Biotransformations of Chlorophenols in River Sediments

The accumulation of chlorophenols, including 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP), from river sediments from southern Taiwan were studied. Through simple or more exhaustive extractions, the results showed that 99% of the samples containing 2,4,6-TCP and PCP could be removed by simple extraction. the concentrations were found to range from non-detectable to 16.60 ngg¹ for 2,4,6-TCP and to 25.02 ngg¹ for PCP. Partition coefficients (K_p) were 0.71, 0.74 mlg¹ for 2,4,6-TCP, 1.35 and 1.41 mlg¹ for PCP. Biodegradation by DCP-adapted or unadapted anaerobes in sediment was carried out. During 21 days' incubation, the complete degradation time for 2,4,6-TCP in DCP-adapted anaerobic, unadapted anaerobic, and unadapted aerobic conditions were found to be 9, 10, 12 days for N3 sediment, and 8, 10, 11 days for N6 sediment, respectively; for PCP it was 19 days, without degradation, 14 days for N3 sediment, and 13, 17, 10 days for N6 sediment, respectively. the biodegradable products were identified as 2,3,4,5-tetrachlorophenol (2,3,4,5-TeCP), 3,4,5-TCP, 3,5-DCP, 3-MCP, phenol, methylphenol, and benzoate for PCP, and 2,4-DCP, 4-MCP, phenol, methylphenol, and benzoate for 2,4,6-TCP.     

Study of Fishes in the Chung-Kung Stream: Species Distribution and Organochlorine Pesticide Residues

Abstract

In this study, we investigated the potential for reductive dechlorination of 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), and pentachlorophenol (PCP) by municipal sewage sludge adapted to 2,4-DCP at different concentrations. 2,4-DCP was completely dechlorinated within 4 weeks. After 18 weeks' incubation, 2,4,6-TCP was also completely dechlorinated and the residue of PCP was 0, 44.46, 51.96% at 0.5, 5, and 50 μg ml¹ respectively. the 2,4-DCP adapted communities initially removed the ortho-chlorine from PCP of 5.0, 50 μg ml¹, following an ortho < para < meta order of chlorine removal. Intermediate products were 3,4,5-TCP, 3,5-DCP, 3-CP (3-chlorophenol), phenol, benzoate and hexanoic acids, whereas PCP (0.5 μg ml¹) indicated a preference for meta-chlorine removal. the intermediate product of 2,4,6-TCP at three concentrations were 2,4-DCP, phenol, benzoate and hexanoic acid. These products were identified by GC-MASS spectrometry. the effects of supplements, including sodium citrate (0.08 mM), sodium pyruvate (0.18 mM), sodium sulphate (0.14 mM) had a direct stimulatory effect on the dechlorination of 2,4,6-TCP and PCP after treatment for 4 weeks, but dechlorination was inhibited after 8 weeks.     

3种氯酚类化合物对河蚬的毒性和氧化应激(Toxicity and Oxidative Stress of Three Chlorophenols to Freshwater Clam Corbicula fluminea)

以河蚬(Corbicula fluminea)为试验生物,研究了2,4-DCP、2,4,6-TCP和PCP的急性毒性和21d慢性毒性,并在同一条件下测试了这3种氯酚类化合物对河蚬软体组织抗氧化系统的影响.结果显示:2,4-DCP、2,4,6-TCP和PCP对河蚬96hLC50值分别为19.25(17.58～21.20)、41.98(35.48～58.24)和0.23(0.18～0.27)mg·L-1;以致死作为观察指标,其21d无观察效应浓度(NOEC)和最低观察效应浓度(LOEC)值分别为1.00、2.00、0.06mg·L-1和2.00、4.00、0.08mg·L-1.3种氯酚类化合物对河蚬软体组织抗氧化系统几种酶的活性均表现出了不同程度的影响,其中对SOD和GSH-Px的影响最为明显.2,4,6-TCP和PCP对SOD均表现为抑制作用,对GSH-Px表现为先诱导后抑制;2,4-DCP对SOD和GSH-Px表现为明显的诱导作用;3种氯酚类化合物对CAT均表现为明显的诱导作用;对GST活性也有一定影响,但与对照组相比变化范围不大(62.95%～137.04%).3种氯酚类化合物暴露后生物体抗氧化酶有不同的响应,表明不同氯酚类化合物所导致的氧化损伤及其应激机制不同.     

