Antifouling agents from marine spongeLissodendoryx isodictyalis carter

The spongeLissodendoryx isodictyalis is an odorous, encrusting, blue-gray sponge found on subtidal flats in North Carolina waters. The strong odor ofL. isodictyalis, coupled with the observation that it is rarely overgrown by fouling organisms, suggested that this sponge may produce metabolites with potent antifouling activity. Ethyl acetate extracts ofL. isodictyalis inhibit larval settlement of the barnacleBalanus amphitrite in laboratory assays at 10 ng/ml. Barnacle settlement bioassays of isolated preparative TLC fractions show thatL. isodictyalis produces at least two pungently scented, antifouling agents with EC₅₀ values of less than 85 μg/ml and less than 250 μg/ml, respectively. The most potent agent inhibits settlement at or below a concentration of 400 ng/ml and kills approximately 25 % of settlement-stage barnacle larvae at 400 μg/ml. The other agent causes 100% mortality of larvae at concentrations greater than 400 μg/ml and inhibits settlement at approximately 40 μg/ml. These metabolites ofL. isodictyalis may inhibit overgrowth of the sponge in nature.     

Norsesquiterpenes as defensive toxins of whirligig beetles (Coleoptera: Gyrinidae)

By use of a minnow bioassay, toxins were detected in the pygidial secretions of the gyrinid beetlesDineutus assimilis (Kirby) andDineutus nigrior Roberts. The active agents, which may be largely responsible for the relative immunity of the Gyrinidae from predation, were isolated and identified as the norsesquiterpenesgyrinidione [(E)-1-methyl-2-formyl-3-(1′-methylhex-3′-ene-2′,5′-dione)-cyclopentane],gyrinidone [(E,Z)-2-hydroxy-5,9-dimethyl-4-(but-1′-ene-3′-one)-3-oxo-bicyclo[4.3.0]-non-4-ene],gyrinidal [(E,E,E)-3,7-dimethyl-8,11-dioxo-2,6,9-dodecatrienal], andisogyrinidal[(E,E,Z)-3,7-dimethyl-8,11-dioxo-2,6,9-dodecatrienal]. Since gyrinidione and isogyrinidal are being reported for the first time, their physical and chemical properties are presented and biosynthetic relationships of the four norsesquiterpene structures are discussed. About 50% of the beetle defensive material was norsesquiterpenes, 25% polar lipids, and 20% could not be extracted from water into chloroform. As quantified by gas-liquid chromatography,D. assimilis contained 245±73 μg andD. nigrior 144±64 μg norsesquiterpenes per individual. The average relative composition of norsesquiterpenes in the pygidial secretions of both beetle species was constant: isogyrinidal, 6%; gyrinidone, 7%; gyrinidione, 36%; and gyrinidal, 48%. When administered externally in solution to fish, isolated norsesquiterpenes possessed narcotic and toxic activity similar to that of the anesthetic steroids deoxycorticosterone (DOC) and testosterone. Minnow dose-response curves demonstrated that gyrinidione and gyrinidal (LC₁₀₀=ca. 2 μg/ml) were as toxic to fish as was DOC (LC₁₀₀=ca. 3 μg/ml). Gyrinidone was less toxic (LC₁₀₀=ca. 15 μg/ml) while isogyrinidal was relatively inactive (LC₁₀₀=ca. 90 μg/ml).     

Bark beetle olfaction. III. Antennal olfactory responsiveness ofDendroctonus frontalis Zimmerman andD. brevicomis Le Conte (Coleoptera: Scolytidae) to aggregation pheromones and host tree terpene hydrocarbons

Electroantennograms from males and females ofDendroctonus frontalis andD. brevicomis in response to serial dilutions of the pheromonesexo brevicomin and frontalin and the host terpene hydrocarbons 3-carene and α-pinene show no significant differences in the threshold concentration for response to the compounds for either sex or species. The intensity of response was greater to higher concentrations of the pheromones (10 to >10⁴ μg) than the terpene hydrocarbons, suggesting the presence of more receptors on the antennae for pheromones than terpene hydrocarbons. Antennal olfactory responses to the compounds did not correlate to published data on behavioral responses by the beetles to the compounds in both field and laboratory studies. Adaptation experiments indicated that forD. frontalis, both frontalin andexo brevicomin share the same receptors on the antennae. The results indicated that the terpene hydrocarbons share some, but not all, of the same receptors.     

