direct combustion of shell material is easier and less time consuming than acidification. In museum collections bivalve shells are traditionally dry stored, whereas soft tissues are preserved in 70% ethanol, sometimes after fixation with 10% formalin. However, often the whole animal is preserved in ethanol and shells are not stored separately. For the application of these preserved specimens in the investigation of past d N values it is essential to know if liquid preservation methods have an effect on the d N values of bivalve shells and if this effect is predictable. The effects of liquid preservation on the d N values of biological tissues have been examined in a variety of For testing the in uence of CaCO 3 content on d N measurements, different mixtures of acetanilide with inorganic pure CaCO 3 were made, containing between 0 and 10.

4 weight % N. Powder calcite samples were loaded into 4 _ 6 mm tin cups and weighed. d N values were measured using an elemental analyzer coupled via a CONFLO III to a ThermoFinnigan Delta V t isotope ratio mass spectrometer. An inline soda lime CO trap was used to scrub MLN8237 CO 2 from the gas stream entering the gas chromatography column of the EA. IAEA N1 was used as a standard, with an accepted value of 0. 4 _ 0. 2% Long term. standard reproducibility is better than 0. 1% for samples nature, even samples between 5 and mg N provided reasonable data. There is also an upper limit to the amount of shell material that can be loaded into the EA, but this was not evaluated here.

This method is robust because calcium carbonate com pletely decomposes around 8258C and the ash combustion Nilotinib in the EA was around 10208C, therefore, all N should be released from the matrix and carried to the IRMS. Moreover, previous studies have used an EA IRMS system to combust Fig. 2 that the narrow and near symmetrical peak shapes are similar for both shell carbonate and synthetic mixtures, which suggests that both matrices are reacting similarly in the EA IRMS. We therefore argue that it is possible to measure carbonates for d C analysis. It is clear from the traces in larger than 30 mg N. d N values are expressed in % vs. atmospheric nitrogen. Pure synthetic CaCO 3 had peaks similar to empty tin cups, empty tin cup 1/4 0. 49 Vs) and therefore did not contribute much to the calculated delta values. The acetanilide standard had a d N value of 2.

12 _ 0. 13% when it was run without PI3K Inhibitors synthetic CaCO 3 and was _2. 02 _ 0. 11% when it was run with 98. 4 to 66. 8% CaCO 3. These values are not significantly different. In addition, during a preliminary trial, we ran 0. 4 mg of the IAEA N1 ammonium sulfate SO 4) standard in. 72 mg CaCO 3 and found no offset from N1 standards run without CaCO3. Our results show that samples with as little as 20 mg N can provide accurate d N values. Prior acidification is not required to eliminate the carbonate matrix to produce accurate results, as has been previously reported. It should be noted that mollusks with very low organic matrix in their shells may require a pre concentration step to reduce the poorer precision of small samples. However, considering the large fractionations associated with nitrogen isotopes in Figure 1.

d N values for acetanilide mixed with 66. 8 to 98. 4 weight % synthetic CaCO 3 powder and pure acetanilide. The solid line represents the mean value of _2. 02% for data above mg N. The error bar represents the 1s of _0. 11%. wileyonlinelibrary. Ion Channel com/journal/rcm Copyright 2011 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 675 680 Letter to the Editor tissue is subject to metabolic turnover and is thus repre sentative for a specific time window, see e. g., Paulet et al,. while the shell samples averaged at least 1 year of growth. This makes comparing soft tissues with shell organic matrix difficult. However, as shown in Delong and Thorp, tissues with slower turnover rates, such as the adductor muscle, are better for comparisons with metabolically inactive shells.

Most previous studies that report differences between skeletal d N and soft tissue d N do not take the different amounts of time being averaged into consider ation. Moreover, HSP many studies compare whole body tissue d N data to shell data while it is known that different organs can have quite different d N values, sometimes as much as 5% in the same animal. This may explain why Dtissue shell values for the same species of clam range from 0. 2 to 2. 4%, see ODonnell et al.. Soft tissue d N data from M. edulis specimens collected at three different periods in 2002 from Knokke show significant changes throughout the year, which would be averaged in the shell samples we analyzed. Taking the average of these 25 soft tissue data results in a Dtissue shell value of _1. 5 _ 1. 0%. In the future it is important to compare tissues and shells that represent the same time period.

xestobii wasalsoshownheretorapidlymineralizeup to 25% of metolachlor after 10 days of growth. Because differ ences in mineralization rates among microorganisms in soils are likely due to both biotic and abiotic factors, more studies are needed to assess the contribution of mineralization to loss of this herbicide in soils. Results of mass balance analyses indicated that 5% of metolachlor in the culture medium was present in C. xestobii and B. simplex cells following incubation with metolachlor. This result indicated that metolachlor was not significantly incorporated into biomass and, thus, metabolites that were not mineralized were likely released into the growth medium. Our results are in contrast to those reported in ref 17, which reported that 80% of ring labeled metolachlor added to a microbial community was removed from the medium and accumulated inside cells.

