Determination of alkylphenol residues in breast and commercial milk by solid-phase extraction and gas chromatography–mass spectrometry

This study presents a feasible and sensitive method to determine alkylphenol residues (i.e., 4-t-octylphenol (4-t-OP) and the isomers of 4-nonylphenols (4-NPs)) in breast milk samples and commercial cow milk products. The matrix interference associated with the constituents in the milk was reduced by extraction with n-hexane and dilution with 50% methanolic solution (v/v, methanol/water), then followed by the Oasis-HLB SPE extraction. The analytes were determined by a GC–MS system in full-scan and selected ion monitoring modes simultaneously. Limit of quantitation was less than 0.05 ng/g in a 20 g (wet weight) milk sample. The 4-NPs were detected in 19 of the 20 breast milk samples at concentrations ranging from 1.7 to 11.6 ng/g, while 4-t-OP was detected in 8 samples at concentrations ranging from 0.4 to 1.1 ng/g. The 4-NPs were detected in all the testing commercial cow milk products at concentrations ranging from 2.9 to 8.8 ng/g.     

Identification and characterization of another 4-nitrophenol degradation gene cluster, nps, in Rhodococcus sp. strain PN1

4-Nitrophenol (4-NP) is a toxic compound formed in soil by the hydrolysis of organophosphorous pesticides, such as parathion. We previously reported the presence of the 4-NP degradation gene cluster (nphRA1A2) in Rhodococcus sp. strain PN1, which encodes a two-component 4-NP hydroxylase system that oxidizes 4-NP into 4-nitrocatechol. In the current study, another gene cluster (npsC and npsRA2A1B) encoding a similar 4-NP hydroxylase system was cloned from strain PN1. The enzymes from this 4-NP hydroxylase system (NpsA1 and NpsA2) were purified as histidine-tagged (His-) proteins and then characterized. His-NpsA2 showed NADH/FAD oxidoreductase activity, and His-NpsA1 showed 4-NP oxidizing activity in the presence of His-NpsA2. In the 4-NP oxidation using the reconstituted enzyme system (His-NpsA1 and His-NpsA2), hydroquinone (35% of 4-NP disappeared) and hydroxyquinol (59% of 4-NP disappeared) were detected in the presence of ascorbic acid as a reducing reagent, suggesting that, without the reducing reagent, 4-NP was converted into their oxidized forms, 1,4-benzoquinone and 2-hydroxy-1,4-benzoquinone. In addition, in the cell extract of recombinant Escherichia coli expressing npsB, a typical spectral change showing conversion of hydroxyquinol into maleylacetate was observed. These results indicate that this nps gene cluster, in addition to the nph gene cluster, is also involved in 4-NP degradation in strain PN1.     

Degradation of synthetic androgens 17alpha- and 17beta-trenbolone and trendione in agricultural soils

17Beta-trenbolone acetate (TBA) is a synthetic androgenic steroid hormone administered as a subcutaneous implant for growth promotion in beef cattle. TBA is converted metabolically to primarily 17alpha-trenbolone and trendione, and excreted in manure from implanted cattle. To predict the persistence of synthetic androgens once land-applied, aerobic degradation rates in two contrasting agricultural soil types (clay loam and a sandy soil) of both trenbolone isomers (17alpha and 17beta) and their primary metabolite trendione were measured and isomer interconversion was assessed. The impact of manure application was also evaluated in the clay loam soil. A pseudo first-order exponential decay model was derived assuming irreversible transformation and no impact of sorption on availability for degradation. The model generally resulted in good fits to the data. Both isomers degraded to trendione in a similar manner with half-lives (t1/2) on the order of a few hours to 0.5 days at applied concentrations of < or = 1 mg/kg. Similar degradation rates were observed in the presence and absence of manure applied at rates typical for land-application of cattle manure. Trenbolone degradation was concentration-dependent with degradation rates decreasing with increasing applied concentrations. Trendione, whether applied directly or produced from trenbolone, persisted longer than trenbolone with t1/2 values of 1 to 4 days. A small amount (1.5%) of conversion of trendione back to 17beta-trenbolone was observed during aerobic incubation regardless of the applied concentration. A small amount of 17alpha-isomer also converted back to 17beta-trenbolone, presumably through trendione. In autoclaved soils, no degradation of 17alpha- or 17beta-trenbolone was observed during the first 3 days, and trendione degradation was relatively small compared to a microbially active soil.     

