Tannins and saponin: Interaction in herbivore diets

Mice allowed to choose between diets containing tannin or saponin did not experience food intake depression, weight loss or high faeces weight to food weight ratios. Diets containing tannin produced all these effects and saponin diets resulted in weight losses and high faeces to food ratios. Mice provided with diets containing both tannin and saponin in predetermined proportions experienced weight losses similar to, or greater than, those of mice fed diets containing either toxin alone. Urinary glucuronide production by mice provided with a choice of tannin and saponin diets was less than that of mice feeding on diets containing either tannin or saponin alone. Simultaneous consumption of tannin and saponin (in the right proportions) may promote chemical interactions that inhibit the toxins' absorption from the intestinal tract. This type of interaction is likely to have influenced the evolution of herbivore feeding behaviour.     

Bitter problems in ecological feeding experiments: Commercial tannin preparations and common methods for tannin quantifications

In the field of plant–herbivore interactions, research methods where plant secondary metabolites are manipulated are becoming more and more popular. Among the most commonly used is tannic acid. However, recent studies have shown that different tannic acid preparations are not comparable in their tannin structures. While tannic acids are meant to contain only gallotannins (GTs), some commercial preparations compose mainly of more simple galloylglucoses (that have, e.g. much lower protein precipitation capacity than GTs) or even of gallic acid (the hydrolysis product of GTs). Another group of tannins used in feeding trials is condensed tannins (CTs), usually in the form of quebracho tannin. Quebracho, however, contains different CT structures than, e.g. leaves of many deciduous trees. Additionally, when analysed with the common acid-butanol assay for total CTs, quebracho tannins give even 30-fold lower absorbance than the CTs of those deciduous trees. In addition to above problems it has been shown that different tannins can give different response even within the same herbivore species, and that the same tannin structure can cause different response in different herbivores. Below we review these problems, as well as some means to deal with them.     

A novel method to evaluate the relative tannin-binding capacities of salivary proteins

Many herbivore species have a diet containing high proportions of polyphenolics, principally lignins and tannins; the latter reduce the conversion of ingested nutrients into biomass and exert systemic toxicity at high levels of intake. It has been proposed that tannin-binding proteins in the saliva might be responsible for minimizing these tannin-related effects by forming soluble protein–tannin complexes. We have developed a method that permits evaluation of the relative tannin-binding properties of salivary proteins at a pH of 8.0–8.5. It is aimed at facilitating both the identification of tannin-binding proteins and the investigation of their relative tannin binding. The principle of the assay is the inhibition of trypsin by tannins and the subsequent reversal of that inhibition when other tannin-binding proteins are added (indirect assay). The method is rapid; large sample numbers may be processed by virtue of its microtiterplate format.     

Feeny revisited: condensed tannins as anti-herbivore defences in leaf-chewing herbivore communities of Quercus

Abstract. 1. Community level oak–tannin–insect patterns have been largely unexplored since Paul Feeny's ground‐breaking research. Two hypotheses were tested for Quercus velutina and Q. alba in the Missouri Ozarks: abundance and richness of leaf‐chewing herbivores are negatively correlated with foliar condensed tannin concentrations and variation in condensed tannin concentrations explains variation in herbivore community structure. 2. In 2001, foliar condensed tannins in the understorey and canopy of these two oak species were quantified simultaneously with censuses of herbivores in May, during leaf expansion, and in June and August, when leaves were fully expanded. Thirty‐eight of the 134 species encountered had densities sufficient to be analysed individually (n = 10). Of those, Acronicta increta (Noctuidae) and Attelabus sp. (Curculionidae), both oak specialists, were negatively correlated with condensed tannins in the canopy of Q. alba. One additional specialist, Chionodes pereyra (Gelechiidae), was marginally negatively correlated with condensed tannins in the understorey of Q. velutina. Understorey species richness of May Q. velutina herbivores was negatively correlated with condensed tannins, as were total canopy insect density and species richness of August herbivores on Q. alba. 3. Principal component analysis (PCA) of insect abundances indicated that understorey and canopy Q. velutina and Q. alba had different communities of leaf‐chewing insects. Furthermore, condensed tannin levels contributed significantly to variation in PCA scores for Q. velutina, explaining 25% of the total variation. 4. Overall, these results indicate that specialists were more likely than generalists both to correlate negatively with condensed tannins and to occur in lower tannin habitats; abundance and richness of both early and late season fauna correlated negatively with tannins; and species were more likely to correlate negatively with condensed tannins when feeding on Q. alba than on Q. velutina and when feeding in the canopy than in the understorey. Future studies of tannin–insect interactions should manipulate leaf quality in combination with manipulations of other factors that likely influence community structure.     

