Biodegradation of Amphipathic Fluorinated Peptides Reveals a New Bacterial Defluorinating Activity and a New Source of Natural Organofluorine Compounds.

Three peptides comprising mono-, di-, and tri-fluoroethylglycine (MfeGly, DfeGly, and TfeGly) residues alternating with lysine were digested by readily available proteases (elastase, bromelain, trypsin, and proteinase K). The degree of degradation depended on the enzyme employed and the extent of fluorination. Incubation of the peptides with a microbial consortium from garden soil resulted in degradation, yielding fluoride ions. Further biodegradation studies conducted with the individual fluorinated amino acids demonstrated that the degree of defluorination followed the sequence MfeGly > DfeGly > TfeGly. Enrichment of the soil bacteria employing MfeGly as a sole carbon and energy source resulted in the isolation of a bacterium, which was identified as Serratia liquefaciens. Cell-free extracts of this bacterium enzymatically defluorinated MfeGly, yielding fluoride ion and homoserine. In silico analysis of the genome revealed the presence of a gene that putatively codes for a dehalogenase. However, the low overall homology to known enzymes suggests a potentially new hydrolase that can degrade monofluorinated compounds. 19F NMR analysis of aqueous soil extracts revealed the unexpected presence of trifluoroacetate, fluoride ion, and fluoroacetate. Growth of the soil consortium in tryptone soya broth supplemented with fluoride ions resulted in fluoroacetate production; thus, bacteria in the soil produce and degrade organofluorine compounds.

Residue analytical method for the determination of trifluoroacetic acid and difluoroacetic acid in plant matrices by capillary electrophoresis tandem mass spectrometry (CE-MS/MS).

In the past years, the technology for trace residue analysis of plant protection compounds in plant and animal matrices, soil, and water has gradually changed to meet changing regulatory demands. Generally, from the '70s to the '90s of the last century, the active compounds and only a few major metabolites had to be determined in a typical "residue definition". Step by step and within the framework of product safety assessments of the enforcement of residues in dietary matrices and in the environment, further metabolites have come into the authorities focus. Many active substances were formerly determined via gas chromatography (GC) based detection techniques. The introduction of liquid chromatography tandem mass spectrometry (LC-MS/MS) technology in the '90s and the acceptance of this technique, by official bodies at the end of the '90s, has led to a major change for residue analytical laboratories all over the world. Most of the medium to non-polar active compounds as well as many of the more polar metabolites can be detected with this technique, and today LC-MS/MS is the "workhorse" in many residue analytical laboratories in the industry as well as official enforcement labs responsible for analyzing registration-related field studies. With the demand to analyze further breakdown products, more and more polar compounds, or even (permanently) charged target compounds, have now come into the focus of the registration authorities. This now brings standard LC-based techniques to their limits and requires the application of approaches such as hydrophilic interaction chromatography (HILIC) MS/MS or ion chromatography, however these techniques often incur related uncertainties and problems with matrix samples. The aim of this study was to develop a new CE-MS/MS-based approach to reduce the impact of matrix on the separation and detection of trifluoroacetic acid (TFA) and difluoroacetic acid (DFA) in agrochemical field trials. This project used 7 representative examples of fruit, grain and vegetables which had undergone homogenization and extraction with acetonitrile water and filtration before CE-MS/MS analysis. The CE-MS/MS developed reached the limit of quantitation (LOQ) requirement of current legislation for both TFA and DFA (0.01 mg/kg) in all 7 matrices tested. The mean relative standard deviation (RSD) obtained from the repeat analysis of control field trail samples in all matrices, for both TFA and DFA, was less than 10% meeting GLP guidelines. When compared with LC-MS/MS, using on column loading amounts, the CE-MS/MS was 17 - 43 times more sensitive than a standard method and less matrix effects were observed. The developed method was validated under GLP conditions to provide a GLP-validated residue analytical method for the charged metabolites TFA and DFA in matrix samples from GLP field residue trials.

Evidence for the Formation of Difluoroacetic Acid in Chlorofluorocarbon-Contaminated Ground Water.