太湖流域3种氯酚类化合物水质基准的探讨

按照美国地面水水质基准制定的程序和规范,筛选了太湖流域广泛存在的水生生物物种并收集了相应的基础毒性数据,探讨了五氯酚(PCP)、2,4-二氯酚(2,4-DCP)和2,4,6-三氯酚(2,4,6-TCP)在我国太湖地区的水生态基准的定值.同时采用蒙特卡罗构建物种敏感度分布(SSD)曲线和生态毒理模型方法预测了3种氯酚类化合物对太湖水生生物的急性基准浓度(CMC)和慢性基准浓度(CCC).结果表明,基于EPA规范方法和急慢性毒性比率得到的PCP、2,4-DCP和2,4,6-TCP3种氯酚类化合物的CMC值分别为25、908和594μg·L-1,CCC值分别为12、176和162μg·L-1;基于SSD曲线得到的CMC值分别为25、818和648μg·L-1,CCC值分别为6、75和198μg·L-1;基于生态毒理模型得到的CCC值分别为4、15和67μg·L-1,显示出3种方法得到的氯酚类化合物的CMC或CCC在同一个数量级上,但在数值上由生态毒理模型得出的CCC要小于其它两种方法,并且除PCP的急慢性基准值与美国EPA推出的水生态基准值相近外,其它两种氯酚类化合物的急慢性基准值均低于美国EPA推出的急慢性基准值.研究结果希望能为我国水质基准的制定提供一些有用的线索.     

典型氯酚类化合物对水生生物的毒性研究进展

氯酚类化合物(CPs)是一类广泛存在于水环境中的有机污染物。这类化合物具有环境稳定性、生物累积性和生物毒性,因而其在水环境中的生态毒理效应一直是人们关注的焦点。在水体中存在最普遍的酚类化合物主要有2,4-二氯酚(2,4-DCP),2,4,6-三氯酚(2,4,6-TCP)和五氯酚(PCP)。本文对近几年来这3种典型氯酚类化合物的水生态毒理学研究进行了总结,主要包括它们对水生生物的急性毒性、氧化损伤、发育毒性、内分泌干扰、遗传毒性、致癌性、免疫毒性、细胞毒性以及复合毒性的效应和机制,同时对目前存在的问题和进一步的研究方向进行了讨论和展望。     

Susceptibility of male and female Japanese medaka (Oryzias latipes) to 2,4,6-trichlorophenol-induced micronuclei in peripheral erythrocytes

2,4,6-trichlorophenol (2,4,6-TCP) is a widespread probable human carcinogen and has been proven to have genotoxicity in in vitro assays. However, little genotoxicity information and no micronuclei induction data for 2,4,6-TCP is available from in vivo tests, especially for sex-specific differences. Following a preliminary test, a piscine peripheral erythrocyte micronucleus assay was conducted on medaka (Oryzias latipes) after a 28-day exposure to 2,4,6-TCP. In the present study, the mean micronuclei (MNC) frequencies of all of the groups increased in a dose-dependent manner, which indicated the potential genotoxicity of 2,4,6-TCP. Moreover, males were found to be more susceptible compared with females after a 28-day exposure to 2,4,6-TCP in all of the dosed groups above 10 μg·L^?1. This is the first report on the potential of micronuclei induction and a sex-susceptible effect in the peripheral erythrocytes of mature fish after 2,4,6-TCP in vivo exposure.     

Pathway of the ozonation of 2,4,6-trichlorophenol in aqueous solution

The reaction mechanism and pathway of the ozonation of 2,4,6-trichlorophenol (2,4,6-TCP) in aqueous solution were investigated. The removal efficiency and the variation of H₂O₂, Cl^? formic acid, and oxalic acid were studied during the semi-batch ozonation experiments (continuous for ozone gas supply, fixed volume of water sample). The results showed that when there was no scavenger, the removal efficiency of 0.1 mmol/L 2,4,6-TCP could reach 99% within 6 min by adding 24 mg/L ozone. The reaction of molecular ozone with 2,4,6-TCP resulted in the formation of H₂O₂. The maximal concentration of H₂O₂ detected during the ozonation could reach 22.5% of the original concentration of 2,4,6-TCP. The reaction of ozone with H₂O₂ resulted in the generation of a lot of OH? radicals. Therefore, 2,4,6-TCP was degraded to formic acid and oxalic acid by ozone and OH? radicals together. With the inhibition of OH? radicals, ozone molecule firstly degraded 2,4,6-TCP to form chlorinated quinone, which was subsequently oxidized to formic acid and oxalic acid. Two reaction pathways of the degradation of 2,4,6-TCP by ozone and O₃/OH? were proposed in this study.     