Field response of the dutch elm disease vectors,Scolytus multistriatus (Marsham) andS. scolytus (F.) (Coleoptera: Scolytidae) to 4-methyl-3-heptanol baits containing α-, β-,δ-, or δ-multistriatin

The field responses of English populations of the Dutch elm disease vectors,Scolytus multistriatus andS. scolytus to baits containing 4-methyl-3-heptanol, a host synergist [(?)-α-cubebene or (?)-limonene] and (±)-α-, (+)-β-, (?)-β-, (±)-γ-, or (±)-δ-multistriatin were examined. (±)-α-Multistriatin, released at 5–10 μg/day, enhanced the response ofS. multistriatus to baits containing 4-methyl-3-heptanol and either of the host synergists but had no effect on the capture ofS. scolytus. The release of larger amounts (57 or 365 μg/day) of (±)-α-multistriatin interrupted the response of both species to the 4-methyl-3-heptanol baits. It appears that α-multistriatin has multiple functions as a behavior-modifying substance for the two beetles. The (+)-β-, (?)-β-, (±)-γ-, and (±)-δ-multistriatins were inactive when released at 5–10 μg/day. The results of these field experiments suggest that one bait can be formulated to capture both species.     

Perfluorinated moth pheromones

Perfluoroalkyl analogs of pheromone constituents were synthesized and responses from male antennal olfactory receptor neurons from three moth species were recorded during stimulation by these analogs. In each analog, the hydrophobic terminus, either a butyl or hexyl substituent on the (Z)-alkenyl chain, was replaced with a perfluorobutyl (Pfb, C₄F₉) or perfluorohexyl (Pfh, C₆F₁₃) moiety. Perfluoroalkyl analogs were more volatile than their hydrocarbon analogs, showing a decrease in gas chromatographic retention time by two to four methylene equivalents (Kovàts retention indices). Specialist neurons of maleHeliothis zea responded to a 0.02-μg dose of (Z)-11-hexadecenal (Z11–16∶Al) and a dose of 200 μg of Pfb-Z11–16∶Al with similar spike discharge rates. The HS(a) neurons ofTrichoplusia ni responded to a dose of 0.02 μg ofZ7–14∶OAc and a dose of 10 μg of Pfb-Z7–12∶Ac with similar discharge rates. The same difference in sensitivity to Pfb-Z7–12∶OH andZ7–12∶OH was observed for the responses of the HS(b) neuron and for the responses of theNS(a) neurons to Pfb-Z9–14∶Ac andZ9–14∶Ac. Sensilla ofDiatraea grandiosella similarly showed 100- to 1000-fold greater sensitivity toZ9–16—Al andZ11–16∶Al than to Pfh-Z9–16∶Al and Pfb-Z11–16∶Al. Thus, replacement of terminal alkyl groups with perfluoroalkyl groups in pheromone components produced biologically active compounds with increased volatility and displaced electrophysiological response profiles. Because of the diminished receptor cell sensitivity, we suggest that the binding of the fluorinated analogs to a putative receptor is reduced as a result of less favorable interaction between the hydrophobic protein binding site and the more rigid and more polar perfluoroalkyl moiety.     

Allelochemical regulation of reproduction and seed germination of two BrazilianBaccharis species by phytotoxic trichothecenes

The potent phytotoxic trichothecene roridins and baccharinoids occur naturally in the Brazilian plants,Baccharis coridifolia andB. megapotamica. Biosynthesis of roridins inB. coridifolia appears to be linked to pollination, and the phytotoxins then accumulate in the seed. The roles of the phytotoxins in pollination, seed maturation, and germination of theBaccharis species were investigated. The high production of roridins occurred only in seeds resulting from intraspecific pollination, and the concentration of the toxins in the seeds generally increased with seed maturity. Removal of seed coats from trichothecene-producing BrazilianBaccharis species (B. coridifolia andB. megapotamica) and non-trichothecene-producing AmericanBaccharis species (B. halimifolia andB. glutinosa) resulted in improved seed germination ofB. halimifolia andB. glutinosa but complete inhibition of seed germination ofB. coridifolia andB. megapotamica. Addition of seed coat extracts of the BrazilianBaccharis species of dilute solutions (10^–6g/ml) of roridins or baccharinoids to the decoated seeds ofB. coridifolia andB. megapotamica resulted in germination, while seeds ofB. halimifolia andB. glutinosa were killed by the phytotoxins. Roridins interacted with gibberellic acid, a germination promoter, but not with abscisic acid, a germination inhibitor. The results from this study suggest that macrocyclic trichothecenes have a regulatory role(s) on reproduction and germination of BrazilianBaccharis species in their natural habitat.     