The concentrations of difluoroacetic acid (DFA) and trifluoroacetic acid (TFA) in rainwater and surface water from Berlin, Germany resembled those reported for similar urban areas, and the TFA/DFA ratio in rainwater of 10:1 was in accordance with the literature. In contrast, nearby ground water historically contaminated with 1,1,2-trichloro-1,2,2-trifluoroethane (R113) displayed a TFA/DFA ratio of 1:3. This observation is discussed versus the inventory of microbial degradation products present in this ground water along with the parent R113 itself. A microbial transformation of chlorotrifluoroethylene (R1113) to DFA so far has not been reported for environmental media, and is suggested based on well-established mammalian metabolic pathways.

Toxicity of the different vegetative stages of Amorimia pubiflora to sheep.

Toxic plants containing monofluoroacetate (MFA) cause sudden death in livestock in Australia, South Africa and Brazil, causing economic losses to producers. The objective of this study was to determine the amount of MFA present in young leaves, mature leaves, senescent leaves, and seeds of Amorimia pubiflora harvested at different times of the year and to determine their toxic effect on sheep. Samples of Amorimia pubiflora were collected during April, August and December of 2015 and March of 2016, separated according to the vegetative stage (young leaves, seeds, mature leaves, and senescent leaves), dried in an oven, and administered in daily doses of 5 g/kg/body weight (bw) of fresh leaves to sheep through ruminal cannulae. The experiment was divided into four stages according to the time of collection of the plant so that each sheep received a different vegetative stage of the plant (young leaves, mature leaves, and senescent leaves). Only in the second stage of the experiment was it possible to collect A. pubiflora seeds, which were administered using the same method used for the administration of the leaves. The sheep were dosed with the plant until they showed clinical signs of toxicosis or until the plant was no longer available. Aliquots of leaves and seeds of A. pubiflora were analyzed for MFA concentration. The seeds and young leaves had higher concentrations of MFA than did the mature (harvested in August and December) and senescent (harvested in December) leaves. However, all vegetative stages of the plant were toxic and caused fatal poisoning. The results of our study showed that A. pubiflora is toxic to sheep even when MFA concentrations are low, demonstrating that the presence of this substance is a risk factor for the occurrence of poisoning. Knowing the toxic principle and its variations allow us to determine the conditions for the occurrence of plant toxicosis as well as possible treatment, control, and prophylaxis methods, contributing significantly to the reduction of economic losses on farms due to plant poisoning.

Sodium fluoroacetate toxicity: a case report of malicious poisoning in dogs across a Phoenix, Arizona neighborhood.

In May 2016, thirteen dogs housed in backyards within a single neighborhood were reported to have developed convulsions and died within a 24 h period. An investigation of the scene by law enforcement resulted in submission of eight dogs for postmortem examination. It was suspected that a rapid acting toxin was the cause of death. A gas chromatography-mass spectrophotometry (GC-MS) protocol combined with thin-layer chromatography that allows screening for common convulsants failed to identify a toxin in either pooled gastric content or liver samples from select cases. After consultation with a veterinary toxicologist, sodium fluoroacetate poisoning was investigated. Sodium fluoroacetate, also known as 1080, is a pesticide that was available in the United States from the 1940's to the 1970's, but since 1972 has been banned or under EPA restricted use. When gastric content was re-tested using a GC-MS protocol with selective fluoroacetate ion monitoring and carbon 14 radiolabeling to facilitate quantification, 379 ppb sodium fluoroacetate was detected in a pooled gastric content sample. In spite of its banned status, sodium fluoroacetate remains a rarely reported cause of malicious poisoning in domestic dogs in the United Sates. This compound is highly toxic and is capable of causing death in dogs, humans, other mammals, and insects in ingested quantities as small as a few droplets. Even when geographic or historical proximity to a source is not evident, this intoxication should be considered in dogs exhibiting compatible clinical signs.

Poisoning in goats by the monofluoracetate-containing plant Palicourea aeneofusca (Rubiaceae).

The epidemiological, clinical and pathological aspects of a spontaneous outbreak of Palicourea aeneofusca poisoning in goats are reported. The main clinical signs were motor incoordination, generalized muscle tremors, broad-based posture, tachypnea, tachycardia, vocalization and respiratory distress. Two goats died 5 and 20 min after the observation of the first clinical signs. Another that was found recumbent died 80 h later. One goat with mild clinical signs recovered. Congestion and hemorrhages were observed macroscopically and histologically in most organs. Pulmonary edema was also observed. The main microscopic findings consisted of cytoplasmic vacuolization and necrosis of the renal tubular epithelium. The average concentration of monofluoroacetate obtained in sixteen samples of P. aeneofusca was 0.29 ± 0.17%. It is concluded that P. aeneofusca is toxic to goats under natural conditions.