Biofilm-electrode process with high efficiency for degradation of 2,4-dichlorophenol

2,4-Dichlorophenol (2,4-DCP) from chemical industry wastewaters has caused serious environmental pollution. Removal of 2,4-DCP using either physico-chemical or biological methods is not very efficient. In this paper, a combination of biological and electrochemical methods gave satisfactory results. By comparisons of the degradation of 2,4-DCP and the removal of chemical oxygen demand (COD) in electrochemical, biological and biofilm-electrode processes, it was found that the biofilm-electrode process possesses the highest degradation efficiency and removal rate; both the pure electrochemical and the pure biological processes were far less efficient. The removal efficiency of 2,4-DCP using the biofilm-electrode process was 100% in 48 h, while that using the pure electrochemical and the pure biological processes were 62 and 42%, respectively. The experiments show that the current of 5 mA for the cathode of 9 cm² and the initial concentration 100 mg/l of 2,4-DCP were the optimal parameters of technology for the biofilm-electrode process. The excellent effects are due to the withdrawing electron action of bacterium, electrochemically anodic oxidation and cathodic dechlorination. It is the first time that the biofilm-electrode method was applied in 2,4-DCP degradation. Here, we demonstrated that biofilm-electrode process is a promising method to remove some aromatic compounds in industrial wastewater.     

典型酚类化合物对土壤跳虫的慢性毒性

以跳虫Folsomia candida为受试物种,基于其28 d繁殖试验评价了12种典型酚类化合物的慢性毒性效应。结果表明,在所设浓度范围内,除间苯二酚外的其他酚类对跳虫繁殖均存在不同程度的抑制效应。氯酚类对跳虫慢性毒性最大,2,4-二氯酚、2-氯酚、2,4,6-三氯酚对跳虫繁殖毒性的EC50分别为5.94、10.2、19.7 mg·kg-1;其次为烷基酚类,2,4-二甲基酚、3-甲基酚、壬基酚对跳虫繁殖毒性的EC50分别为21.7、35.1、50.5 mg·kg-1。苯酚的毒性较上述烷基酚低,EC50值为71.7 mg·kg-1。其他取代酚—包括2-萘酚、4-硝基酚、邻苯二酚对跳虫繁殖的EC50分别为95.4、133、306 mg·kg-1。双酚A仅在最高浓度(500 mg·kg-1)处理下对跳虫繁殖有显著影响。     

氯酚在胡敏酸上的吸附

用静态平衡法了白洋研究泥中胡敏酸对氯酚的吸附情况,当温度升高或ＰＨ值降低时,ＨＡ对４－氯酚的吸附量增大,对４－氯酚和３,５－二氯酚两种吸附质组成的混合溶液,ＨＡ对３,５－二氯酚的吸附量大于ＨＡ对４－氯酚的吸附量;     

Chlorophenol tolerant and degradative bacteria isolated from a river receiving pulp mill discharges

A screening was carried out in a river receiving pulp mill discharges (BioBio river, central Chile) to study the bacterial population able to tolerate and degrade chlorophenols. In four selected stations and at different seasons, water samples were taken from the river. Total bacterial population and the number of chlorophenol tolerant bacteria were counted. Bacterial tolerance to 25, 50, 100, 200 and 400 μg/ml of 2,4‐dichlorophenol (DCP), 2,4,6‐trichlorophenol (TCP) and pentachlorophenol (PCP) was determined. Strains able to tolerate at least 400 μg/ml chlorophenols were found. Eighteen bacterial strains able to use chlorophenols as the sole carbon source were isolated: five of them were Pseudomonas fluorescens, two Alcaligenes sp., one Citrobacter freundii, one Aeromonas sp. and nine unidentified Gram negative bacilli. A good correlation (r ² = 0.758) between the logarithm of DCP tolerant bacteria and the content of adsorbable organic halogen (AOX) at low dilution in the river water was established. Four groups of tolerant bacteria were found: growing on PCP and DCP, growing on PCP and TCP, growing only on PCP and growing only on TCP.     