Mechanism of Degradation. The mechanism by which metola chlor is transformed by C. xestobii is not clear. Because Pazopanib analytical standards of possible metolachlor metabolites were not available, we used the University of Minnesota Biocatalysis/Biodegrada tion Database Pathway Prediction System to predict plausible pathways for the microbial degradation of metola chlor. The PPS identi fied 22 possible molecules with molecular ions 190. Comparison of the possible molecular ions from the total ion current plot of culture medium obtained following growth of C. xestobii on metolachlor resulted in no positive matches. Also, HPLC fractionation of the spent medium following growth of C.

xestobii in uniformly ring labeled metolachlor did not result in any peaks that had 2% of the applied C, other than the metolachlor Pazopanib peak, leading to difficulty in extrapolating a degra dation pathway. Although it is tempting to speculate that dechlorination was not a major mechanism for the degradation of metolachlor by the isolated yeast, too few data are available to accurately determine this. Consequently, the pathway by which metolachlor is transformed by C. xestobii is currently unknown and awaits further analyses. In summary, in this study we report on the isolation of a bacte rium and yeast that have the ability to catabolize metolachlor. We also show that the yeast C. xestobii uses metolachlor as a sole C and energy source for growth and is able to mineralize t. this compound under controlled laboratory conditions.

Although otherfungalandbacterialstrainshavebeenisolatedthatareableto Cannabinoid Receptor partially transform metolachlor, most attempts to isolate pure or mixed microbial cultures capable of mineralizing metolachlor have been unsuccessful. Whereas the degradation of metola chlor has been previously studied with a pure culture of the fungus Ch. globosum, which also used this herbicide as a sole source of C and energy, gas liquid chromatographic analysis of the concen trated extract from resting cell experiments with this fungus showed that at least eight extractable products were produced fromtheoriginalcompound. TiedjeandHagedorn reported that the major product of alachlor degradation by this fungus was likely 2,6 diethyl N aniline, and McGahen and Tiedje reported that the co metabolism of metolachlor by Ch.

globosum is thought to occur by removal of one or both R groups from the nitrogen atom and subsequent dehydrogenation of the ethyl substituent. These authors also postulated that the HDAC-42 fungus may eventually remove the chloro, methoxy, or ethoxy substituent from the R groups. In addition to fungi, bacteria have also been reported to transform alachlor. For example, Sette et al. reported that a Streptomycetes sp. strain degraded ??60 75% of the alachlor within days to produce indole and quinoline deriv atives, and Villareal et al. reported that Moraxella sp. strain DAK3 respired and grew on N substituted acylanilides containing methyl, ethyl, or isopropyl substitutions, but failed to grow on alachlor and metolachlor. In contrast to previous studies with fungi, the isolated C.

xestobii strain degraded 50% of metolachlor after 4 days of growth, and no metabolites, such as the ethanesulfonic acid and oxanilic acid, were detected in the growthmediumbyHPLCanalysis. A. flavus and A. terr ??cola PARP have been also described as metolachlor degrading fungi, reducing the half life of this herbicide from 189 to 3. 6 and 6. 4 days, respec tively. Coupled with data showing that some fungicides significantly reduce metolachlor dissipation in soils, results from our studies are consistent with the notion that soil yeast and other fungi may be responsible for significant transformation of metolachlor in soils. Moreover, because degradation of metola chlor by C. xestobii was fairly rapid and resulted in the miner alization of this herbicide, our data suggest that this yeast may eventually prove to be useful for metolachlor bioremediation efforts. More studies, however, are needed to determine whether this yeast is also able to metabolize and mineralize other aniline herbicide compounds and to identify metabolites produced dur ing the degradation process.