Degradation of racemic and enantiopure metalaxyl in tropical and temperate soils

The degradation of the racemic mixture and the enantiomers of metalaxyl in typical soils from Germany and Cameroon has been studied. Formulated and unformulated R-metalaxyl were studied as well as racemic (rac) metalaxyl in controlled incubation experiments. The kinetics of the degradation or transformation were determined by means of reversed phase HPLC, while the enantiomeric ratios were measured by HPLC with a chiral Whelk O1 column. The degradation followed first-order kinetics (R2 > or = 0.96). Higher metalaxyl acid metabolite concentrations were found in German soil than in Cameroonian soil. The enantiomers of the fungicide each had different degradation rates in both soils, with half-lives ranging from 17 to 38 days. All forms of metalaxyl had lower degradation rates in the Cameroonian soil than in the German soil. The degradation. of the R-enantiomer was much faster than the S-enantiomer in the German soil and slower than the S-enantiomer in the Cameroonian soil suggesting that different microbial populations, which may be using different enzymes, have different degradation preferences. The results for the major differences in the degradation of the enantiomers may have some implication for the frequency of use as well as the environmental assessment for chiral pesticides.     

Determination of nonylphenol ethoxylate metabolites in vegetables and crops by high performance liquid chromatography-tandem mass spectrometry

A method has been developed for the simultaneous determination of the concentration of nonylphenol (4-NP), nonylphenol monoethoxylates (NP₁EO) and nonylphenol diethoxylates (NP₂EO) in vegetables and crops by liquid chromatography-tandem quadrupole mass spectrometry (HPLC-MS/MS). These target compounds were extracted from vegetable and crop samples with acetonitrile, and then the extracts were cleaned using solid phase extraction with graphitised carbon black tandem primary secondary amine (PSA) cartridges. The MS method enabled highly reliable identification by monitoring the corresponding ammonium adduct [M+NH₄]⁺ in the positive mode for NP₁EO and NP₂EO, and the deprotonated molecule [M−H]⁻ in the negative mode for 4-NP. Recoveries for the spiked samples ranged from 65% to 118%. The limit of detection (LOD) of 4-NP, NP₁EO and NP₂EO was 3, 5 and 0.1 μg kg⁻¹, respectively. This method would be useful for the quick and routine detection of the residues of 4-NP, NP₁EO and NP₂EO in vegetables and crops.     

Biodegradation of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) by Phanerochaete chrysosporium: new insight into the degradation pathway

Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) is a recalcitrant energetic chemical that tends to accumulate in soil, close to the surface. The present study describes the aerobic biodegradability of HMX using Phanerochaete chrysosporium. When added to 7 day old static P. chrysosporium liquid cultures, HMX (600 nmol) degraded within 25 days of incubation. The removal of HMX was concomitant with the formation of transient amounts of its mono-nitroso derivative (1-NO-HMX). The latter apparently degraded via two potential routes: the first involved N-denitration followed by hydrolytic ring cleavage, and the second involved alpha-hydroxylation prior to ring cleavage. The degradation of 1-NO-HMX gave the ring-cleavage product 4-nitro-2,4-diazabutanal (NDAB), nitrite (NO2 -), nitrous oxide (N2O), and formaldehyde (HCHO). Using [14C]-HMX, we obtained 14CO2 (70% in 50 days), representing three C atoms of HMX. Incubation of real soils, contaminated with either HMX (403 micromol kg(-1)) (military base soil) or HMX (3057 micromol kg(-1)), and RDX (342 micromol kg(-1)) (ammunition soil) with the fungus led to 75 and 19.8% mineralization of HMX (liberated 14CO2), respectively, also via the intermediary formation of 1-NO-HMX. Mineralization in the latter soil increased to 35% after the addition of glucose, indicating that a fungus-based remediation process for heavily contaminated soils is promising. The present findings improve our understanding about the degradation pathway of HMX and demonstrate the utility of using the robust and versatile fungus P. chrysosporium to develop effective remediation processes for the removal of HMX.     