Remediation of 2,4-dichlorophenol-contaminated groundwater using nano-sized CaO2 in a two-dimensional scale tank

• Nano CaO₂ is evaluated as a remediation agent for 2,4-DCP contaminated groundwater. • 2,4-DCP degradation mechanism by different Fe²⁺ concentration was proposed. • 2,4-DCP was not degraded in the system for solution pH>10. • The 2,4-DCP degradation area is inconsistent with the nano CaO₂ distribution area. This study evaluates the applicability of nano-sized calcium peroxide (CaO₂) as a source of H₂O₂ to remediate 2,4-dichlorophenol (2,4-DCP) contaminated groundwater via the advanced oxidation process (AOP). First, the effect and mechanism of 2,4-DCP degradation by CaO₂ at different Fe concentrations were studied (Fenton reaction). We found that at high Fe concentrations, 2,4-DCP almost completely degrades via primarily the oxidation of •OH within 5 h. At low Fe concentrations, the degradation rate of 2,4-DCP decreased rapidly. The main mechanism was the combined action of •OH and O₂^•−. Without Fe, the 2,4-DCP degradation reached 13.6% in 213 h, primarily via the heterogeneous reaction on the surface of CaO₂. Besides, 2,4-DCP degradation was significantly affected by solution pH. When the solution pH was>10, the degradation was almost completely inhibited. Thus, we adopted a two-dimensional water tank experiment to study the remediation efficiency CaO₂ on the water sample. We noticed that the degradation took place mainly in regions of pH<10 (i.e., CaO₂ distribution area), both upstream and downstream of the tank. After 28 days of treatment, the average 2,4-DCP degradation level was ≈36.5%. Given the inadequacy of the results, we recommend that groundwater remediation using nano CaO₂: (1) a buffer solution should be added to retard the rapid increase in pH, and (2) the nano CaO₂ should be injected copiously in batches to reduce CaO₂ deposition.     

沉积物中五氯酚对底栖生物的急慢性毒性效应

以淡水底栖动物花翅羽摇蚊幼虫和淡水单孔蚓为研究对象,研究了沉积物中五氯酚对底栖生物的急慢性毒性效应。五氯酚对花翅羽摇蚊幼虫96 h及10 d的半数致死浓度(LC50)分别为20.6 mg·kg-1和12.5 mg·kg-1,28 d羽化半数抑制浓度(EC50)为0.79 mg·kg-1。沉积物中五氯酚对花翅羽摇蚊幼虫的羽化具有延滞作用,而且对雌性摇蚊羽化的延滞作用大于雄性,最终导致羽化摇蚊的性别失衡。淡水单孔蚓对五氯酚的耐受力较摇蚊幼虫强。五氯酚对淡水单孔蚓的96 h及14 d的LC50分别为37.6 mg·kg-1和20.2 mg·kg-1,对淡水单孔蚓21 d生长抑制的EC50为1.39 mg·kg-1。研究结果对推导五氯酚沉积物质量基准和进行沉积物生态风险评价提供依据。     

Transfer of 65Zn from sediments by marine polychaete worms

Silty marine sediments spiked with ⁶⁵Zn lose only small fractions of their radioactivity when exposed to slowly flowing seawater for several weeks. However, polychaete worms (Nereis diversicolor), burrowing through the sediment, cause ⁶⁵Zn losses 3 to 7 times higher than in sediment without worms. Long-term experiments on the uptake and loss of ⁶⁵Zn by the polychaete Hermione hystrix indicate that 60 or more days exposure are required for this worm to approach steady state with ⁶⁵Zn in the sediment. Biological half-life estimates for ⁶⁵Zn accumulated from sediment by H. hystrix are extremely variable (52 to 197 days), depending on the loss-time interval chosen for the calculation. Following 5 days exposure to 16 cm³ of radioactive sediment, N. diversicolor individuals contained an average of 0.2% of the total ⁶⁵Zn in the sediment. When these worms were transferred to non-radioactive sediment, estimates of biological half-life for ⁶⁵Zn averaged 14 to 17 days during the loss period Day 3 to Day 15. Based on these experimental results, it is estimated that a population of N. diversicolor could cause an annual loss of 3% or more of the ⁶⁵Zn in the upper 2 cm of the sediment of a hypothetical radioactive estuary.     