Tannins in nutrient dynamics of forest ecosystems - a review

Tannins make up a significant portion of forest carbon pools and foliage and bark may contain up to 40% tannin. Like many other plant secondary compounds, tannins were believed to function primarily as herbivore deterrents. However, recent evidence casts doubts on their universal effectiveness against herbivory. Alternatively, tannins may play an important role in plant–plant and plant–litter–soil interactions. The convergent evolution of tannin-rich plant communities on highly acidic and infertile soils throughout the world, and the intraspecific variation in tannin concentrations along edaphic gradients suggests that tannins can affect nutrient cycles. This paper reviews nutrient dynamics in forest ecosystems in relation to tannins. Tannins comprise a complex class of organic compounds whose concentration and chemistry differ greatly both among and within plant species. Because the function and reactivity of tannins are strongly controlled by their chemical structure, the effects of tannins on forest ecosystem processes are expected to vary widely. Tannins can affect nutrient cycling by hindering decomposition rates, complexing proteins, inducing toxicity to microbial populations and inhibiting enzyme activities. As a result, tannins may reduce nutrient losses in infertile ecosystems and may alter N cycling to enhance the level of organic versus mineral N forms. The ecological consequences of elevated tannin levels may include allelopathic responses, changes in soil quality and reduced ecosystem productivity. These effects may alter or control successional pathways. While a great deal of research has addressed tannins and their role in nutrient dynamics, there are many facets of tannin biogeochemistry that are not known. This lack of information hinders a complete synthesis of tannin effects on forest ecosystem processes and nutrient cycling. Areas of study that would help clarify the role of tannins in forest ecosystems include improved characterization and quantification techniques, enhanced understanding of structure-activity relationships, investigation of the fate of tannins in soil, further determination of the influence of environmental factors on plant tannin production and decomposition, and additional information on the effects of tannins on soil organisms.     

Deciduous sapling responses to season and large herbivores in a semi‐arid African savanna

Impacts of large herbivores (>5 kg) on woody plants in African savannas are potentially most severe among plants shorter than 1.6 m. It is well established that severe browsing leads to longer shoots, yet prevents saplings from recruiting into adult size‐classes in African savannas. Increased shoot length, indicating faster shoot growth, is often associated with reduced concentrations of tannins and increased nutrient concentrations, suggesting carbon limitation. We hypothesized that, on average, large herbivores suppress stem height or circumference, but increase shoot length. We also hypothesized that if there were concomitant positive effects on nutrients, or negative effects on tannin concentrations, they would be greatest early in the wet season. We sampled saplings of four deciduous woody species (Acacia grandicornuta, Dichrostachys cinerea, Combretum apiculatum and Grewia flavescens) at different stages of the wet season in a large‐scale, long‐term herbivore exclusion experiment in Kruger National Park, South Africa. Plant height, shoot length and stem circumference were generally not adversely affected by large herbivores, suggesting C limitation is rarely present among deciduous saplings in semi‐arid African savannas, allowing them to tolerate browsing. Time since first rainfall emerged as a predominant factor consistently affecting nutrient and tannin concentrations, rather than large herbivores. Nitrogen and phosphorus generally decreased (by 20–50%), while condensed tannin concentration increased (150–350%) during the wet season, except for one species. We postulate that A. grandicornuta is less prone than other species to accumulating tannins during the wet season because of high investment of C in spines. Although nutrient and tannin concentrations were generally not affected by large herbivores, species‐specific responses were evident very early in the wet season, which is when herbivore populations are most likely to be affected by differential forage quality among plants.     