Bioaccessibility of environmentally aged 14C-atrazine residues in an agriculturally used soil and its particle-size aggregates

After 22 years of aging under natural conditions in an outdoor lysimeter the bioaccessibility of 14C-labeled atrazine soil residues to bacteria was tested. Entire soil samples as well as sand-sized, silt-sized, and clay-sized aggregates (>20, 20-2, and <2microm aggregate size, respectively) were investigated under slurried conditions. The mineralization of residual radioactivity in the outdoor lysimeter soil reached up to 4.5% of the total 14C-activity after 16 days, inoculated with Pseudomonas sp. strain ADP. The control samples without inoculated bacteria showed a mineralization maximum of only about 1% after 44 days of incubation. Mineralization increased in the clay-sized aggregates up to 6.2% of the total residual 14C-activity within 23 days. With decreasing soil aggregate sizes, residual 14C-activity increased per unit of weight, but only minor differences of the mineralization in the soil and soil size aggregates using mineral-media for incubation was observed. Using additional Na-citrate in the incubation, the extent of mineralization increased to 6.7% in soil after 23 days following incubation with Pseudomonas sp. strain ADP. These results show that long-term aged 14C-atrazine residues are still partly accessible to the atrazine degrading microorganism Pseudomonas sp. strain ADP.     

混合价态铁基金属有机框架催化过氧乙酸高效降解对硝基苯酚

为提升铁基金属有机框架(Fe-MOFs)对印染废水深度处理的效能,采用原位掺杂溶剂热技术制备了混合Fe(Ⅱ)/Fe(Ⅲ)价态MIL-53(Fe)(MV-MIL-53(Fe))催化剂。借助X射线粉末衍射仪、场发射扫描电镜、氮气吸附仪和吡啶吸附红外光谱仪等对MV-MIL-53(Fe)晶体结构、微观形貌、孔结构和表面酸位等本征结构进行了测试分析。选取对硝基苯酚(4-NP)作为印染废水模型污染物,以过氧乙酸(PAA)为氧源,研究了MV-MIL-53(Fe)催化PAA降解4-NP的性能和关键活性物种。结果表明:Fe(Ⅱ)的引入提高了MIL-53(Fe)表面Lewis酸位密度,为PAA催化活化提供了更丰富更高效的活性位点;MV-MIL-53(Fe)/PAA体系对4-NP的降解速率常数高达0.052 1 min^-1,分别是MV-MIL-53(Fe)/H₂O₂、MIL-53(Fe)/PAA和MIL-53(Fe)/H₂O₂体系的2.05、1.45和6.68倍,且MV-MIL-53(Fe)重复循环使用5次后仍保持良好的结构稳定性和催化降解性能;羟基自由基(·OH)是MV-MIL-53(Fe)催化PAA快速降解4-NP的关键活性物质。     

Sorption behavior of nonylphenol in terrestrial soils

Nonylphenol (NP) as an intermediate from anaerobic degradation of widely used nonionic surfactants occurs widespread in the environment. Partition behavior of this toxic and endocrine-disrupting chemical between soil and water was not examined until yet. The objective of this investigation was to quantify sorption and desorption behavior of 4-nonyl[14C]phenol in a set of 51 soils using the batch equilibrium approach. Kinetic studies indicated apparent equilibrium within 20 h. Sorption was influenced by sorbate structure as could be shown with branched 4-nonyl[14C]phenol and the linear 4-n-NP, respectively. Linear 4-n-NP behaves differently from the branched isomers of 4-NP. Sorption of 4-nonyl[14C]phenol tested with five different initial concentrations resulted in linearly fitted isotherms that provided calculation of sorption partition coefficients (KP). Desorption partition coefficients (KP-des) revealed hysteresis independent of soil properties but decreasing with decreasing initial NP concentrations. KP values were correlated with organic carbon content of the soils yielding a log KOC of 3.97.     

