Chemical and biological characterization of non-extractable sulfonamide residues in soil

For sulfonamides, the formation of non-extractable residues has been identified by laboratory testing as the most relevant concentration determining process in manured soil. Therefore, the present study has been focused on the chemical and biological characterization of non-extractable residues of ¹⁴C-labeled sulfadiazine or sulfamethoxazole. In laboratory batch experiments, the test substances were spiked via standard solution or test slurry to microbially active soil samples. After incubation periods of up to 102 d, a sequential extraction technique was applied. Despite the exhaustive extraction procedure, sulfadiazine residues mainly remained non-extractable, indicating the high affinity to the soil matrix. The remobilization of non-extractable ¹⁴C-sulfadiazine residues was monitored in the activated sludge test and the Brassica rapa test. Only small amounts (<3%) were transferred into the extractable fractions and 0.1% was taken up by the plants. In the Lumbricus terrestris test A, the release of non-extractable ¹⁴C-sulfamethoxazole residues by the burrowing activity of the earthworms was investigated. The residues mainly remained non-extractable (96%). The L. terrestris test B was designed to study the immobilization of ¹⁴C-sulfamethoxazole in soil directly after the test slurry application. The mean uptake by earthworms was 1%. Extractable and non-extractable residues amounted to 5% and 93%, respectively. Consequently, the results of all tests confirmed the high affinity of the non-extractable sulfonamide residues to the soil matrix.     

Physico-chemical versus microbial release of c-atrazine bound residues from a loamy clay soil incubated in laboratory microcosms

A loamy clay soil containing unextractable ¹⁴C-ring labeled atrazine residues was incubated in microcosms under abiotic and biotic conditions. The mineralization activity of the soil microflora was evaluated by the release of total CO₂ and ¹⁴C0₂. After 63 days of sample incubation the total organic carbon mineralization was of 1.71%, that of ¹⁴C-residues was of 0.72% of the initial radioactivity. No direct relationship was established between the mineralization of atrazine residues and the global mineralization. The contribution of soil microorganisms in the release of ¹⁴C-residues was weak. The availability of non-extractable residues was mainly controlled by physico-chemical factors. The low value of the reextractability rate and the distribution of bound residues during the soil sample incubation shown the active role of organic matter in detoxification procedure. Ninety percent of the residues remained bound after 63 days of incubation and were thus, potentially available without biocide activity.

The fractionation of soil organic matter allowed to specify the distribution of bound residues within the organic compartments. After a long-stay of pesticides in soils, approximately 65% of bound residues were associated with humin.     

Correlation of gas chromatographic properties of chlorobenzenes and polychlorinated biphenyls with the occurrence of reductive dechlorination by untamed microorganisms

To understand the dechlorination ability of chlorobenzenes (CBs) and polychlorinated biphenyls (PCBs) by untamed microorganisms under anaerobic condition and to correlate gas chromatographic properties with the occurrence of reductive dechlorination, introduction of CBs and PCBs in the culture medium inoculated with microorganisms from sludge and sediment, respectively, were performed. Three kinds of culture media preparing from sludge, river water and a synthetic medium were used in the experiments. HCB was degraded to 1,3,5-trichlorobenzene (1,3,5-TCB) and 1,3-dichlorobenzene (1,3-DCB) in both sludge medium and synthetic medium with inoculated microorganisms. Three PCB congeners including 2,3,4-, 3,4,5- and 2,3,4,5-CBp (chlorinated biphenyl) were not found to be dechlorinated in the river water medium with inoculation culture but to be dechlorinated in the synthetic medium. MNDO methodology was used to compute theoretical dechlorination reaction heats and GC-ECD techniques were used to estimate chromatographic data of CB and PCB congeners. Both CB and PCB congeners showed that dechlorination by untamed microorganisms under anaerobic mixed cultures were more likely to occur when larger amounts of energy were released and greater deltaln RRT value between the parent congener and the daughter product was observed. Deltaln RRT provided a more precise information on the singularity of PCBs ortho-dechlorination in an aspect of thermodynamic favorable rule.     