Wound-induced accumulations of condensed tannins in turtlegrass, Thalassia testudinum

Turtlegrass, Thalassia testudinum, produces high concentrations of proanthocyanidins (condensed tannins) which we hypothesized are induced by grazing, as a component of a general wound response. To test this we quantified condensed tannins in a variety of turtlegrass tissues following simulated fish grazing, grazing by the urchin Lytechinus variegatus, and treatment with the natural plant wound hormone jasmonic acid. We observed that simulated fish grazing triggered rapid induction of condensed tannins by an average of 10 mg tannin g⁻¹ dry mass (DM) after 5 days. Condensed tannin accumulations were correlated with a reduction of blade extension rates. Further, we observed that constitutive tannin levels in developing first-rank leaves were strongly correlated with the width of second-rank leaves on each shoot, with an increase of 7.7 mg tannin g⁻¹ tissue dry mass per millimeter blade width on average. We propose that wider source leaves provide additional resources for phenolic biosynthesis. There was no induction of tannins in leaves, meristematic or sheath tissues in response to grazing by the urchin L. variegatus, the presence of potential waterborne cues from nearby grazed plants, or to treatment with 5 mM jasmonic acid. However, urchin grazing did induce tannin production in root/rhizome tissues, where they accumulated to levels 3–4 times higher than in blades (up to 350 mg tannin g⁻¹ tissue dry mass). These results confirm the potential for rapid wound-induced condensed tannin accumulations in T. testudinum. The link between blade widths and the tannin content of new leaves indicates that leaf morphology may be a useful bioindicator for predicting herbivore and disease-resistance in the field.     

Phenolic synthesis and phenylalanine ammonia-lyase activity in suspension cultures of Acer pseudoplatanus L.

Summary Phenolic metabolism is influenced by the levels of sucrose, nitrogen and 2,4 dichlorophenoxyacetic acid (2,4-D) in the growth medium. Chromatographic evidence suggests that the principle products are polymers of leucocyanin, (-) epicatechin and (+) catechin, constituting condensed tannins. Comparison of ethanolic cell extracts with extracts from plant organs shows that although these compounds are present in parts of the plant they are not the major phenolics.Cells maintained in a modified Heller's medium containing 9.0×10^–7 M 2,4-D produce increased levels of tannins from mid passage (day 12) onwards. The presence of 2,4-D at 9.0×10^–6 M supresses this response and increased initial sucrose levels cause the amount of tannins to be greater. At the period when tannin levels increase the standard medium is exhaused of its nitrogen sources, urea and nitrate. Increased initial nitrogen levels delay the beginning of increased tannin production and the addition of urea or 2,4-D to cultures already containing high levels of tannins causes the tannin content per gram fresh weight and per culture to decline. These results indicate an antagonism between tannin synthesis and nitrogen metabolism. The activity of phenylalanine ammonia-lyase EC 4.1.1.5. (PAL) estimated by a spectrophotometric method in acetone powders derived from Acer cells increases three to four fold at the onset of increased tannin synthesis and then declines sharply. The phase of high PAL activity correlates with the exhausion of the medium nitrogen sources.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid One of the authors (R.J.W.) was supported by a Science Research Council Studentship during the course of this work.     

Rumen Fermentation and Nitrogen Balance of Lambs fed Diets Containing Plant Extracts Rich in Tannins and Saponins,and Associated Emissions of Nitrogen and Methane

Tannins were added to experimental diets at levels of 1 and 2g/kg DM (hydrolysable tannins; Castanea sativa wood extract) and saponins at 2 and 30mg/kg DM (sarsaponin; Yucca schidigera extract). These levels were far below thresholds expected to be adverse in ruminants. Effects were measured in lambs by comparison with unsupplemented control diets calculated to be either deficient (10%) or adequate in protein. The diets consisted of hay, concentrate (1:1) and extra wheat starch with increasing body weight. Ruminal pH, VFA concentration, protozoa count and apparent digestibilities of organic matter and fibre did not differ among treatments. The low tannin dose significantly decreased bacteria count compared to the high saponin dose. Saponin supplementation and the high tannin dose showed some potential to reduce ruminal ammonia concentration. This was associated with weak trends towards lower urine N excretion (only tannins) and ammonia emission from manure. Methane release was increased by the low tannin dose compared to the unsupplemented control. Diet effects on heat production were not systematic. In conclusion, the extracts rich in tannins or saponins gave only slight indications for either increased body nitrogen retention or reduced nitrogen emission. However, effects might have been larger with more pronounced dietary protein deficit.     