Enantioselective degradation of metalaxyl in soils: chiral preference changes with soil pH

Chiral pesticides are often degraded enantio-/stereoselectively in soils. Degradation is typically studied with one or a small number of soils so that it is not possible to extrapolate the findings on chiral preference to other soils. For this study, the fungicide metalaxyl was chosen as a "chiral probe" to investigate its enantioselective degradation in 20 different soils, selected primarily to cover a wide range of soil properties (e.g., acidic/alkaline, aerobic/ anaerobic) rather than to consider soils of agricultural importance. Racemic metalaxyl was incubated in these soils under laboratory conditions, and the degradation of the enantiomers as well as the enantioselective formation/ degradation of the primary major metabolite, metalaxyl acid, was followed over time, using enantioselective GC-MS after ethylation with diazoethane. In aerobic soils with pH > 5, the fungicidally active R-enantiomer was degraded faster than the S-enantiomer (k(R) > k(S)), leading to residues with a composition [S] > [R]. However, in aerobic soils with pH 4-5, both enantiomers were degraded at similar rates (k(R) approximately k(S)), and in aerobic soils with pH < 4 and in most anaerobic soils, the enantioselectivity was reversed (k(R) < k(S)). These considerable soil-to-soil variations were observed with soils from locations close to each other, in one case even within a single soil profile. Liming and acidification of a "nonenantioselective" soil prior to incubation resulted in enantioselective degradation with k(R)> k(S) and k(R) < k(S), respectively. While the enantioselectivity (expressed as ES = (k(R) - k(S))/(k(R) + k(S))) of metalaxyl degradation in aerobic soils apparently correlated with soil pH, no such correlation was found for metalaxyl acid. Reevaluation of published kinetic data for the herbicides dichlorprop and mecoprop indicated similar correlations between soil pH and ES as for metalaxyl.     

Effect of duration of feeding under free-range conditions on production results and carcass and fat quality in Iberian pigs

This experiment was undertaken to provide information on the effect of feeding system applied during the finishing period (100-150kg) on the quality of Iberian pig meat. Four feeding systems were applied: pigs fed under free-range conditions with acorns and grass fully available during 111 days (FR(111)), pigs fed concentrate diet in confinement during 28 days and free-range with acorns and grass fully available during 83 days (CDC(28)+FR(83)), pigs fed concentrate diet in confinement during 65 days and free-range with acorns and grass fully available during 46 days (CDC(65)+FR(46)) and pigs fed concentrate diet in confinement with a feed average daily amount of 3.1kg during 111 days (CDC(111)). The CDC(111) pigs had more muscular carcasses characteristics than the remaining groups of pigs. However, the higher concentration of C18:1 n-9 in subcutaneous backfat was found in FR(111) pigs. In the neutral lipids from intramuscular fat of Longissimus dorsi only the C18:2 n-6 and C18:3 n-3 proportions were affected by feeding system, and the n-6/n-3 ratio observed was lower in FR(111) and CDC(28)+FR(83) pigs than in CDC(111) pigs. In the neutral lipids from hepatic fat the proportion of n-6 fatty acids was lower in FR(111) pigs than in CDC(65)+FR(46) and CDC(111) pigs.     

Atmospheric formation of hydroxynitropyrenes from a photochemical reaction of particle-associated 1-nitropyrene