Fluoroacetamide Moieties as NMR Spectroscopy Probes for the Molecular Recognition of GlcNAc-Containing Sugars: Modulation of the CH-π Stacking Interactions by Different Fluorination Patterns.

We herein propose the use of fluoroacetamide and difluoroacetamide moieties as sensitive tags for the detection of sugar-protein interactions by simple 1 H and/or 19 F NMR spectroscopy methods. In this process, we have chosen the binding of N,N'-diacetyl chitobiose, a ubiquitous disaccharide fragment in glycoproteins, by wheat-germ agglutinin (WGA), a model lectin. By using saturation-transfer difference (STD)-NMR spectroscopy, we experimentally demonstrate that, under solution conditions, the molecule that contained the CHF2 CONH- moiety is the stronger aromatic binder, followed by the analogue with the CH2 FCONH- group and the natural molecule (with the CH3 CONH- fragment). In contrast, the molecule with the CF3 CONH- isoster displayed the weakest intermolecular interaction (one order of magnitude weaker). Because sugar-aromatic CH-π interactions are at the origin of these observations, these results further contribute to the characterization and exploration of these forces and offer an opportunity to use them to unravel complex recognition processes.

A Gas Chromatography-Mass Spectrometry Method for the Detection and Quantitation of Monofluoroacetate in Plants Toxic to Livestock.

Monofluoroacetate (MFA) is a potent toxin that occurs in over 50 plant species in Africa, Australia, and South America and is responsible for significant livestock deaths in these regions. A gas chromatography-mass spectrometry (GC-MS) method for the analysis of MFA in plants based on the derivatization of MFA with n-propanol in the presence of sulfuric acid to form propyl fluoroacetate was developed. This method compared favorably to a currently employed high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for the analysis of MFA in plants. The GC-MS method was applied to the analysis of MFA in herbarium specimens of Fridericia elegans, Niedenzuella stannea, N. multiglandulosa, N. acutifolia, and Aenigmatanthera lasiandra. This is the first report of MFA being detected in F. elegans, N. multiglandulosa, N. acutifolia, and A. lasiandra, some of which have been reported to cause sudden death or that are toxic to livestock.

Studies in regard to the classification and putative toxicity of Fridericia japurensis (Arrabidaea japurensis) in Brazil.

Numerous plant species worldwide including Palicourea marcgravii (Rubiaceae) and Tanaecium bilabiatum (Bignoniaceae) in Brazil cause acute cardiac failure (sudden death) and are known to contain monofluoroacetate (MFA). Other Bignoniaceae species including Fridericia japurensis (Arrabidaea japurensis) are reported to cause sudden death in livestock in the Brazilian state of Roraima and are suspected to contain MFA due to the similarity of clinical signs. In this study herbarium specimens of Fridericia japurensis and field collections suspected to be F. japurensis were analyzed for MFA, and plant material from the field collections was dosed to rabbits. No MFA was detected in the herbarium specimens authoritatively identified as F. japurensis; however, MFA was detected in the field collections, which were identified as T. bilabiatum. Rabbits dosed orally with T. bilabiatum died acutely. Voucher toxic specimens initially described as F. japurensis were incorrectly identified, and the correct botanical name for this plant is T. bilabiatum (Arrabidaea bilabiata). Based on this study we conclude that there are no data to support the toxicity of F. japurensis and that the plant previously reported under this name as causing acute cardiac failure in cattle in Roraima is T. bilabiatum. This research highlights the importance of voucher specimens as part of any toxic plant investigation and corrects the literature regarding these toxic plants.

Determination of Sodium Fluoroacetate in Dairy Powders by Liquid Chromatography-Tandem Mass Spectrometry: Single-Laboratory Validation, First Action 2015.02.