Biological Activity of Resveratrol, a Stilbenic Compound from Grapevines, Against Botrytis cinerea, the Causal Agent for Gray Mold

The biological activity of resveratrol, a stilbenic compound synthesized by grapevines in response to various stresses, was reevaluated against Botrytis cinerea using a novel in vitro system that enabled direct observation of the fungus with an inverted microscope. We determined that 90 μg resveratrol/ml reduced germination of B. cinerea conidia by ca. 50%. Moreover, resveratrol was shown to significantly reduce mycelial growth of B. cinerea at concentrations ranging from 60 to 140 μg/ml. Exposure to resveratrol at concentrations ranging from 60 to 140 μg/ml resulted in cytological changes in B. cinerea, such as production of secondary or tertiary germ tubes by conidia, cytoplasmic granulations, protoplasmic retractation in the hyphal tip cells, and formation of curved germ tubes. These data reinforce the role played by this compound in the B. cinerea–grapevine interaction.     

Biocidal activity of algal toxins against immature mosquitoes

Extracts of two species of green algae, filamentousRhizoclonium hieroglyphicum Kütz and a phytoplankton,Chlorella ellipsoidea Gerneck, obtained with solvents in the laboratory were assayed againstAedes aegypti L.,Culex quinquefasciatus Say, andCuliseta incidens (Thomson). On extraction with petroleum ether, groundR. hieroglyphicum yielded an active crude extract which was chromatographed on a neutral alumina column and eluted consecutively with petroleum ether, benzene, and methanol. All three eluted fractions were found to induce significant mortality in test mosquito species. The benzene-eluted fraction was the least toxic. The methanol-eluted fraction was the most toxic to all species and exhibited juvenile hormone-like activity; it also caused morphogenetic changes in emerging adults. All three fractions delayed the rate of development of mosquito larvae by 2–5 days. Three supernatants ofC. ellipsoidea obtained on different occasions were tested against first instars ofC. quinquefasciatus. After the confirmation of their activity, all supernatants were extracted with diethyl ether, combined, and assayed against first- and fourth-stage larvae of the three mosquito species. The first-stage larvae ofC. quinquefasciatus andC. incidens were approximately three times more suspectible than those ofA. aegypti. However, fourth-stage larvae of the former two species were about twice as susceptible to the extract as those of the latter species. Dead first-stage larvae of all the species had a shrunken appearance. In general,C. ellipsoidea extract was quicker acting than that ofR. hieroglyphicum.     

Inhibiting biofilm formation of Enterobacter sp. prevented premature withering in cut flowers

Early withering and premature flower fall are a growing menace to the cut flower industry, the reasons of which were considered to be varied from loss of water uptake, bacterial proliferation, decay in tap water etc. In the present study, we identified the bacterial biofilm formation by Enterobacter sp. and subsequent water uptake blockage as the root cause of early withering in cut flowers using Chrysanthemum, Yellow Daisy and Maroon Rose as model plants. The biofilm-forming Enterobacter sp was identified by 16 S rDNA sequencing data. Studies on biofilm were conducted by using field emission scanning electron microscope electron back scattering diffraction (FE-SEM-EBSD), Atomic force microscopy (AFM) and fluorescence microscopy. In vitro and in vivo studies were conducted with different antimicrobials to prevent biofilm formation in both conditions. Most antimicrobials were toxic to plants, but we found citric acid 1,000 μg/ml and calcium hypochlorite 50 μg/ml to be most effective in preventing biofilm formation and extending the vase life of cut flowers. We studied the synergistic action of different combinations in vivo and suggest citric acid 1,000 μg/mL, Ca hypochlorite 50 μg/mL and glucose 1,000 μg/mL as the best combination to be used for prolonging vase life of cut flowers from 10 days (non-treated) to 30 days (treated).     