The formation of hydroxynitropyrene (OHNP) via a photochemical reaction of 1-nitropyrene (1-NP) was demonstrated using a UV irradiation system. The photoreaction of 1-NP in methanol gave products that were hydroxy-substituted at position 1 and mononitro-substituted at positions 2, 3, 5, 6, and 8 [1-hydroxy-x-nitropyrenes (1-OH-x-NPs); x = 2, 3, 5, 6, and 8]. 1-OH-2-NP and 1-OH-5-NP have been identified in ambient airborne particles for the first time. On the contrary, these two OHNP isomers were not found in standard reference materials (SRM) 1650b and SRM 1975, which are typical samples of diesel exhaust particles (DEPs). The concentrations of the other OHNP isomers in the DEP samples were much lower than the concentration of 1-NP, which is a representative nitro-derivative polycyclic aromatic hydrocarbon that is emitted directly from combustion sources. On the other hand, significantly higher concentration ratios of ∑OHNP (=1-OH-3-NP + 1-OH-6-NP + 1-OH-8-NP) to 1-NP were observed in ambient airborne particles than in the DEP samples. In ambient airborne particles, the mean ∑OHNP/1-NP concentration ratio of 1.4 was 35 times higher than that in SRM 1650b and 470 times higher than that in SRM 1975. The diurnal concentration of 1-NP, which was observed at a typical residential area in Osaka, Japan, increased early in the morning and late in the evening, suggesting that automotive emissions contributed to the occurrence of 1-NP. The OHNP concentrations also rose in the morning, and variations of OHNP concentrations similar to those of 1-NP were observed during the daytime. However, the concentrations of OHNPs did not increase in the evening rush hour, and were low at night, i.e., in the absence of sunlight. These results support the idea that atmospheric OHNPs are predominantly formed via secondary formation processes; i.e., photochemical reactions of 1-NP are expected to have a significant effect on the occurrence of OHNPs in the atmosphere.     

Aflatoxin B1 degradation by flavobacterium aurantiacum in the presence of reducing conditions and seryl and sulfhydryl group inhibitors

This study was undertaken to determine the effects of reducing conditions (L-cysteine) and seryl (phenylmethylsulfonyl fluoride) and sulfhydryl (divalent cadmium) group inhibitors on aflatoxin B1 (AFB1) degradation by Flavobacterium aurantiacum. High-performance liquid chromatography was used to determine AFB1 concentrations in 72-h cultures of F. aurantiacum. The addition of 0.1, 1, or 10 mM L-cysteine did not have any significant effect on AFB1 degradation by these cultures after incubation for 4, 24, or 48 h (P > 0.05). The addition of 0.1 mM phenylmethylsulfonyl fluoride did not significantly decrease AFB1 degradation (P > 0.05), but 1 mM phenylmethylsulfonyl fluoride significantly decreased AFB1 degradation after 4, 24, and 48 h of incubation (P < or = 0.05). No significant difference in AFB1 degradation was obtained with 0.1 mM Cd2+ after 4, 24, or 48 h of incubation (P > 0.05). The addition of 1 and 10 mM Cd2+ significantly decreased AFB1 degradation compared with the cells containing AFB1 alone after 4 and 24 h (P < or = 0.05). The addition of chelators, 1 mM EDTA and 1 mM o-phenanthroline, did not result in removal of inhibition of AFB1 degradation observed with 1 and 10 mM Cd2+. Higher concentration of chelators (>1 mM) are necessary to overcome the inhibitory effect. Further work on the cellular fractions and/or crude enzyme preparations is necessary to determine if indeed sulfhydryl and seryl groups of the enzymes are involved in AFB1 degradation (by maintaining either the structure or function of the enzyme).     

Cloning and characterization of a 4-nitrophenol hydroxylase gene cluster from Rhodococcus sp. PN1

A 4-nitrophenol (4-NP)-degrading bacterium was isolated from activated sludge and identified as a Rhodococcus sp. This bacterium, designated as strain PN1, could utilize 4-NP as a sole carbon, nitrogen and energy source. Degradation tests of 4-NP using cell suspensions of strain PN1 revealed that the degradation was induced by 4-NP and that 4-nitrocatechol (4-NC) was one of the metabolites. A gene library was constructed from the total DNA of strain PN1 and introduced into Rhodococcus rhodochrous ATCC 12674. Two recombinant strains showed 4-NP hydroxylase activity, and a 9.1-kb DNA fragment encoding the activity was isolated from one of the strains. In addition, a 2.4-kb smaller fragment expressing the activity was subcloned from the 9.1-kb fragment and sequenced. The sequence analysis showed that the fragment encodes a two-component 4-NP hydroxylase, the predicted amino acid sequence of which exhibits significant similarity to those of phenol hydroxylases and 4-hydroxyphenylacetate 3-hydroxylases belonging to the two-component flavin diffusible monooxygenase (TC-FDM) family proposed by Galán et al. (J. Bacteriol., 182, 627-636, 2000).     