Separation of polymeric tannins from white wine by a combination of 2-butanol extraction and column chromatography

A new procedure was developed for the separation of polymeric tannins from a high-tannin white table Koshu wine by extraction with organic solvent and adsorption chromatography. The wine was adjusted to pH 1.0, sodium chloride was added to give a final concentration of 12.5% NaCl, then phenolic compounds including phenolic acids and tannin phenolics were extracted with 2-butanol. About 95% of the total phenolics was extractable.Polymeric tannins were separated from the extract by chromatography on Bio-Gel P-2, which adsorbs polymeric tannins in the presence of 10% acetic acid-50% ethanol, and recovered quantitatively by subsequent elution with 50% acetone. The phenolic acids and flavonoid tannins of low molecular weight that were eluted first using 10% acetic acid-50% ethanol, and the tannin phenolics of high molecular weight subsequently eluted using 50% acetone, were both free of potassium and sodium ions.This method, including the extraction and chromatography, is useful for rough separation of polymeric tannins and other phenolics from white wines.     

食物单宁和皂苷对小白鼠食物选择的影响

通过饲喂自然种子及人工饲料块等单宁和皂苷含量不同的食物,研究了食物中单宁或皂苷水平对小白鼠(Mus musculus domesticus)食物选择的影响,以及取食经历在其食物选择中的作用。结果表明,(1)无论有无取食经历,小白鼠均优先取食次生物质含量低的锥栗(Castanea henryi)或花生(Arachis hypogaea),而很少取食单宁含量高的栓皮栎(Quercus variabilis)或皂苷含量高的油茶(Camelia oleifera);(2)小白鼠的取食经历能增强其对种子中单宁或皂苷水平的识别,从而减少其摄入;(3)小白鼠的食物摄入量随单宁或皂苷含量的增加而显著降低。本研究说明单宁或皂苷均可显著影响动物的食物选择,且取食经历能增强动物对食物的识别能力。     

Defence,growth and nutrient allocation in the tropical shrub Bauhinia brevipes (Leguminosae)

Herbivory in natural communities can be high, reducing the growth and reproduction of individual plants. To diminish the impact of herbivores, plants use a variety of defensive mechanisms. It is now recognized that the plant world is characterized by a proliferation of secondary metabolites, but models of the evolution of defence strategies assume that plants are constrained by finite resources, and that defences are costly. In the present study, the relationships between defence, growth and nutrient concentration in Bauhinia brevipes Vog. (Leguminosae) in south‐eastern Brazil were investigated. Patterns of herbivory and defence were examined for 170 B. brevipes individuals in 1997 and 1998. Leaf age influenced tannin concentration and herbivore attack on B. brevipes. Mature leaves had higher concentrations of tannins than young leaves, and young leaves were approximately 60% more likely to be attacked than mature leaves. Carbon and nitrogen content explained 36% of the variation in the percentage of attacked leaves in B. brevipes (y = 16.29 – 0.31 C + 0.25 N; r² = 0.36; F = 9.56; P < 0.0001). Tannins acted as defensive compounds on B. brevipes leaves against free‐feeding herbivores, but no clear pattern was observed against galling herbivores. The amounts of nitrogen, carbon, potassium, and aluminium explained approximately 69% of the variation in tannin concentration on B. brevipes leaves (y = 0.62 – 0.16 N – 0.36 K – 0.26 Al + 0.74 C; r² = 0.69; F = 21.18; P < 0.0001). We found an inverse relationship between shoot growth rates and tannin concentration in B. brevipes, indicating that the production of tannins may be costly, therefore offering some evidence for the trade‐off hypothesis. Nevertheless, the weak relationship observed between herbivore damage and tannin concentration indicates that other factors are also involved with herbivore pressure.     

Cattle and sheep develop preference for drinking water containing grape seed tannin