A single-laboratory validation (SLV) study was conducted for the determination of sodium fluoroacetate in dairy powders by LC-tandem MS (LC-MS/MS). Linearity of response was confirmed by analysis of samples fortified over the concentration range 0.10-100 µg/kg. The LOD was estimated to be 0.028 µg/kg (0.028 ppb) from the SD of the measured concentrations of infant formula samples fortified at 0.10 µg/kg. The corresponding LOQ calculates at 0.085 µg/kg (0.085 ppb), which ensures excellent reliability of quantification at the limit of reporting of 1.0 µg/kg (1 ppb). Repeatability and intermediate precision were estimated from the SD of the recovery of samples fortified at 0.075, 0.10, 0.20, 0.50, 1.0, and 10.0 µg/kg. The previously mentioned method performance values were established using a representative stage 2 (6-12 months) bovine infant formula, and the robustness of the method was tested by the analysis of 107 unique dairy powders and formulations fortified at 1.0 µg/kg. The data collected in this study satisfy the requirements of SLV studies established by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN), and the method was awarded First Action Official Method(SM) status by the AOAC Expert Review Panel on SPIFAN Nutrient Methods (Contaminants) on March 17, 2015.

High-Throughput Quantification of Monofluoroacetate (1080) in Milk as a Response to an Extortion Threat.

As a food defense measure against an extortion threat to poison infant formula with monofluoroacetate, a robust methodology for monofluoroacetate analysis in fluid milk and powdered dairy products was developed and optimized. Critical challenges posed by this situation required that the analytical methodology provide (i) high specificity, (ii) high throughput capable of analyzing thousands of samples of fluid milk per day, and (iii) trace-level detection of 1 ng/g or lower to achieve the maximum residue limit. Solid-phase extraction-purified acetone extracts of fluid milk were derivatized with aniline, and after ultrahigh-performance liquid chromatography using a Kinetex-C18 column packed with 1.3-µm shell particles, the resulting N-phenyl 2-fluoroacetamide could be determined by liquid chromatography-tandem mass spectrometry in a highly specific manner and with a limit of quantification of 0.5 ng/ml. By using 4-(4-chlorophenoxy)aniline as a derivatizing agent, the method could be extended to powdered dairy products with the same limit of quantification. Between January and July 2015, some 136,000 fluid milk samples were tested using this method. This analytical testing of fluid milk formed one element in a larger program of work by multiple agencies to ensure that consumers could continue to have confidence in the safety of New Zealand milk and dairy products.

Amino Acid and Peptide Utilization Profiles of the Fluoroacetate-Degrading Bacterium Synergistetes Strain MFA1 Under Varying Conditions.

Synergistetes strain MFA1 is an asaccharolytic ruminal bacterium isolated based on its ability to degrade fluoroacetate, a plant toxin. The amino acid and peptide requirements of the bacterium were investigated under different culturing conditions. The growth of strain MFA1 and its fluoroacetate degradation rate were enhanced by peptide-rich protein hydrolysates (tryptone and yeast extract) compared to casamino acid, an amino acid-rich protein hydrolysate. Complete utilization and preference for arginine, asparagine, glutamate, glycine, and histidine as free amino acids from yeast extract were observed, while the utilization of serine, threonine, and lysine in free form and peptide-bound glutamate was stimulated during growth on fluoroacetate. A predominant peptide in yeast extract preferentially utilized by strain MFA1 was partially characterized by high-liquid performance chromatography-mass spectrometry as a hepta-glutamate oligopeptide. Similar utilization profiles of amino acids were observed between the co-culture of strain MFA1 with Methanobrevibacter smithii without fluoroacetate and pure strain MFA1 culture with fluoroacetate. This suggests that growth of strain MFA1 could be enhanced by a reduction of hydrogen partial pressure as a result of hydrogen removal by a methanogen or reduction of fluoroacetate.

Quantitative determination of sodium monofluoroacetate "1080" in infant formulas and dairy products by isotope dilution LC-MS/MS.

A fast and easy-to-use confirmatory liquid-chromatography tandem mass-spectrometry (LC-MS/MS) based-method was developed for the analysis of the pesticide sodium monofluoroacetate (MFA, also called "1080") in infant formulas and related dairy products. Extraction of the compound encompassed sample reconstitution and liquid-liquid extraction under acidic conditions. Time-consuming solid-phase extraction steps for clean-up and enrichment and tedious derivatisation were thus avoided. Resulting sample extracts were analysed by electrospray ionisation (ESI) in negative mode. Quantification was performed by the isotopic dilution approach using (13)C-labelled MFA as internal standard. The procedure was validated according to the European document SANCO/12571/2013 and performance parameters such as linearity (r(2) > 0.99), precision (RSD(r) ≤ 9%, RSD(iR) ≤ 11%) and recovery (96-117%) fulfilled its requirements. Limit of quantifications (LOQ) was 1 µg kg(-1) for infant formulas and related dairy products except for whey proteins powders with a LOQ of 5 µg kg(-1). Method ruggedness was further assessed in another laboratory devoted to routine testing for quality control.

