The identification of five unreported lindane metabolites recovered from rat urine

Summary Previously unreported metabolites from the urine of rats fed lindane have been identified as 3,4-dichlorophenol, 2,4,6-trichlorophenol, 2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol and 2,3,4,5,6-pentachloro-2-cyclohexen-1-ol. While the 3,4-dichlorophenol is a minor metabolite, the others are excreted in greater quantities than either 2,3,5- or 2,4,5-trichlorophenol, previously identified lindane metabolites. The excretion of tetrachlorophenols and PCCOL require a revision in the currently accepted theory regarding the metabolism of lindane by mammals.     

Urinary excretion of chlorinated phenols in saw-mill workers

Summary The excretion and conjugation of chlorophenols were studied in workers exposed to 2,4,6-tri-, 2,3,4,6-tetra-, and pentachlorophenolates, the main components of the chlorophenolate product manufactured by direct chlorination of phenol. The workers were exposed in two different saw mills in which sodium chlorophenolate was used for treatment of lumber during the warm season. Urine specimens were collected at the end of the treatment season as well as at the start of a new treatment period in the spring. Serum specimens were collected towards the end of the treatment period. Total and unconjugated chlorophenols were analyzed with a gas chromatographic method. The maximal concentrations of urinary 2,4,6-tri-, 2,3,4,6-tetra- and pentachlorophenol at the end of the lumber-treatment period were 1–11.8, 3.4–17.3, and 0.2–0.9 mol/l, respectively, and the average apparent half-times calculated using a one-compartment model were 18 h, 4.3 days and 16 days, respectively. For 2,3,4,6-tetrachlorophenol, the data of some subjects showed a better fit with a two-compartment model; the corresponding half-times were 5.3 and 26 days. During the continuous-exposure period the average serum levels of tetra- and pentachlorophenol were rather similar before and after the working day: 2.79 ±1.78 mol/l for tetrachlorophenol and 0.85 ± 0.4 mol/l for pentachlorophenol. Renal clearance values for tetra-and pentachlorophenol were related to urine flow and indicated tubular reabsorption. At low concentrations, sulfate conjugation was dominant. With increasing chlorophenol concentrations the proportion of glucuronide conjugation was increased, especially for pentachlorophenol.     

Estimation of selected phenols in drinking water with In situ acetylation and study on the DNA damaging properties of polychlorinated phenols

Gas chromatography/mass spectrometry with selected-ion monitoring (GC/MS-SIM) method were used to analyze 23 selected phenols in natural and drinking waters by an in situ acetylation technique. This method was suitable for determining phenol concentrations at the ng/L level because of less background interference and better recoveries. The application range for all these phenols was from 0.01 or 0.04 to 10 g/L, using a 800 ml water sample. The levels of trace phenols in four Taiwan water treatment plants were in the range of 12–312 ng/L.The polychlorinated phenols, namely 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol, were evaluated for their ability to induce deoxyribonucleic acid (DNA) damage using a DNA precipitation assay employing mouse embryonic fibroblast cells (C3H10T1/2) with or without a liver microsomal activation system. These agents exhibited a weak positive response when microsomal activation enzymes were present in this assay. When the tetrachlorohydroquinone, a toxic metabolite of pentachlorophenol, was measured by the same method without the activation system, a significant and dose-dependent DNA damage was found. This result indicates that in the evaluation of the carcinogenic potential of these agents, their corresponding metabolites should be taken into consideration. DNA strand breakage caused by these active metabolites may play an important role in the tumorigenetic process of polychlorinated phenols.     

Characterization of Pentachlorophenol Degrading Bacterial Consortium from Chemostat

A microbial consortium was developed by continuous enrichment of bacterial population isolated from sediment core of pulp and paper mill effluent in mineral salts medium (MSM) supplemented with pentachlorophenol (PCP) as sole source of carbon and energy in the chemostat. The enriched consortium contained three bacterial strains identified as Escherichia coli (PCP1), Pseudomonas aeruginosa (PCP2) and Acinetobacter sp. (PCP3) by morphological and biochemical tests, further confirmation was done using 16S rDNA sequence analysis. The potency of bacterial isolates in degradation of PCP was monitored in terms of growth and utilization of PCP as substrate with spectrophotometer and gas chromatograph-mass spectrometer (GC-MS) analysis. The strains were tested for their utilization of various organic compounds. The strain PCP3, showed higher potency to utilize PCP as sole source of carbon and energy than PCP1 and PCP2. The bacterial strain were able to utilize PCP through an oxidative and reductive route as indicated with the formation of tetrachloro-p-hydroquinone (TeCH), 2-chloro-1,4-benzenediol and 2,3,4,6-tetrachlorophenol, respectively.     