Trophodynamic behavior of 4-nonylphenol and nonylphenol polyethoxylate in a marine aquatic food web from Bohai Bay, north China: comparison to DDTs

4-Nonylphenol (4-NP) is of particular concern because of its ubiquity in aquatic environment and its endocrine-disrupting effects in aquatic organisms. On the basis of its octanol-water partition coefficient (104.6), it has a potential to bioaccumulate in aquatic food webs. However, there are no reported field studies on the trophodynamics of 4-NP and its precursor, nonylphenol polyethoxylate (NPEOs) surfactants, in aquatic food webs. This study reports the trophodynamics of 4-NP and NPEOs (4 < s < 16) in a marine aquatic food web from Bohai Bay, North China. 4-NP and NPEOs (4 < s < 16) were determined in 14 marine species including plankton, benthic invertebrates, fish, and marine birds. This paper provides the first report on the occurrence of NPEOs with s > 5 in marine biota. Co-analysis of DDTs in all samples allowed a direct comparison of the bioaccumulation behavior of DDTs with that of NP and NPEOs. The lipid equivalent concentration of DDE and 2,2-bis(chlorophenyl)-1-chloroethylene (DDMU) increased with increasing trophic level, and the trophic level was determined by stable isotope ratios. The trophic magnification factors (TMFs) of DDE and DDMU were 3.26 and 3.7, respectively. Lipid equivalent concentrations of 4-NP and of all NPEOs did not exhibit a statistically significant correlation with trophic levels in the food web, and the TMF of NP was 0.83, which was similar to those of all NPEOs (0.45-1.22). These results show that in the studied aquatic food web, there was no trophic magnification for 4-NP and NPEOs, whereas DDE and DDMU biomagnified.     

Degradation of aflatoxin B(1) by cell-free extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp. nov. DSM44556(T)

Biological degradation of aflatoxin B(1) (AFB(1)) by Rhodococcus erythropolis was examined in liquid cultures and in cell-free extracts. Dramatic reduction of AFB(1) was observed during incubation in the presence of R. erythropolis cells (17% residual AFB(1) after 48 h and only 3-6% residual AFB(1) after 72 h). Cell-free extracts of four bacterial strains, R. erythropolis DSM 14,303, Nocardia corynebacterioides DSM 12,676, N. corynebacterioides DSM 20,151, and Mycobacterium fluoranthenivorans sp. nov. DSM 44,556(T) were produced by disrupting cells in a French pressure cell. The ability of crude cell-free extracts to degrade AFB(1) was studied under different incubation conditions. Aflatoxin B(1) was effectively degraded by cell free extracts of all four bacterial strains. N. corynebacterioides DSM 12,676 (formerly erroneously classified as Flavobacterium aurantiacum) showed the lowest degradation ability (60%) after 24 h, while >90% degradation was observed with N. corynebacterioides DSM 20,151 over the same time. R. erythropolis and M. fluoranthenivorans sp. nov. DSM 44,556(T) have shown more than 90% degradation of AFB(1) within 4 h at 30 degrees C, whilst after 8 h AFB(1) was practicably not detectable. The high degradation rate and wide temperature range for degradation by R. erythropolis DSM 14,303 and M. fluoranthenivorans sp. nov. DSM 44,556(T) indicate potential for application in food and feed processing.     

烯酰吗啉在人参和土壤中的残留动态及膳食风险评估

采用高效液相色谱-质谱技术研究质量分数80%的烯酰吗啉水分散粒剂在人参根、茎、叶和土壤中的消解动态及最终残留量,并对其可能产生的膳食安全风险进行评估。样品经丙酮提取,GPR固相萃取柱净化后以液相色谱分离,采用电喷雾电离源,以质谱正离子扫描多反应监测模式进行定量分析,外标法定量。在0.01～0.20 mg/kg添加范围内,烯酰吗啉在人参根、茎、叶及土壤中的回收率为82.0%～95.5%,相对标准偏差为5.01%～7.67%。施药剂量为800 g a.i./hm2时,烯酰吗啉在人参根、茎、叶和土壤中的降解半衰期为9.13～16.35 d。在一个生长季节使用1 次,施药剂量为533.33～800 g a.i./hm2时,烯酰吗啉在人参根、茎、叶和土壤中的最终残留量分别为未检出～0.024 5、0.023 3～0.138 7、0.121 5～0.618 2 mg/kg和0.008 4～0.073 8 mg/kg,风险商值为3.56×10-7（远小于1）,风险较低,处于安全水平。建议我国烯酰吗啉在人参中的最大残留限量值可暂定为0.05 mg/kg,安全间隔期为28 d。     