Simultaneous quantification of trihalomethanes and haloacetic acids in cheese by on-line static headspace gas chromatography-mass spectrometry.

Trihalomethanes (THMs) and haloacetic acids (HAAs) are the two most prevalent classes of disinfection by-products (DBPs) that are present in treated water. Four THMs and six HAAs are regulated by several countries in drinking waters but no regulation for these DBPs has been established in foods. THMs are volatile species that can easily be determined by static headspace (SHS)-GC-MS, but HAAs require a derivatisation step to make them suitable for GC due to their polar and hydrophilic nature. This paper describes the first analytical method that performs the simultaneous determination of 10 THMs and 13 HAAs (chlorinated, brominated and iodinated) in cheeses by SHS in one unique GC-MS run. Parameters controlling leaching, centrifugation, derivatisation and volatilisation were optimised taking into account the high volatility of THMs and the thermal instability of HAAs. To increase sensitivity, 3g of cheese was extracted with 10mL of water at pH 4.5-7.7, and after centrifugation the supernatant (∼8mL) was introduced into an HS vial for the derivatisation (HAAs) and volatilisation (HAA esters and THMs) of the species in an automatic SHS unit coupled to GC-MS. Detection limits within the range of 0.05-0.50 and 0.15-0.85µg/kg for THMs and HAAs, respectively, were obtained, and the relative standard deviation was lower than 10% for all the target analytes. Recoveries throughout the whole method were between 85-90% and 92-97% for THMs and HAAs, respectively. The SHS-GC-MS method was applied for the determination of THMs and HAAs in 3 groups of Spanish cheeses, which can be contaminated through contact with treated water during the manufacturing steps. Up to 2 THMs and 4 HAAs were found at µg/kg levels in the samples analysed.

Monofluoroacetate-containing plants that are potentially toxic to livestock.

Many plants worldwide contain monofluoroacetate and cause sudden death in livestock. These plants are primarily found in the southern continents of Africa, Australia, and South America, where they negatively affect livestock production. This review highlights past and current research investigating (1) the plants reported to contain monofluoroacetate and cause sudden death; (2) the mode of action, clinical signs, and pathology associated with poisoning by monofluoroacetate-containing plants; (3) chemical methods for the analysis of monofluoroacetate in plants; (4) the coevolution of native flora and fauna in Western Australia with respect to monofluoroacetate-containing plants; and (5) methods to mitigate livestock losses caused by monofluoroacetate-containing plants.

Unusual amino acids and monofluoroacetate from Dichapetalum michelsonii (Umutambasha), a toxic plant from Rwanda.

In the course of our investigations on Umutambasha in order to identify its convulsant principles, small quantities of monofluoroacetate were observed in stem bark, leaves, and fruits of this plant newly identified as Dichapetalum michelsonii Hauman. Conclusive evidence for a monofluoroacetate presence came from its isolation from the freeze-dried extract of stem bark. Three free unusual amino acids, named N-methyl-α-alanine, N-methyl-ß-alanine, and 2,7-diaminooctan-1,8-dioic acid, described for the first time in a plant, and known trigonelline were also isolated from the stem bark of D. michelsonii. Structure elucidations were mainly achieved by spectroscopic methods (1H-NMR, 2D-NMR, MS) and by comparison with authentic references. These unusual amino acids were detected by a fast, reliable TLC analysis in all our batches of Umutambasha, suggesting that they could be used for identification purposes in case of human or livestock intoxications. Finally, EEG recordings and behavioural observations performed in mice suggested that the convulsive patterns produced by Umutambasha are the consequence of monofluoroacetate presence in D. michelsonii.

Detection of monofluoroacetate in Palicourea and Amorimia species.

Numerous plant species worldwide including Palicourea marcgravii and Tanaecium bilabiatum in Brazil cause sudden death and are known to contain monofluoroacetate (MFA). Other species in Brazil including some species traditionally assigned to Mascagnia but now properly called Amorimia species and other Palicourea species are reported to cause sudden death in livestock and are suspected to contain MFA due to the similarity of clinical signs. In this study, an HPLC-APCI-MS method to detect and quantify MFA was developed and was used to investigate plant material from field collections and/or herbarium specimens of Mascagnia, Amorimia, and Palicourea species suspected of causing sudden death. MFA was detected in Amorimia amazonica, Amorimia camporum, Amorimia exotropica, Amorimia pubiflora, Amorimia rigida, and Amorimia septentrionalis as well as Palicourea aeneofusca. MFA concentrations differ greatly between Palicourea species and Amorimia species, which may explain the incidence of poisoning and the amount of plant material required to cause sudden death between these taxa.