Detoxification of hexachlorobenzene by dechlorination with potassium-sodium alloy

Dechlorination of hexachlorobenzene (HCB) was achieved by a liquid potassium–sodium (K–Na)-alloy. HCB in a cyclohexane/benzene solution (22 mmol/l, 4.67 g/l as chlorine) was dechlorinated by almost 100% after a 30-min reaction, indicating high reactivity of K–Na alloy and high proton donating power of cyclohexane. Decreasing orders of chlorobenzenes identified after a 15-min reaction, by amount were 1,2,3,4- > 1,2,3,5- > 1,2,4,5- for tetrachlorobenzenes, 1,2,4- > 1,2,3- > 1,3,5- for trichlorobenzenes, and 1,4- > 1,3- > 1,2- for dichlorobenzenes. It was hypothesized that once one chlorine atom in HCB was replaced with a proton, the adjacent chlorine atom to the proton tended to be replaced with another hydrogen atom. A total of 63 PCBs formed via the Wurtz–Fittig reaction were identified as by-products in the sample after a 15-min reaction. Among PCBs found, 2,3^′,4^′,5-tetrachlorobiphenyl, which was a product from 1,2,4-trichlorobenzene formed via the Wurtz–Fittig reaction, was detected in relatively high concentration (48.9 nmol/ml). The sample obtained from a reaction mixture after 30 min contained only 14 PCBs in trace amounts, indicating that the PCBs formed were also further dechlorinated by K–Na alloy. Non-chlorinated compounds––such as methylbenzene, dimethylbenzene, dimer of tetrahydrofuran, and dicyclohexyl (dimer of cyclohexane)––were also identified in the samples. A method using K–Na alloy developed in the present study dechlorinated satisfactorily HCB at room temperature.     

Fate of imidacloprid in soil and plant after application to cotton seeds

This study aimed to investigate the persistence of imidacloprid in soil after application to cotton seeds and to obtain a complete picture on the mass balance of this compound in soil and cotton plants. The study was carried out as a pot culture experiment under laboratory conditions using a Gaucho formulation containing ¹⁴C-labeled imidacloprid. Three treatments of cotton seeds were made in sandy loamy soil: live seeds grown in autoclaved soil, dead seeds put in live soil and live seeds grown in live soil. Results showed that total ¹⁴C recoveries decreased by time ranging 93.8–96.2, 77.1–88.4 and 53.5–62.4% of the applied radioactivity at 7, 14, and 21 d after application, respectively. The reduction in the extracted ¹⁴C from soil coincided with the increase of non-extracted ones. Levels of bound ¹⁴C was always less in autoclaved soil than in live ones. Results revealed also that only 1.8–6.8% of the applied ¹⁴C was taken up by the plants and fluctuated within the test period. ¹⁴C levels were higher in plants grown in autoclaved soil than those in live ones and the radioactivity tended to accumulate on the edges of cotton leaves. Most of the radioactivity in the soil extracts was identified as unchanged ¹⁴C-imidacloprid.     

Biomineralisation of 1,2,4-trichlorobenzene in soils by an adapted microbial population

In laboratory experiments the mineralisation of 14C-labelled 1,2,4-trichlorobenzene (1,2,4-TCB) in soils was studied by direct measurement of the evolved 14CO2. The degradation capacity of the indigenous microbial population was investigated in an agricultural soil and in a soil from a contaminated site. Very low mineralisation of 1% within 23 days was measured in the agricultural soil. Whereas in the soil from the contaminated site the mineralisation occurred very fast and in high rates; up to 62% of the initially applied amount of 1,2,4-TCB were mineralised within 23 days. The transfer of the adapted microbial population into the agricultural soil significantly enhanced the mineralisation of 1,2,4-TCB in this soil, reflecting, that the transferred microbial population survived and maintained its degradation ability in the new microbial ecosystem. Additional nutrition sources ((NH4)2HPO4) increased the mineralisation rates in the first days significantly in the contaminated soil. In the soil from the contaminated site high amounts of non extractable 14C-residues were formed.     

Determination of trichloroacetic acid in environmental studies using carbon 14 and chlorine 36

Radioisotopes carbon 14 and chlorine 36 were used to elucidate the environmental role of trichloroacetic acid (TCA) formerly taken to be a herbicide and a secondary air pollutant with phytotoxic effects. However, use of ¹⁴C-labeling posed again known analytical problems, especially in TCA extraction from the sample matrix. Therefore—after evaluation of available methods—a new procedure using decarboxylation of [1,2-¹⁴C]TCA combined with extraction of the resultant ¹⁴C-chloroform with a non-polar solvent and its subsequent radiometric measurement was developed. The method solves previous difficulties and permits an easy determination of amounts between 0.4 and 20 kBq (10–500 ng g⁻¹) of carrier-less [1,2-¹⁴C]TCA in samples from environmental investigations. The procedure is, however, not suitable for direct [³⁶Cl]TCA determination in chlorination studies with ³⁶Cl. Because TCA might be microbially degraded in soil during extraction and sample storage and its extraction from soil or needles is never complete, the decarboxylation method—i.e. 2 h TCA decomposition to chloroform and CO₂ in aqueous solution or suspension in closed vial at 90 °C and pH 4.6 with subsequent CHCl₃ extraction—is recommended here, estimated V < 7%. Moreover, the influence of pH and temperature on the decarboxylation of TCA in aqueous solution was studied in a broad range and its environmental relevance is shown in the case of TCA decarboxylation in spruce needles which takes place also at ambient temperatures and might amount more than 10–20% after a growing season. A study of TCA distribution in spruce needles after below-ground uptake shows the highest uptake rate into current needles which have, however, a lower TCA content than older needle-year classes, TCA biodegradation in forest soil leads predominatingly to CO₂.     