Fate of chloroalkylene-9-14C in carrots,sugar beets,and soil under outdoor conditions

Immediately after application of chloroalkylene-9-¹⁴C to soil (1.32 ppm, based on dry weight of soil in the upper layer of 0 to 10 cm) under outdoor conditions, carrots were sown; in the following year, sugar beets were grown. About 80% of the radioactivity applied volatilized within one vegetation period. Most of the remaining radioactivity was still in the upper soil layer; 0.8% had dispersed to a depth of 40 cm, and 3.3% was taken up by the carrot plants. In the second year, no more decrease of soil residues was observed; uptake by sugar beets was 0.1% of the applied radioactivity.In the first year, the residues in the upper soil layer consisted of 41% unchanged chloroalkylene-9, 19% soluble metabolites, and 40% unextractable residues; the amount of unextractable residues rose to 68% in the second year. The following conversion products were characterized in the soil extracts: a monohydroxylated dichlorobiphenyl, a monomethoxylated dichlorobiphenyl, and two isomeric monohydroxylated, monoisopropylated dichlorobiphenyls; in carrot roots, a monomethoxylated dichlorobiphenyl was detected. Conjugates occurring in the soil yielded, after acid hydrolysis, a monohydroxylated dichlorobiphenyl among other compounds.     

Efficiencies of three common lipid extraction methods evaluated by calculating mass balances of the fatty acids

Efficiencies of three common lipid extraction methods have been evaluated by analyzing fatty acids in residues and extracts and calculating the mass balance for the fatty acids. Fatty acids were analyzed by an acid catalyzed direct methylation procedure followed by gas chromatography of fatty acid methyl esters. This procedure was also used as the benchmark for the calculation of mass balances. The three extraction principles investigated were Soxhlet extraction with petroleum ether, Soxhlet extraction after acid hydrolysis, and the Bligh and Dyer method. All samples were dry powders of marine origin; most samples had high ratios of polar to nonpolar lipids. Significant amounts of fatty acids were detected in the residues after extraction. The lowest extraction efficiencies were 30% for the Soxhlet method, 83% for the acid hydrolysis method and 90% for the Bligh and Dyer extraction. The lowest extraction efficiencies were typically found in samples with high ratio of polar to nonpolar lipids.     

Mutagenic hazards of complex polycyclic aromatic hydrocarbon mixtures in contaminated soil

The objective of the present study was to evaluate hazard/risk assessment methods for complex environmental mixtures that involve a targeted, priority chemical approach based on the cumulative hazard/risk of known mixture components or analyses of sufficiently similar mixtures. Ten polycyclic aromatic hydrocarbon (PAH)-contaminated soils were separated into nonpolar and semipolar fractions, and both fractions elicited positive responses on the Salmonella reverse mutation assay. Targeted and nontargeted methods of hazard prediction routinely overestimated mutagenic activities for the nonpolar soil fractions, suggesting nonadditive interactions of PAHs in complex mixtures. This suggests that current risk assessment methods for complex mixtures may provide conservative estimates regarding soils contaminated with priority PAHs alone. Significant underestimations of total risk, however, will be obtained if the soils also contain unidentified PAHs as well as polycyclic aromatic compounds and related compounds that contribute to the total mutagenic activity. Furthermore, estimates of excess lifetime cancer risk associated with the nondietary ingestion of the PAH-contaminated soils studied here indicate that a traditional risk assessment model based on identified priority PAHs and an assumption of additivity generally underestimates the risk associated with the nonpolar soil fractions (in comparison to bioassay-derived risk estimates). Additional cancer risk may be associated with the more polar compounds that also are found at these contaminated sites and that rarely are included in the standard risk assessment methodology.     