Treatment of fluoroacetate by a Pseudomonas fluorescens bioflm grown in membrane aerated biofilm reactor.

Fluorinated organic compounds have widespread applications, and their accumulation in the environment is a concern. Biofilm reactors are an effective technology for the treatment of contaminated wastewater, yet almost no research has been conducted on the effectiveness of biofilms for the biodegradation of fluorinated aliphatic compounds. In this paper we describe experiments undertaken to investigate the degradation of fluoroacetate using a membrane aerated biofilm reactor (MABR) by Pseudomonas fluorescens DSM8341. The concentration of fluoroacetate in the medium influenced biofilm structure, with less dense biofilm observed at lower fluoroacetate loading rates. As biofilm thickness increased, oxygen utilization decreased, probably as a consequence of increased resistance to oxygen transfer. Furthermore, most of the biofilm was anaerobic, since oxygen penetration depth was less than 1000 microm. Biofilm performance, in terms of fluoroacetate removal efficiency, was improved by decreasing the fluoroacetate loading rate, however increasing the intramembrane oxygen pressure had little effect on biofilm performance. A mathematical model showed that while fluoroacetate does not penetrate the entire biofilm, the defluorination intermediate metabolite glycolate does, and consequently the biofilm was not carbon limited at the biofilm-membrane interface where oxygen concentrations were highest The model also showed the accumulation of the free fluoride ion within the biofilm. Overflow metabolism of glycolate was identified to be most likely a result of a combination of oxygen limitation and free fluoride ion inhibition. The study demonstrated the potential of MABR for treating wastewater streams contaminated with organofluorine compounds.

Simultaneous determination of two acute poisoning rodenticides tetramine and fluoroacetamide with a coupled column in poisoning cases.

A coupled column system was developed for the simultaneous determination of both rodenticides fluoroacetamide and tetramine in this paper by gas chromatography/mass spectrometry (GC/MS). A short length of strong polar column (1.5 m of Innowax) was coupled to the top of a 30 m of DB-5 ms with a quartz capillary column connector. Peak width at half height (W(h)) was used to evaluate the band broadening of the coupled column system. The length of the short couple column and oven temperature program were discussed according to W(h). The precisions of the coupled column were analyzed with peak area and retention time. Good linear correlations were found for both rodenticides. Typical samples were discussed for each rodenticide and some poisoning cases were presented.

Yield of trihalomethanes and haloacetic acids upon chlorinating algal cells, and its prediction via algal cellular biochemical composition.

The major objective of the present study was to investigate the contribution of major biomolecules, including protein, carbohydrates and lipids, in predicting DBPs formation upon chlorination of algal cells. Three model compounds, including bovine serum albumin (BSA), starch and fish oil, as surrogates of algal-derived proteins, carbohydrates and lipids, and cells of three algae species, representing blue-green algae, green algae, and diatoms, were chlorinated in the laboratory. The results showed that BSA (27 microg mg(-1) C) and fish oil (50 microg mg(-1) C) produced more than nine times higher levels of chloroform than starch (3 microg mg(-1) C). For the formation of HAAs, BSA was shown to have higher reactivity (49 microg mg(-1) C) than fish oil and starch (5 microg mg(-1) C). For the algal cells, Nitzschia sp. (diatom) showed higher chloroform yields (48 microg mg(-1) C) but lower HAA yields (43 microg mg(-1) C) than Chlamydomonas sp. (green algae) (chloroform: 34 microg mg(-1) C; HAA: 62 microg mg(-1) C) and Oscillatoria sp. (blue-green algae) (chloroform: 26 microg mg(-1) C; HAA: 72 microg mg(-1) C). The calculated chloroform formation of cells from the three algal groups, based on their biochemical compositions, was generally consistent with the experimental data, while the predicted values for HAAs were significantly lower than the observed ones. As compared to humic substances, such as humic and fulvic acids, the algal cells appeared to be important precursors of dichloroacetic acid.