Growth of Candida xestobii in minimal medium in the presence of 50 g mL metolachlor and in MM with metolachlor plus sucrose y east extract, or sucrose plus yeast extract. Metolachlor degradation by Candida xestobii in MM in the presence of 50 g mL metolachlor i n MM plus sucrose, yeast extract, and sucrose plus yeast extract, and in control medium containing metolachlor but without added inoculum. Metolachlor MRM transitions were as follows: 283. 8 M t H 284. 2 252. 2 and 284. 2 176. 2. Minimal matrix effects were observed. RESULTS AND DISCUSSION Metolachlor, a member of the chloroacetanilide class of herbicides, contains 15 carbon atoms and one nitrogen atom per molecule and, thus, can potentially serve as a nutrient source for microbial growth.

Nilotinib However, despite its use over the past 30 years, only a relatively few microorganisms that can incompletely transform metolachlor have been identified. This was thought to be due, in part, to its sorptive behavior, lack of bioavailability, and requirements for co meta bolism in the presence of microbial consortia. In the study reported here, we describe the isolation and identification of two microorganisms that were capable of using metolachlor as the sole source of C for growth. Both microbes were isolated, via enrichment, from the same Spanish soil with a history of metolachlor application. Microscopic and molecular analyses showed that the isolated organisms were a bacterium and a yeast. The bacterium was a Gram positive, spore forming, microorganism, and 16S rRNA sequence analysis confirmed the isolate was B.

simplex, with 99% nucleotide sequence similarity. The identification of the yeast was much more difficult, in part due to incomplete and complicated taxonomy of yeasts isolated from natural substrates, such as soil. Consequently, they are extremely difficult to differentiate phenotypically and are very often misidentified. Sequence analysis of 18S Entinostat and 26S rDNA and the ITS region led to the conclusion that the isolated yeast was C. xestobii, with 99% nucleotide similarity in the GenBank CBS Yeast databases. Because only 2 bp differentiate C. xestobii and Pichia guillier mondii in the D1/D2 and ITS regions, species identity was confirmed by using biochemical analyses. The isolated yeast grew in MM containing glucose, sucrose, D xylose, trehalose, maltose, starch, and galactose, but failed to grow on rhamnose, inositol, lactose, D mannitol, and D arabinose.

Results of these analyses were consistent with taxonomic assignment of the yeast to C. xestobii. Growth and Degradation of Metolachlor by C. xestobii and B. simplex. The influence of culture media and carbon sources on the degradation of 50 g mL metolachlor was examined. The dis appearance of metolachlor PI3K Inhibitors was determined to be due to microbial metabolism. Results in Figure 2A show that as C. xestobii grew in MM amended with metolachlor, with or without other added amendments, the concentration of metolachlor decreased to 40% of the initial concentration after 6 days of incubation. No further degradation of metolachlor was observed after this time.

Control media, which were not inoculated, did not exhibit metolachlor disappearance, in agreement with previous reports that metolachlor degradation is mainly due to biological rather than chemical processes. The greatest amount and fastest rate of metolachlor PI3K Inhibitors degradation wereobservedinmetolachlormediumamendedwith 0. 04% of yeast extract. In contrast, whereas growth of the yeast was faster and greatest in metolachlor medium amended with sucrose and yeast extract, only about 20% of metol achlor was degraded after 9 days of incubation. Taken together, these results indicated that the yeast has the ability to catabolize metolachlor as a sole source of nutrients for growth, but preferred other nutrient sources, suchas yeast extract and sucrose, whichare probablyeasiertometabolize. Because the yeast also grew in MM amended only with metolachlor, data presented in Figure 2 also show that C.

xestobii uses metolachlor as a sole C source for growth. To our knowledge, this is the first reported yeast that has the ability to catabolize metolachlor and use this compound as sole C metolachlor. metolachlor, these cultures demonstrated a faster rate of degradation than that seen with the initial degradation of the compound. This indicated that C. FDA xestobii more actively degraded metolachlor following initial growth on this substrate, perhaps due to either the presence of more cells or the induction of enzymes required for metolachlor degradation. Results in Figure 4 show that B. simplex also grew in metola chlor medium, with or without added amendments. The initial concentration of metolachlor decreased 65% after 6 days of incubation, after which time no further degradation of the compound was observed.

The degradation of metolachlor by B. simplex was approximately 25% less than that observed with the yeast under the same conditions. The degradation rate of metolachlor was similar in the different culture media used, despite the greater growth observed when the growth medium containing metolachlor was amended with yeast extract or with sucrose plus yeastextract.