Ingestion of small amounts of some types of condensed tannins (CTs) by ruminant livestock can provide nutritional, environmental and economic benefits. However, practical methods are needed to make these tannins more available to ruminant livestock. Results from previous trials with crude quebracho and black wattle tannin indicated that cattle and/or sheep would not preferentially drink water containing these tannins. Therefore, we conducted preference trials to determine if cattle and sheep would learn to prefer water containing purified grape seed tannin (GST) that provided up to 2% of their daily dry matter (DM) intake. After gradual exposure to increasing amounts of this tannin in water during a pre-trial period, five adult ewes and five yearling heifers fed lucerne (Medicago sativa) pellets (19% CP) were offered water and several concentrations of GST solutions for either 15 (sheep trial) or 20 days (cattle trial). We measured intake of all liquids daily. Concentrations of blood urea were also measured for heifers when they drank only tannin solutions or water. Both sheep and cattle developed preferences for water with GST in it over water alone (P < 0.01) although this preference appeared earlier in the trial for sheep than for cattle. For the sheep, mean daily intake of water alone and all tannin solutions (in total) was 0.6 and 6.1 l, respectively. For the cattle, mean daily intake of water and all tannin solutions in total was 21.8 and 20.6 l, respectively, in the first half of the trial and 10.8 and 26.1 l, respectively, in the second half of the trial. Compared with the other tannin solutions, both sheep and cattle drank more of the solution with the highest tannin concentration (2% of daily DM intake as GST) than of water on more trial days (P < 0.05). Ingestion of water with the highest concentration of GST reduced blood plasma urea concentration in the cattle by 9% to 14% (P ⩽ 0.10) compared with ingestion of water alone. Results from the trials suggest that providing grape seed and perhaps other CTs via drinking water may be a practical way to introduce CTs into sheep and cattle diets.     

Polyvinylpyrrolidon als Schutzstoff bei der Untersuchung gerbstoffgehemmter Enzymreaktionen

Summary Investigations on various enzyme systems showed that addition of polyvinylpyrrolidone protected enzymatic reactions against inhibition by tannins. Furthermore, polyvinylpyrrolidone reactivated enzymatic reactions already inhibited by tannins. Addition of polyvinylpyrrolidone during the isolation of enzymes from tannin containing tissues prevented inhibition by tannins.     

Effects of tannins on digestion in the common ringtail possum (Pseudocheirus peregrinus), a specialized marsupial folivore

Digestibility trials using common ringtail possums ( Pseudocheirus peregrinus ), small folivorous marsupials, were used to determine effects of tannins on a herbivore which has a specialized hind gut and which normally consumes tannin-rich eucalypt leaves. In one group, untreated leaves were fed to possums, and in a second group polyethylene glycol (PEG) was added to the eucalypt leaves to inactivate tannins. Animals maintained weight, and intakes were not different between the two dietary groups. Digestibility was determined chemically. Digesta were examined histologically. The presence of bacterial rafts in the stomachs of possums fed either diet indicated that PEG did not alter the coprophagic behaviour of possums. Cell wall digestion did not appear to be inhibited by tannins. Tanning of cell contents was predominant, rather than tanning of cell walls. PEG increased faecal excretion of tannins, suggesting that some tannins from normal leaves were digested during gut passage. Dry matter digestibility was higher when animals were fed normal tannin-rich leaves than when they were fed leaves in which tannins were inactivated. The difference could be explained in part by the high digestibility of tannins. The presence of tannin did not reduce nitrogen digestion. We suggest that dissociation of tannin-protein complexes may take place in the specialized caecum. We propose that some other folivorous marsupials may have a similar capacity to overcome tannins. This capacity may allow them to consume a tannin-rich diet.     

The Effect of Tannins on the Morphology and Hydrogenase Activity of Sulphate Reducing Bacteria

Two strains of Desulphovibrio desulphuricans were found to have much reduced hydrogenase activity for sulphate reduction and methylene blue reduction after repeated subculture in medium containing mimosa tannin. The loss of activity was not reversed on transfer back to a medium containing no tannin. Changes in the morphology and motility of these organisms, and of the related organism Desulphovibrio orientis , brought about by culture in media containing tannins are reported.     

Formation of vacuolar tannin deposits in the chlorophyllous organs of Tracheophyta: from shuttles to accretions

Most Tracheophyta synthesize-condensed tannins (also called proanthocyanidins), polymers of catechins, which appear in the vacuole as uniformly stained deposits—termed tannin accretions—lining the inner face of the tonoplast. A large body of evidence argues that tannins are formed in recently described thylakoid-derived organelles, the tannosomes, which are packed in membrane-bound shuttles (Brillouet et al. 2013); it has been suggested that shuttles agglomerate into tannin accretions. The aim of the study was to describe the ontogenesis of tannin accretions in members of the Tracheophyta. For this purpose, fresh specimens of young tissues from diverse Tracheophyta were cut, gently lacerated in paraformaldehyde, and examined using light, epifluorescence, confocal, and transmission electron microscopy. Fresh samples were also incubated with gelatin-Oregon Green, a fluorescent marker of condensed tannins. Our observations showed that vacuolar accretions (1?→?40 μm), that constitute the typical form of tannin storage in tannin-producing Tracheophyta, are formed by agglomeration (not fusion) of shuttles containing various proportions of chlorophylls and tannins.     