Preliminary Evidence Supporting the Ability of Hermatypic Corals to Affect Adversely Larvae and Early Settlement Stages of Hard Coral Competitors

Crude, aqueous extracts of five species of hermatypic coral from three scleractinian families were assayed for bioactivity against the early life stages of scleractinian corals. At concentrations between 62.5 and 250 μg/ml, extracts of all species were lethal to at least three of the five species of coral planulae larvae tested. To test for behavioral changes, extracts from four species were tested on planulae of the scleractinian Pocillopora damicornis. All four caused immediate but reversible behavioral aberrations in the larvae at concentrations as low as 7.8 μg/ml. To determine the effects on newly settled corals, postmetamorphosis juveniles (spat) of P. damicornis were exposed to extracts from four hermatypic species. They were either killed or suffered a reduction in growth rates. If released under natural conditions, substances with lethal or sublethal activities against the early life stages of other scleractinians could allow hermatypic corals to influence patterns of scleractinian recruitment, thus playing a role in ordering coral communities.     

Enrichment of eicosenoic and docosadienoic acids fromLimnanthes oil

The long chain (>C₁₈) monoene and diene acids ofLimnanthes oil are useful in synthesizing diene and tetraene wax ester intermediates for prospective lubricant additives and PVC plasticizers. Low-temperature crystallization of theL. alba acids (61% eicosenoic, 19% docosadienoic, and 20% others) in acetone (initial concentration=0.05 g/ml) at −50 C enriches eicosenoic acid (74%) in the precipitated fraction while concentrating docosadienoic (70%) in the supernatant fraction. This simple and efficient process is well suited for large-scale laboratory separations.     

Cytotoxicity of Selected Water-Soluble Polymer–cis-Diaminedichloroplatinum(II) Conjugates Against the Human HeLa Cancer Cell Line

Several polymer-platinum conjugates comprising the square-planar cis-diaminedichloroplatinum(II) complex system of cisplatin-type anticancer drugs are screened for antiproliferative activity in cell culture tests. The water-soluble conjugates prepared in this study or taken from preceding investigations are obtained by platination of aliphatic polyamide carriers containing ethylenediamine segments as side-group or main-chain components. These segments, coordinating to the metal as cis-diamine chelating ligands, are bound to, or into, the carrier backbone through biofissionable amide links permitting drug release from the carrier. In vitro tests are performed against a HeLa human cervix carcinoma cell line. IC₅₀ data, expressed as the concentration of Pt in the conjugates (μg/ml), causing 50% inhibition of cell growth, show the highest activity, with IC₅₀=14 μg/ml, to be associated with a conjugate derived from a polyaspartamide carrier that contains the platinum complex as a side group in proximity to the main chain and, additionally, contains dimethylaminopropyl side groups as solubilizing functions. At the low end of the performance spectrum is a conjugate, with IC₅₀>120 μg/ml, possessing a similar backbone and metal-binding structure, yet comprising long poly(ethylene oxide) grafts. The latter apparently shield the complex-binding tether from enzymatic attack and thus prevent efficient intracellular release of the monomeric complex. Selected conjugates will be submitted for toxicological evaluation.     

Identification of the defensive secretion from soldiers of the North American termite,Amitermes wheeleri (Desneux) (Isoptera: Termitidae)

Head capsule extracts ofAmitermes wheeleri soldiers yielded an isomeric mixture (67 μg/ soldier) of three sesquiterpene hydrocarbons identified by EIMS, [¹³C]-, and [¹H]NMR as (+)-(S,Z)-α-bisabolene (53%), (+)-(R)-β-bisabolene (16%), and (?)-(Z)-α₂-bisabolene (31%). When alarmed, the termite soldiers secreted the fluid onto the head surface surrounding the efferent pore of the frontal gland reservoir. A defensive function for the soldier secretion was indicated by the avoidance behavior displayed toward alarmed soldiers by the antagonistic ants,Pogonomyrmex rugosus andIridomyrmex humilis. Laboratory studies demonstrated that the sesquiterpene mixture is repellent to foragingI. humilis workers.     