Investigation of organic xenobiotic transfers,partitioning and processing in air-soil-plant systems using a microcosm apparatus. Part II: comparing the fate of chlorobenzenes in grass planted soil

A microcosm system was used to investigate and compare transfers of 14C labeled-1,2-dichlorobenzene (DCB), 1,2,4-trichlorobenzene (TCB) and hexachlorobenzene (HCB) in an air-soil-plant system using single grass tillers planted into spiked soil. This study was the second phase of a development investigation for eventual study of a range of xenobiotic pollutants. Recoveries from the system were excellent at >90%. The predominant loss pathway for 14C labeled-1,2-DCB and 1,2,4-TCB was volatilisation with 85% and 76% volatilisation of parent compound and volatile metabolites over 5 weeks respectively. Most of the added label in the hexachlorobenzene spiked system remained in soil. Mineralisation was <1% for all compounds. 14C plant burdens expressed as microg parent compound/g plant fresh weight were significant and suggest that plant uptake of chlorobenzenes from soil may be an important exposure pathway for grazing herbivores. Both shoot and root uptake of 14C was detected, with foliar uptake of volatilised compounds dominating shoot uptake, and being greatest in TCB spiked systems. The microcosm is shown as potentially an ideal system with which to investigate organic xenobiotic partitioning in air-soil-plant systems to improve understanding of the equilibria and kinetics of exchanges. However, limitations imposed by the lab based conditions must be recognized and data should be compared with field based data sets as a consequence.     

Synergic effect of calcium oxide and iron (III) oxide on the dechlorination of hexachlorobenzene

Dechlorination of hexachlorobenzene (HCB) was achieved by a mixture of commercial CaO and alpha-Fe2O3 (CaO/alpha-Fe2O3) in closed systems at temperatures of 300 degrees C and 350 degrees C, which exhibited a synergic effect compared to CaO or alpha-Fe2O3 alone, and the dechlorination efficiency was dramatically enhanced. When CaO and alpha-Fe2O3 coexisted, HCB was dechlorinated by about 98% after 0.5 h reaction at 300 degrees C. All TeCB-, TrCB-, and DCB-isomers were detected, showing the presence of more than one dechlorination pathway. The pathway to form 1,2,4-TrCB must be a major pathway because the greatest amount of 1,2,4-TrCB was detected. There existed discrepancy of the material balance between the starting and dechlorinated materials. It implies that besides dechlorination other decomposition processes may be present. The mechanism of synergic effect of calcium and iron oxides was investigated.     

Reductive dechlorination of 1,2,4-trichlorobenzene with palladized nanoscale Fe(zero-valent) particles supported on chitosan and silica

In this study, nanoscale Pd-Fe particles, with diameters less than 100 nm, were synthesized and dispersed over the chitosan and silica supports. Three different Pd-Fe particles were synthesized, namely 0.1% Pd-Fe, 0.5% Pd-Fe and 1.0% Pd-Fe. SEM images confirmed that the Pd-Fe particles were dispersed over the surface of the supports while SEM-EDX confirmed evenly distribution of Pd over Fe(zero-valent). alpha-Fe(zero-valent) crystallites were identified by means of XRD and observed in TEM. Reductive dechlorinations of 1,2,4-trichlorobenzene (1,2,4-TCB) with the nanoscale Pd-Fe/chitosan and Pd-Fe/silica were carried out in the batch experiment system. Disappearance of the parent species and formation of the reaction intermediates and end product were monitored at discrete times. The results show that the nano-scale Pd-Fe particles were able to completely dechlorinate the chlorinated benzenes within a very short timescale. Complete dechlorinations of 1,2,4-TCB to benzene were achieved within 60 min with the 1.0% Pd-Fe/chitosan and within 100 min with the 1.0% Pd-Fe/silica. Reaction rates were observed to increase with increasing Pd content of the Pd-Fe/support. The reactions apparently followed pseudo-first-order kinetics with respect to the 1,2,4-TCB transformation. A kinetic model is constructed to fit the experimental results for the reactions, enabling identification of the major and minor dechlorination pathways of 1,2,4-TCB. The model suggests that the 1,2,4-TCB transformation mainly followed the primary pathway of direct reductive dechlorination to benzene and secondary pathway of sequential hydrogenolysis to 1,2-dichlorobenzene (1,2-DCB) and then chlorobenzene (CB) or benzene.     