Bactericidal effect of hydrolysable and condensed tannin extracts on <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> in vitro

Strategies are sought to reduce intestinal colonisation of food-producing animals by Campylobacter jejuni, a leading bacterial cause of human foodborne illness worldwide. Presently, we tested the antimicrobial activity of hydrolysable-rich blackberry, cranberry and chestnut tannin extracts and condensed tannin-rich mimosa, quebracho and sorghum tannins (each at 100 mg/mL) against C. jejuni via disc diffusion assay in the presence of supplemental casamino acids. We found that when compared to non-tannin-treated controls, all tested tannins inhibited the growth of C. jejuni and that inhibition by the condensed tannin-rich mimosa and quebracho extracts was mitigated in nutrient-limited medium supplemented with casamino acids. When tested in broth culture, both chestnut and mimosa extracts inhibited growth of C. jejuni and this inhibition was much greater in nutrient-limited than in full-strength medium. Consistent with observations from the disc diffusion assay, the inhibitory activity of the condensed tannin-rich mimosa extracts but not the hydrolysable tannin-rich chestnut extracts was mitigated by casamino acid supplementation to the nutrient-limited medium, likely because the added amino acids saturated the binding potential of the condensed tannins. These results demonstrate the antimicrobial activity of various hydrolysable and condensed tannin-rich extracts against C. jejuni and reveal that condensed tannins may be less efficient than hydrolysable tannins in controlling C. jejuni in gut environments containing high concentrations of amino acids and soluble proteins.     

Interactions among white spruce tannins, Bacillus thuringiensis subsp. kurstaki, and spruce budworm (Lepidoptera: Tortricidae), on larval survival, growth, and development

The interactions among white spruce, Picea glauca (Moench) Voss, purified acetone tannin extracts (hydrolyzable and condensed tannin), Bacillus thuringiensis subsp. kurstaki Cry1A(c) delta-endotoxin strain HD-73 (Btk), and spruce budworm, Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae) on larval survival, growth, and development were investigated over the whole larval feeding period by using artificial diet supplemented with three concentrations of Btk toxins per milliliter of diet (0, 0.021, and 1.72 microg/ml) and three concentrations of foliar tannin extract (0, 8, and 15% dry mass basis). At high Btk concentration, tannin antagonized Btk potency against spruce budworm by lowering Btk-related larval mortality from 83 to 43%. At moderate Btk concentration tannin did not affect Btk potency. Host tree tannins antagonized not only the lethal effects of Btk toxin but also sublethal Btk-related impacts in terms of larval development, pupal weight, relative consumption rate, and growth rate. When alone in the diet, tannin negatively affected larval survival, growth, and development. Maximum potency of tannins against spruce budworm larvae (60% mortality) was reached at dietary concentrations corresponding to what is found in the plant (8% dry mass). The addition of Btk toxin in food containing tannin reduced percentage of larval mortality by one-third, indicating that Btk toxin can antagonize tannin potency against the insect. Development of Btk transgenic spruce trees should consider the antagonistic effect the toxin may have on the resistance conferred by tannins that have evolved naturally in spruce trees.     

Measurement of low molecular weight tannins: Indicators of methanogenic toxic tannins

The effectiveness of several low molecular weight (MW) tannin measurement methods for indicating the tannin toxicity to methan bacteria was evaluated. The methanogenic toxicity of the low and high MW tannins from autoxidized bark extracts was studied by selective removal of MW fractions from the extract with active carbon adsorption and calcium precipitation treatments. The toxicity of the low MW tannin fraction and the nontoxicity of the high MW tannin fraction were demonstrated. The low MW tannin concentration, measured by HPLC and a method based on the loss of tannins by treatment with granular active carbon (AC), had a very close relationship with the methanogenic toxicity, but a poor relationship was found with the total tannin concentration. The low MW tannins detected by the HPLC and AC methods had similar peak area positions in HPLC chromatograms as the tannins that were adsorbed by polyamide (trisacryl GF05) gel beads. These gel beads have an exclusion limit of 3000 g·mol⁻¹, indicating that this is the approximate MW boundary between toxic and non-toxic tannins.