Linalool in mandibular gland secretion ofColletes bees (Hymenoptera: Apoidea)

Uinalool was identified as the main component of the mandibular gland secretion in females and males of four species ofColletes bees, viz.,C. cunicularius, C. daviesanus, C. impunctatus andC. succinctus. It was also shown to be the dominant volatile compound in the same gland in maleC. floralis (the female of this species has not yet been investigated). Further, in another species,C. similis, linalool is present in the mandibular gland secretions of females and males, but the dominant volatile component in these secretions is geranial, together with neral. Females of the speciesC. fodiens seem to lack monoterpenes altogether; nonadecane is the dominant volatile compound of the cephalic secretion (based on analysis of a single individual). When linalool is put out in the area of nest aggregation ofC. cunicularius, where the males are patrolling, a distinct increase in flight activity is noted. The function of linalool is discussed on the basis of field observations.This report forms part XXI in the series Studies on Natural Odoriferous Compounds.     

Ratio of in vivo incorporation of3H arachidonic acid and14C linoleic acid into liver lipids from normal and diabetic rats

Normal and streptozotocin diabetic rats were injected via the portal vein with a labeled solution containing³H arachidonic acid and¹⁴C linoleic acid (³H/¹⁴C ratio, 0.5) during a 1 min period. Livers were quickly frozen, pulverized, and the lipids extracted and fractioned by thin layer chromatography. The incorporation of³H and¹⁴C into liver lipids was measured and the percentage distribution of radioactivity into the different lipid fractions was determined. The incorporation of¹⁴C linoleic acid and³H arachidonic acid into liver lipids is apparently reduced in rats with severe diabetes. The higher³H/^14C ratio found in the 1,2 diglycerides from diabetic rats may be explained by the apparently smaller incorporation of¹⁴C linoleic acid or by an isotopic dilution attributable to the great availability of this acid in diabetic rats. On the other hand, the higher³H/¹⁴C ratio observed in triglycerides and phospholipids from diabetic rats, due to a relatively large incorporation of³H arachidonic acid into this fraction, may be explained by the affinity of the enzymes involved in their synthesis for some 1,2-diglyceride units. Insulin was unable to correct the changes observed in the diabetic rats.     

Cuticular hydrocarbons of eight species of north american cone beetles,Conophthorus hopkins

A study to determine the degree of similarity and/or diversity among eight of the 15 described species ofConophthorus is reported. Cuticular hydrocarbons were evaluated forC. conicolens, C. ponderosae, C. cembroides, C. edulis, C. radiatae, C. coniperda, C. resinosae, andC. banksianae. Seventy-eight individual and isomeric mixtures of hydrocarbons were identified by gas chromatography-mass spectrometry, includingn-alkanes, alkenes, alkadienes, 2- or 4-methylalkanes, 3-methylalkanes, and single-component and isomeric mixtures of internally branched mono-, di-, and trimethylalkanes. Differences in alkenes and mono-, di-, and trimethylalkanes can be used easily to separate the eight species.Conophthorus conicolens andC. ponderosae contain the most complex blends. Hydrocarbon patterns in three geographically separated populations ofC. ponderosae, each from a different host, are qualitatively identical with the exception of a homologous series of 3,7-dimethylalkanes from adults collected fromPinus lambertiana cones. The latter could comprise a sibling species. Hydrocarbon mixtures of two eastern species,C. resinosae andC. banksianae, are qualitatively identical, supporting the suspicion thatC. banksianae may not be a valid species. Closely relatedC. cembroides andC. edulis have similar combinations of hydrocarbons except for a unique and abundant alkene (C₂₇₁) inC. edulis and two dimethyhexacosanes inC. cembriodes.Coleoptera: Scolytidae.     

