarget effects of rotenone to aquatic invertebrates have been repeatedly investigated ever since the 1950s.49,50 For instance, 21% of the investigated aquatic macroinvertebrate taxa were still missing five years after a 48 h application of 0.15 mg/ L rotenone aiming to remove fish species competing with trout in the Stawberry River, Utah.49 Such a concentration seems unrealistically high under natural conditions, but actual (and probably more chronical) surface water exposures in agricultural catchments, or such covered with rotenone producing plants, are currently missing. Azadirachtin from neem tree (Azadirachta indica) is a limonoid insecticid with repellent, antifeedant, molt regulating activity. The first commercial product of neem, Margosan-O, was registered by the U.S. EPA for nonfood crop insect pest control in 1985.51 Neem oil is still registered as a biopesticide in the U.S.52 and the EU.44 Kreuzweiser and colleagues have been investigating ecological implications of azadirachtin applications for years. For instance, copepodes and zooplankton food web stability in Ontario forest ponds were affected by concentrations of 10 to 28 μg/L, that is, below the expected environmental concentration of 35 μg/L, calculated according to the Canadian pesticide regulatory guideline.53 Conti et al.54 showed that 1,8-cineole, a major constituent (i.e., 59%) of the essential oil from tea tree (Melaleuca alternifolia) exhibited an over three times lower lethal effect concentration to the nontarget organism water flea (Daphnia magna) than to the target organism Asian tiger mosquito (Aedes albopictus). Tea tree oil is listed as a fungicide in the U.S. EPA’s Biopesticide Active Ingredients Workplan for 2014,55 and in the Biopesticide Database.44
Probably the most thorough study ever on phytotoxins from agricultural crops was conducted within the EU Framework 5 project “Fate and toxicity of allelochemicals in relation to environment and consumer (FATEALLCHEM)”.56 FATEALLCHEM aimed at an environmental and human risk assessment on benzoxazinone derivatives in wheat, rye, and maize, considered to be used as biopesticides, because if alternative strategies for suppressing weeds, insects, pathogens, and other pests are exploited extensively, these strategies cannot automatically be considered harmless to the environment and the consumer“.56 Accordingly, the project dealt with isolation and synthesis of benzoxazinones, development of analytical methods and determination of benzoxazinone levels, degradation in soil, target and nontarget effects, and molecular modelling. Key findings were that ecotoxicological effects of benzoxazinone allelochemicals to various aquatic non-target organisms were sometimes comparable with those of conventional pesticides,57 and that the soil transformation products suppressing weeds and pathogenic fungi also were the most toxic to beneficial organisms.56 A second example comprises phytoestrogenic isoflavones from legumes such as soy and red clover. We investigated the emission of formononetin and biochanin A from red clover grassland and found them to be permanently present in drainage waters at concentrations up to a few μg/L.58 We could also show that red clover actually was the main contributor leading to the frequent occurrence of isoflavones in river waters.14 Other studies reported on the leaching of cyanogenic glucosides and cyanide from white clover (Trifolium repens) green manure,13 on potato glycoalkaloid occurrence in soils,15 or on juglone in soils under walnut trees.59 Several phytotoxins from nonagricultural crop plants have been studied as well. A well investigated example is ptaquiloside from bracken fern (Pteridium aquilinum), one of the five most common plants on earth.60 Ptaquiloside is a norsesquiterpene glucoside produced in amounts up to several kg/ha. It is highly toxic to husbandry animals (carcinogenic), and may be transferred via milk to humans.61 Its hydrolysis half-life is