Simultaneous determination of 7 kinds of preservatives and saccharin in foods with HPLC, and identification with LC/MS/MS

A simultaneous determination method of saccharin (SA), sorbic acid (SOA), benzoic acid (BA), p-hydroxybenzoic acid ethyl (PHBA-Et), p-hydroxybenzoic acid isopropyl (PHBA-isoPr), p-hydroxybenzoic acid propyl (PHBA-Pr), p-hydroxybenzoic acid isobutyl (PHBA-isoBu) and p-hydroxybenzoic acid butyl (PHBA-Bu) in foods by HPLC was examined. A mixture of acetonitrile-water (1:1) was used to extract these additives from foods excluding liquid foods, while acetonitrile was used to extract them from liquid foods. HPLC was performed using a TSKgel ODS80Ts (4.6 mm i.d. x 150 mm) column with a mobile phase of 0.01% formic acid solution containing 2 mmol/L-di-n-butyl (or amyl) ammonium acetate (A) and acetonitrile (B) under the following conditions: A/B = 8: 2 (0-8 min) --> 6: 4 (15-32 min). Recoveries of these additives spiked in foods were 78-120%. The determination limits were 10 microg/g. As the identification method, examination by liquid chromatography with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used. Unknown compounds were identified by detection of product ions from their precursor ions in the negative mode with multiple reaction monitoring, m/z 182 > 106 for SA, m/z 121 > 77 for BA, m/z 111 > 67 for SOA and m/z 165 > 92 for PHBA-Et. Ratios of intensity of m/z 179 > 137 to m/z 179 > 92 were used for identification of isomers PHBA-isoPr and PHBA-Pr, and the ratios of intensity of m/z 193 > 137 to m/z 193 > 92 were used for isomers PHBA-isoBu and PHBA-Bu, because these isomers have very similar (Received December 12, 2006)     

Combined bioaugmentation and biostimulation to cleanup soil contaminated with high concentrations of atrazine

We developed a joint bioaugmentation and biostimulation approach for the clean up of soil contaminated with high (168.7 and 337.4 microg g(-1)) concentrations of the herbicide atrazine (2-chloro-4-(ethylamino)-6-isopropylamino-s-triazine). Pseudomonas sp. strain ADP (P. ADP) was used for bioaugmentation (approximately 10(7) cells g(-1) soil), and citrate (concentration range 5.8-40 mg g(-1) soil) and succinate (6.2-30.8 mg g(-1)) were used for biostimulation. The study soil had indigenous potential for atrazine mineralization (54.4 +/- 2% of 168.7 microg g(-1) mineralized after 67 day), but rapid mineralization only took place after a prolonged acclimation phase (approximately 28 days). Inoculation with P. ADP alone resulted in a limited improvement in mineralization (e.g., 30.6 +/- 1% mineralization of 168.7 microg g(-1) of atrazine in inoculated soil cf. < 0.5% in noninoculated in 7 days). Quantification of surviving numbers of P. ADP revealed a 10-fold decline from initial levels. However, bioaugmentation together with citrate or succinate biostimulation markedly increased P. ADP cell survival and atrazine mineralization (e.g., addition of 11.6 mg g(-1) of citrate increased mineralization of 337.4 microg g(-1) of atrazine from < 2 to 79.9 +/- 1% in 13 days). A critical parameter in determining the extent of atrazine mineralization by P. ADP was C(s):N(atz) (soluble carbon to atrazine nitrogen ratio): C(s):N(atz) > 40 was required for maximal atrazine mineralization. We suggest our observations may be used as a framework for rational bioremediation of field soils contaminated with atrazine.