This chemical acts by inhibiting elongases andthebiosynthesisofgibberellicacid,resultinginplantdeath when absorbed through the roots and shoots just above the seed of the target plants. TheUSEPAestimatedthat59 64millionpoundsofmetolachlor was applied in 1995, and its use has been steadily declining duringrecentyears. Recommendedapplicationlevelsofthechemical were 1. 2 5 lb/acre in 1995. In 1999, however, Syngenta Crop Protection, one of the main manufacturers of this herbicide, dis continued sales of metolachlor and replaced it with the reduced risk compound S metolachlor. This enantiomer is more effective in weed control than racemic metolachlor, providing the same weed control but requiring 35% less applied chemical.

Meto lachlor use in the United States was subsequently reduced by 15 24 million pounds in 2001, as herbicides containing this chemical were replaced SNX-5422 with S metolachlor, of which 20 24 million pounds wasappliedduringthatyear. Thisisthelargestreductionofpesticide use in the United States to date. Since atrazine was banned in Europe in 2003, there had been increasing use of metolachlor combinedwithpostemergence herbicidesuntil S metolachlorwas substituted for use of the mixed enantiomer. The European Union presently allows application of only S metolachlor for weed control. In Spain, it has been estimated that 5000 t of S metola chlor is applied on 1. 3 million hectares per year Metolachlorisslightlysolubleinwater and is moderately sorbed by most soils, with greater sorption occurring on soils having greater organic matter and clay contents.

Extensive leaching of 2010 American Chemical Society Published on Web 12/29/2010 PDE Inhibitors pubs. acs. org/JAFC metolachlor is reported to occur,especially insoilswithlow organic content. Metolachlor is relatively more persistent in soils as compared to other widely used chloroacetanilide herbicides, such as alachlor and propachlor. Metolachlor half lives ranging from 15 to 70 days have been observed in different soils. The herbicide is highly persistent in water, over a wide range of pH values, with reported half life values of g200 and 97 days in highly acid and basic conditions, respectively. Metolachlor is also relatively stable in water, and under natural sunlight, only about 6. 6% was degraded in 30 days. Because very little metolachlor volatilizes from soil, photodegradation is thought to be a pathway for loss, but only in the top few centimeters of soil.

On the basis of these observations, it has been postulated that metolachlor dissipation in soil mainly occurs via biological degrada tion, rather than chemical processes. The degradation of metolachlor Cannabinoid Receptor in soils has been proposed to occur via co metabolic processes that are affected by soil texture, microbial activity, and bioavailability. The limited number of reports on the micro bial degradation of metolachlor, and its long half life, led to contrasting hypotheses that microbial consortia are likely needed for metolachlor catabolism in soils or that metolachlor is not readily metabolized bysoilmicroorganisms. More over, previous attempts to enhance metolachlor degradation in natural fields have generally not been successful.

This was, in part, attributed to the low bioavailability of this herbicide to microorganisms. However, the half life of metolachlor in sterile soil was reduced from 97 to 12 days after the addition of an active HDAC-42 microbial community, indicating that other biotic factors influence metolachlor degradation in soils. Whereas pure cultures of an actinomycete, a streptomycete, and a fungus capableofmetabolizingmetolachlorhavebeenreported,degradation times were long, and only small amounts of the herbi cide were degraded or mineralized. Similarly, low rates of mineralization of the chloroacetanilide herbicide alachlor have also been reported, only 3 % of the herbicide was mineralized after 30 122 days. Pure microbial cultures have also been reported to be relatively ineffective in mineralizing acetochlor, a related herbicide, with maximum rates of 24%.

Recently, Xu et al. reported that 89, 63, and 39% of the chloroacetanilide HSP herbicides propachlor, alachlor, and meto lachlor were degraded, respectively, after 21 days of incubation. The major dissipation routes for both alachlor and acetochlor appear to be due to microbiologically mediated degradation, runoff, and leaching. Most chloroacetanilide degrading microorganisms reported to date are fungi, and metolachlor is thought to be more persistent and recalcitrant to degradation thanthe other chloroacetanilide herbicidesinsoils and water. In this study, we examined Spanish soils with a history of metolachlor application for the presence of pure microbial cultures capable of catabolizing this herbicide. Here we report the isolation and characterization of a pure culture of a yeast, Candida xestobii, and a bacterium, Bacillus simplex, that have the ability of catabolize metolachlor and use this herbicide as a sole source of carbon for growth. We also report that the yeast is also capable of rapidly catabolizing other chloroacetanilide herbi cides, such as acetochlor and alachlor.