Pharmacokinetics of the thioether phospholipid analogue BM 41.440 in rats

BM 41.440 (1-hexadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine) is a cytotoxic thioether phospholipid analogue that recently has entered phase I trials in cancer patients. The objective of this study was to evaluate the pharmacokinetics of this compound in female rats after administration of a single oral dose (15 mg/kg body weight [bw]). Furthermore, BM 41.440 serum concentrations were determined under a daily oral treatment of up to 13 weeks. Blood samples were obtained via permanent catheters from the femoral arteries before and after drug administration for a total of 120 hr. Urine was collected in 24 hr-intervals for 120 hr; the volume was measured, and aliquots were stored at ?20 C until analytical determination of the thioether derivative. BM 41.440 was assayed in serum and urine by means of a specific, newly developed reverse-phase high pressure liquid chromatography technique. Mean maximum serum concentrations (1.7 μg/ml, n=4 animals) were attained after seven hr. A terminal half-life of ca. 27 hr was calculated from the rate constant for the terminal elimination phase (λ _z ～ 0.026/hr). The mean serum BM 41.440 concentration-time-area-under-the-curve was 52.9 mg × hr/l. The ratio of total body clearance to absorption fraction was 4.7 ml/min × kg bw. Only a small amount of the drug was found in the urine. The quantity excreted in the urine during a 24 hr-interval never exceeded 1.5% of the administered dose. Under a daily oral schedule (15 mg/kg bw × day) up to 13 weeks, mean BM 41.440 serum concentrations of 3.3±0.5 μg/ml and 5.2±1.2 μg/ml (mean ±S.D., n=10 animals) were found after five and 13 weeks, respectively. Taken together, the data indicate that BM 41.440 was absorbed from the gastrointestinal tract after oral administration and that accumulation of BM 41.440 can occur in rats.     

Specificity of response to pheromones in the genusIps (Coleoptera: Scolytidae)

Seventeen species ofIps were laboratory or field tested for the specificity of their response to male-produced aggregating pheromones. In the laboratory, some species appeared not to differentiate among their own pheromones and those of closely related species, whether the pheromones were bioassayed individually or in direct competition. Other species showed strong preference for their own pheromones in competition with those of closely related species, even though they had demonstrated strong attraction to pheromones of the related species. Cross-responsiveness amongI. confusus, I. montanus, andI. paraconfusus, and betweenI. mexicanus andI. concinnus, was confirmed in field tests. Moreover, wildI. paraconfusus females entered the nuptial chambers of males ofI. montanus andI. confusus but not those of the more distantly relatedI. mexicanus. It is hypothesized that specificity of response to aggregating pheromone is important in the maintenance of reproductive isolation among sympatricIps and that the lack of specificity among closely related species enforces the parapatric distributions characteristic of these species.     

Allelopathic research of subtropical vegetation in Taiwan. IV

The phytotoxicity of plant leachates was evaluated from four subtropical grasses:Brachiaria mutica, Digitaria decumbens, Imperata cylindrica var. Major, andPanicum repens. The aqueous leachate of each grass was used to water the growth of the four grasses in pots. The leachates exhibited variable inhibition of grass growth as compared to the tap water control. By the 41st day after treatment, the leachate ofD. decumbens significantly suppressed the growth of itself and retarded that ofB. mutica andP. repens. The growth ofB. mutica was inhibited by its own leachate, but that ofI. cylindrica was not affected by any of the grass leachates. In crop growth rate (CGR) analysis, the four grass leachates exhibited a similar inhibition pattern. In laboratory bioassays, the leachates showed a significant phytotoxic effect on the radicle growth of ryegrass and lettuce. Six phytotoxic phenolics were quantitatively compared by high-performance liquid chromatography, and the amount of compounds varied with species. The highest total amount of phytotoxic phenolics occurred inD. decumbens, followed, in decreasing order, byP. repens, B. mutica, andI. cylindrica. These findings show that the leachates of four grasses possess phytotoxic compounds that may play a significant role in grass dominance in the field.     

Isolation and some Properties of Bacteria that Degrade Polycyclic Aromatic Hydrocarbons

Four bacteria, which could grow on pyrene as the sole source of carbon, were isolated from soil from an urban area in Tokyo. One of them, strain H2-5, was a rod bacterium that was positive in gram staining and in acid-fast staining. The optimum growth temperature was 34-35°C, and the upper limit temperature for the growth was around 45°C. At a concentration of 1.3 μg/ml polycyclic aromatic hydrocarbon (PAH), H2-5 cells (45 μg dry weight/ml) grown on Tryptic Soy Broth made disappear 90% of pyrene in 12 hr, and Benzo(a)pyrene (BaP), Benz(a)-anthracene (BaA), and Benzo(ghi)perylene individually disappeared 60%, 25%, and 8%, respectively, in 3 days. PAHs in the extract by dichloromethane from airborne particles in approximately 5 m³ of air disappeared by the action of H2-5 as follows: pyrene, 100% in 3 days; BaA and BaP, 70% and 71%, respectively, in 4 days. Pyrene in tarry matter extracted from soil disappeared 88% in 4 days.