Influence of the amendment of corn straw on the degradation behaviour of the fungicide dithianon in soil

Degradation studies were conducted with the fungicide (14)C-dithianon under standard conditions for 64 days in soil. The compound is characterized by mineralization losses of approx. 33% and the formation of non-extractable (bound) residues of approx. 63% in 64 days. The microbial activity of the soil was stimulated by an amendment of corn straw simulating post-harvest conditions. This addition of straw decreased the mineralization of the compound initially. At the end of the incubation period, however, the mineralization rate was higher in the straw amended soil compared to the control. The addition of straw increased the amount of radiocarbon in the desorption solutions. Thus higher amounts of incorporated radiocarbon could be found in the biomass of the amended soil. Model calculations show that the straw amendment has a sustained influence on the mineralization of the compound. Potential mechanisms of the effect of dissolved organic matter on the sorption/desorption equilibrium are discussed.     

Localization of atrazine non-extractable (bound) residues in soil size fractions

Three different soils were incubated under field conditions with ¹⁴C-ring labelled atrazine. After six months, the soils were exhaustively extracted with methanol and sonicated in water. The dispersed material was then fractionated by sieving, sedimentation and centrifugation, and each fraction was separated into humin, fulvic and humic acids. In all soils, the well humified organic matter and the atrazine residues were mainly located in the 20-2 and 2-0.2 μm fractions. There was a very large concentration of bound residues in the coarsest fractions, especially in the 200-50 μm fraction. These could be related to the active degradation of coarse plant residues, or to bioconcentration by soil actinomycetes and fungi.     

Pollution topography of chlorobenzenes and hexachlorobutadiene in sediments along the Kaohsiung coast, Taiwan--a comparison of two consecutive years' survey with statistical interpretation

Forty sediments were analyzed for chlorobenzenes (CBs) and hexachlorobutadiene (HCBD) to investigate their pollution topography along the Kaohsiung coast, Taiwan. Maximum CB concentrations found, varied from 15.4 ng/g dw for 1,2,3-TCB to 56.8 ng/g dw for 1,2,4-TCB, while higher HCBD concentrations were detected among north-bound stations (around the exit of the Tsoying outfall pipe) and its concentration decreases from north to south. Compared to a previous survey executed a year before, there is no statistically significant difference in CB and HCBD congeners between these two surveys (p = 0.68–0.87, two-tailed paired t-test). The spatial distribution of toxic index reveals that biological effects might occur near Tsoying and Dalinpu outfall pipe outlets which dispose of petro-chemical wastewater. Evidence found in this study also shows distinct CB patterns from the two nearby sources, the Dalinpu outfall pipe outlet and the Kaoping estuary. Principal component analysis shows that four principal components conducting CBs and HCBD distribution were extracted. Both the first two components (accounted for 58.8% of the total variance), comprised all of the CBs except HCB (DCBs to PeCB), were found not capable of differentiating any distinct pollution source. On the other hand, HCBD and HCB were extracted as third and fourth components, respectively, pointing out their distinct sources in this area.     

Pharmacok inetics and metabolism of 1,2,3,7,8-pentachlorodibenzo-p-dioxin in the rat

¹⁴C-1,2,3,7,8-Pentachloroaibenzodioxin (P₅CDD), administered to rats as single oral dose (1.69–1.75 μg/animal, 8.42–10.06 μg/kg) was eliminated with a half life of 29.5±2.7 days from the body of the animals. Residual P₅CDD was located mainly in the liver and the adipose tissue. In the bile, polar metabolites of P₅CDD were detected but no unmetabolized P₅CDD.     