Aliphatic and aromatic halocarbons as potential mutagens in drinking water

Chlorinated phenols are either products of industrial chemical processes or the result of chlorination of drinking water. Often, the formation of chlorinated phenols is based upon naturally occurring phenol. The following chlorinated phenols have been selected for testing in the Ames‐test for their mutagenic activity: 3‐chlorophenol, 4‐chlorophenol, 2,3,6‐, 2,4,5‐, 2,4,6‐trichlorophenol, 4‐chloro‐2‐methylphenol and 4‐chloro‐3‐methylchlorophenol. The tester strains TA97, TA98, TA100 and TA104 were employed. All tested compounds produced mutagenic activity at least in one tester strain. The highest numbers of revertants were detected for 2,3,6‐ and 2,4,6‐trichlorophenol. But in contrast to the other substances, these two induced only frameshift mutations in presence of a metabolizing system. The evidence of their presence in drinking water and of their mutagenic activity makes them to a potential health hazard.     

Recycle of buried macroalgal detritus in sediments: use of dual-labelling experiments in the field

In intertidal sediments, burial and decomposition of macroalgal detritus can fuel the sediment of carbon (C) and nitrogen (N), which can be either promptly mineralised or assimilated to enter the food web. This study investigates the transfer of algal-derived C and N to the sediment and to the infauna feeding primarily on benthic diatoms. Thalli of Ulva spp. were ¹³C- and ¹⁵N-labelled in the mesocosm and frozen to create detritus. Thawed macroalgae were, then, buried in the sediment of an intertidal sand-flat forming a mosaic of small patches (50 × 50 cm²) enriched with the macroalgae interspersed with bare sediment. The area was dominated by Corophium volutator and Hydrobia ulvae. The uptake of ¹³C and ¹⁵N was measured in the residual macroalgae, in the sediment and in those animals. Decomposition of detritus was rapid and after 4 weeks the residual biomass was 3% the amount added. Algal-derived ¹³C and ¹⁵N were moved to the sediment. The total amount of ¹³C and ¹⁵N retained in the sediment after completing the decomposition was 3.4 ± 0.5% ¹³C and 2.7 ± 0.6% ¹⁵N the amount decomposed. During the first 2 weeks more N than C was assimilated (1.7% ¹³C and 13.5% ¹⁵N). During the remaining two weeks, N was released from the sediment, while there was little accumulation of C (+6.4 ± 2.0 % ¹³C and −7.7 ± 3.8% ¹⁵N). At the end of the decomposition, animals were ¹⁵N- and ¹³C-labelled. Considering the total accumulation of label in the sediment, they accounted for 3.5 ± 1.8% ¹³C and 25.8 ± 12.9 % ¹⁵N. Similarly, considering the mass of the heavy isotopes gained (¹³C) or lost (¹⁵N) during the remaining 2 weeks, the animals accounted for 4.7 ± 2.1% of the ¹³C in excess and for 18.6 ± 9.1% of the ¹⁵N loss. The transfer of C and N to the sediment and to the surface deposit-feeders can be a relevant mechanism to remove the excess of detritus from the sediment.     

Impact of the polychaete Capitella sp. I on microbial activity in an organic-rich marine sediment contaminated with the polycyclic aromatic hydrocarbon fluoranthene