Leaching and degradation of 21 pesticides in a full-scale model biobed

Filling and cleaning of pesticide sprayers presents a potential risk of pollution of soil and water. Three different solutions for handling sprayers have been suggested: Filling and cleaning in the field, filling and cleaning on hard surfaces with collection of the waste water, and filling and cleaning on a biobed, which is an excavation lined with clay and filled with a mixture of chopped straw, sphagnum and soil with turf on top, and with increased sorption capacity and microbial activity for degradation of the pesticides. In the present study the degradation and leaching of 21 pesticides (5 g of each) was followed in an established full-scale model biobed. Percolate was collected and analysed for pesticide residues, and the biobed material was sampled at three different depths and analysed by liquid chromatography double mass spectrometry (LC-MSMS). During the total study period of 563 days, no traces of 10 out of 21 applied pesticides were detected in the percolate (detection limits between 0.02 and 0.9 μg l⁻¹) and three pesticides were only detected once and at concentrations below 2 μg l⁻¹. During the first 198 days before second application, 14% of the applied herbicide bentazone was detected in the leachate with maximum and mean concentrations of 445 and 172 μg l⁻¹, respectively. About 2% of the initial mecoprop and fluazifop dose was detected in the percolate, with mean concentrations of 23 μg l⁻¹, while MCPA and dimethoate had mean concentrations of 3.5 and 4.7 μg l⁻¹, respectively. Leachate concentrations for the remaining pesticides were generally below the detection limit (0.02–0.9 μg l⁻¹, below 1% of applied). Sorption studies of five pesticides showed that compounds with a low K_d value appeared in the leachate. After 169 days, all pesticides in the biobed profile were degraded to a level below 50% of the calculated initial dose. Pesticides with K_oc values above 100 were primarily found in the uppermost 10 cm and degraded slowest due to the low bioavailability. The 11 most degradable pesticides were all degraded such that less than 3% remained in the biobed after 169 days.

Following second pesticide application of the biobed, leachate was sampled 215 and 365 days after the treatment. This showed the same pesticides to be leached out and at concentrations comparable to those of the first treatment. The same pesticides as after the first treatment were retained in the biobed.     

Organochlorine pesticides, PCBs and mercury in antarctic and subantarctic seabirds

Between 1978 and 1983, eggs from 15 species of Antarctic and Subantarctic seabirds were collected and analysed for organochlorine pesticides, PCBs, and mercury residues. The lowest levels of these chemicals were detected in the eggs of Adélie Penguins from Prydz Bay. Antarctica. On a wet weight basis, the mean concentrations in eggs of these penguins were 0.01 μg/g HCB, 0.005 μg/g DDE, less than 0.1 μg/g PCBs (1260) and 0.02 μg/g mercury. Such values indicate that the environmental levels of these contaminants in the biotypes of these species are extremely low, and present no significant threat.

The highest levels of contaminants were found in the eggs of species which breed in Antarctica and migrate to regions well north of the Antarctic Convergence in the non-breeding season. On a wet weight basis, Northern Giant-Petrel eggs contained 0.11 μg/g HCB, 0.95 μg/g DDE and 1.8 μg/g PCB (1260), and 1.8 μg/g mercury. There are indications that DDE levels are increasing for both Southern and Northern Giant-Petrels, and it is possible that reports of a decreasing number of breeding pairs of these species at several colonies are due to one or more of these contaminants reaching toxic levels.

Analysis of penguin tissues show that preen gland lipid is suitable for monitoring the body burden of organochlorine insecticides and PCBs in seabirds.

Variation in DDE and mercury residue levels for some of these species suggest that the biotypes in which they live are quite different, and that the Antarctic Convergence may have a major influence in protecting the Antarctic ecosystem from these pollutants.     

Isolation and characterization of 1,2,4-trichlorobenzene mineralizing Bordetella sp. and its bioremediation potential in soil

A soil which has been polluted with chlorinated benzenes for more than 25 years was used for isolation of adapted microorganisms able to mineralize 1,2,4-trichlorobenzene (1,2,4-TCB). A microbial community was enriched from this soil and acclimated in liquid culture under aerobic conditions using 1,2,4-TCB as a sole available carbon source. From this community, two strains were isolated and identified by comparative sequence analysis of their 16S-rRNA coding genes as members of the genus Bordetella with Bordetella sp. QJ2-5 as the highest homological strain and with Bordetella petrii as the closest related described species. The 16S-rDNA of the two isolated strains showed a similarity of 100%. These strains were able to mineralize 1,2,4-TCB within two weeks to approximately 50% in liquid culture experiments. One of these strains was reinoculated to an agricultural soil with low native 1,2,4-TCB degradation capacity to investigate its bioremediation potential. The reinoculated strain kept its biodegradation capability: (14)C-labeled 1,2,4-TCB applied to this inoculated soil was mineralized to about 40% within one month of incubation. This indicates a possible application of the isolated Bordetella sp. for bioremediation of 1,2,4-TCB contaminated sites.     