Polychaetes belonging to the genus Capitella are often present in high numbers in organic-rich sediments polluted with, e.g., oil components, and Capitella spp. may have a great impact on the biogeochemistry of these sediments. We examined the influence of Capitella sp. I on microbial activity in an organic-rich marine sediment contaminated with the polycyclic aromatic hydrocarbon, fluoranthene. Capitella sp. I were added to microcosms (10 000 ind m⁻²) and the impact of a pulse-sedimentation of fluoranthene-contaminated sediment (3 mm layer) was studied for a period of 12 d after sedimentation. The sediment oxygen uptake and total sediment metabolism (TCO₂ production) increased in cores with worms (71 to 131%), whereas the anaerobic activity, measured as sulfate reduction rate 12 d after sedimentation, was lower compared to cores without worms. The effect of fluoranthene on sulfate reduction was most pronounced in the presence of worms, with a 34% reduction versus 16% in cores without worms. The reduced sulfur pools in cores with worms were smaller than in cores without worms, suggesting that the reduced anaerobic activity was caused by increased oxidation of the sediment, which may favor O₂ and other electron-acceptors (e.g. NO₃ ⁻, Fe³⁺, Mn⁴⁺) in organic matter decomposition. The sediment oxygen uptake and TCO₂ production did not show significant changes due to fluoranthene treatment, indicating that these parameters were either less sensitive to fluoranthene stress or recovered more rapidly (i.e. within 48 h) than sulfate reduction rates. Bioturbation by Capitella sp. I altered the depth profile of fluoranthene such that fluoranthene was found in deeper sediment layers (down to 2 cm) where diffusional loss and microbial breakdown probably are reduced relative to surface layers. In cores without worms, fluoranthene was found down to 1 cm, with 75% remaining in the upper 5 mm. Received: 5 December 1996 / Accepted: 11 February 1997     

Fe~0体系降解2,4二氯酚的影响因素及其反应机理

采用铁屑、炉渣及河砂混合介质降解2,4-二氯酚（2,4-DCP）模拟废水,研究铁屑粒径、铁屑投加量、铁屑与炉渣配比、pH值等因素对2,4-DCP脱氯效果的影响,探讨Fe0体系降解2,4-DCP的反应机理。结果表明,铁屑粒径、铁屑投加量、铁屑与炉渣配比、pH对2,4-DCP脱氯效果均有显著影响,在铁屑粒径为2～5 mm、不改变废水pH、铁屑与炉渣质量比为31∶9条件下,Fe0体系对2,4-DCP去除率高达97%。2,4-DCP经脱氯后主要产物为2-氯酚、4-氯酚和苯酚,反应后废水的可生化性明显提高,利于后续的生物处理。     

Rates of ammonium turnover and the role of amino-acid deamination in seagrass (Zostera capricorni) beds of Moreton Bay,Australia

Samples of sediments from Australian seagrass (Zostera capricorni Aschers.) beds were taken in June to August 1983 (for¹⁵N experiments) and November 1982 to January 1983 (¹⁴N experiments). The ammonium pool turned-over every 0.4 to 0.8 d, as determined with a¹⁵N isotope-dilution technique. The ammonium pool in subtidal bare areas turned-over two to three times more slowly than in adjacent seagrass beds. Gross rates ofin situ ammonium regeneration equalled those of utilization, and ranged from 0.04 to 0.35 mol cm^-3 d^-1, or from 50 to 490 mg N m^-2 d^-1 over the upper 10 cm of the sediment. The potential rate of glycine utilization, measured with a large excess of glycine added to anaerobic incubations, ranged from 0.21 to 0.39mol cm^-3 d^-1, butin situ rates were probably much lower. Between 35 and 65% of added¹⁵N-glycine was deaminated over 12 h, and the remainder was most likely assimilated by microbes. Evidence for the seagrasses taking up glycine was equivocal, owing to the rapid deamination of the amino acid and the likelihood that they assimilated the labelled ammonium produced from the glycine.     

Biodegradation of N,N-bis(carboxymethyl)-L-glutamate and its utilization as sole source of carbon,nitrogen, and energy by a Rhizobium radiobacter strain in seawater

Biodegradation of N,N-bis(carboxymethyl)-L-glutamate and its utilization by micro-organisms in marine environments were studied in Organization of Economic Development and Cooperation (OECD) screening tests and with pure bacterial cultures. Biodegradation in excess of 60% was achieved in closed bottle tests in less than 60 days, suggesting complete degradation. The bacterial strain isolated from marine sandy sediment and utilizing N,N-bis(carboxymethyl)-L-glutamate as sole source of carbon, nitrogen, and energy was identified as Rhizobium radiobacter. The strain was capable to mineralize N,N-bis(carboxymethyl)-L-glutamate in seawater and in synthetic media containing 0–60 g L^?1 sea salts. Growth and adaptation studies indicated that N-carboxymethyl-L-glutamate and L-glutamate are main metabolites.