Thermal degradation of hexachlorobenzene in the presence of calcium oxide at 340–400 °C

Hexachlorobenzene (HCB) in the milligram range was co-heated with calcium oxide (CaO) powder in sealed glass ampoules at 340–400 °C. The heated samples were characterized and analyzed by Raman spectroscopy, elemental analysis, gas chromatography/mass spectrometry, ion chromatography, and thermal/optical carbon analysis. The degradation products of HCB were studied at different temperatures and heated times. The amorphous carbon was firstly quantitatively evaluated and was thought to be important fate of the C element of HCB. The yield of amorphous carbon in products increased with heating time, for samples treated for 8 h at 340, 380 °C and 400 °C, the value were 17.5%, 34.8% and 50.2%, respectively. After identification of the dechlorination products, the HCB degradation on CaO at 340–400 °C was supposed to through dechlorination/polymerization pathway, which is induced by electron transfer, generate chloride ions and form high-molecular weight intermediates with significant levels of both hydrogen and chlorine, and finally form amorphous carbon. Higher temperature was beneficial for the dechlorination/polymerization efficiency. The results are helpful for clarifying the reaction mechanism for thermal degradation of chlorinated aromatics in alkaline matrices.     

Studying soil organic matter using 13C CP-MAS NMR: the effect of soil chemical pre-treatments on spectra quality and representativity

¹³C CP-MAS NMR spectroscopy is a technique that has proved to be useful in studying soil organic matter (SOM). Nevertheless, NMR spectra exhibit a weak signal and have very low resolution due to: the low natural abundance of ¹³C (1.1 % of C) in SOM, the generally low SOM content of soils, and the presence of paramagnetic impurities. This paper studies the effects of soil chemical pre-treatments on ¹³CP-MAS NMR spectra quality and spectra representativity i.e. soil C mass balance.

After chemical pre-treatment to increase total organic carbon (TOC) content and C/Fe ratio, eight soils characterized by different levels of organic carbon content and C/Fe ratios were studied using ¹³CP-MAS NMR. Moreover, where chemical treatments were not applicable due to high carbon losses, the number of ¹³CP-MAS NMR scans was increased in order to obtain satisfactory spectra.

Results show that chemical pre-treatment of soils with C/Fe > 1 caused high C losses. Bulk soils were therefore studied by increasing the number of ¹³CP-MAS NMR scans. Acceptable spectra were obtained from 8K scans (1K = 1024 transient). On the other hand, even when a large number of scan (32K) are used, soil with C/Fe < 1 cannot be studied. As these soils are characterized by low C losses after HCl treatments (range of 2.9–25.4%), a pre-treatment of at least 1.39 mol l⁻¹ HCl removes excess Fe and at the same time increases C/Fe ratio resulting in 32K scans providing good spectra.     

Relationship between electron donor and microorganism on the dechlorination of carbon tetrachloride by an anaerobic enrichment culture

An investigation involving the supplement of different concentrations of substrates and microorganisms was carried out under anaerobic condition to assess the feasibility of bioremediation of carbon tetrachloride (CCl₄) with the amendment of low concentrations of auxiliary substrate and microorganisms. The concentrations of substrate and microorganisms ranged from 10 to 100 mg/l and from 3.7 × 10⁴ to 3.7 × 10⁶ cell/ml, respectively. The biotransformation rate of CCl₄ increased progressively with the increase in the concentrations of the substrate and microorganisms. In the low biomass-amended system (3.7 × 10⁴cells/ml), 28–71% and 57–96% of CCl₄ removals were exhibited when 10–100 mg/l of acetate or glucose was supplemented, respectively, whereas nearly complete degradation of CCl₄ was observed in the heavily inoculated systems (3.7 × 10⁶ cells/ml). An addition of electron donor in the low microbial activity batches enhanced greater efficiency in dechlorination than in the high microbial activity batches. The second-order rate constants ranged from 0.0059 to 0.0092 l/mg/day in high biomass input system, while a two- to four-fold increase in rate constant was obtained in the low microbial activity system. This study indicates that biomass was the more important environmental parameter than substrate affecting the fate of CCl₄. The addition of auxiliary substrates was effective only in low biomass-amended batches (0.56 mg-VSS/l) and diminished inversely with the increase of microbial concentration.