Toxicity of wide-range polarity fractions from wood smoke and diesel exhaust particulate obtained using hot pressurized water

Epidemiological and toxicological studies correlate the adverse health effects of particulate matter (PM) with the available information regarding their chemical characterization, which has focused on nonpolar organics (e.g., polycyclic aromatic hydrocarbons [PAHs]), which are limited to 15 to 50% of total organic carbon. To study both polar and nonpolar species, we have employed hot pressurized (liquid) water for the fractionation of diesel exhaust and wood smoke PM. In agreement with the results of previous studies, nonpolar fractions from both PM samples showed strong cytotoxicity (cell viability decreased to 50-60%) corresponding to the presence of PAHs. Surprisingly, similar decreases in cell viability were also found in polar fractions (50 degrees C) from both diesel exhaust and wood smoke PM. The midpolarity fractions (100-150 degrees C) from wood smoke PM also displayed high cytotoxicity corresponding to methoxyphenols and oxy-PAHs. Although the midpolarity fractions from diesel exhaust PM showed no cytotoxicity, genotoxicity was found in the 150 degrees C fraction, possibly corresponding to nitroaromatics. The present study demonstrates the suitability of hot pressurized water (as a single, nontoxic solvent) for the fractionation and toxicological characterization of wide-range polarity constituents of PM and, possibly, other environmental matrices.     

Lethal body residue of chlorophenols and mixtures of chlorophenols in benthic organisms

The lethal body residue (LBR) of a few chlorophenol congeners were measured in the oligochaete worm Lumbriculus variegatus, and the LBR of pentachlorophenol was measured also in a midge, Chironomus riparius larvae. LBR is defined as the concentration of the compound in the organism, on molar basis, to cause death, and the LBR₅₀ is defined as the calculated LBR value to cause a 50% mortality in population after a given time. Groups of 30 or 40 organisms were exposed to different chlorophenol concentrations in artificial soft fresh water to achieve differential mortality. Exposure times were either 24 h or 48 h. In addition to exposures with individual congeners, mixtures of chlorophenols were also tested. After each exposure, the surviving organisms were collected and the body burden of chlorophenols was measured by gas chromatography with electron capture detection. The measured body burden was related to the percent mortality in the group. The trichlorophenols and pentachlorophenol have a 48-h LBR₅₀ of 0.45–0.66 μmol/g wet weight in L. variegatus. The 48-h LBR₅₀ of pentachlorophenol for C. riparius was 0.15 μmol/g wet weight, indicating a slight difference in the sensitivity of these two species. The 48-h LBR₅₀ of 2,3,4,6-tetrachlorophenol is 0.91 μmol/g wet weight, and the value for 2,6-dichlorophenol is 1.2 μmol/g wet weight in L. variegatus. The 48-h LBR₅₀s of the chlorophenol mixtures ranged from 0.50 to 0.83 μmol/g wet weight, demonstrating an additive toxicity. Received: 13 August 2001/Accepted: 6 March 2002     

Mercury fractionation, bioavailability, and ecotoxicity in highly contaminated soils from chlor-alkali plants

Mercury (Hg) fractionation, speciation, bioavailability, and ecotoxicity were investigated in three highly contaminated soils from chlor-alkali plants. Single extractions and a validated four-step sequential extraction scheme were used. Total, volatile, and methyl-Hg concentrations were determined. Mercury was then separated in fractions defined as water-soluble (F1), exchangeable (F2), organic (F3), and residual (F4). Germination and growth inhibition of barley (Hordeum vulgare) and mortality of earthworms (Eisenia andrei) were assessed, and tissue-Hg concentrations of exposed organisms were determined. Results revealed highly (295 +/- 18-11,500 +/- 500 microg Hg/g) contaminated soils, but extracted fractions indicated relatively low mobility of Hg. Nevertheless, the water-soluble and the CaCl2-extractable fractions represented significant Hg concentrations (299 +/- 18 microg/g in soil 3, 67.4 +/- 2.3 microg/g in soil 1, and 9.5 +/- 0.3 microg/g in soil 2), and volatile Hg ranged between 14 and 98% of total Hg. Overall, Hg concentrations reached 6,560 +/- 240 microg/g in roots, 4,200 +/- 1,070 microg/g in aerial plants, and 1,410 +/- 120 microg/g in E. andrei. Earthworm mortality was 100% after exposure to the soil with the highest concentration of mobile Hg. In the latter soil, earthworm fragmentation and chlorotic plants were observed. Bioconcentration factors (BCFs) were higher in barley compared to earthworms, but BCFs yielded misleading values after exposure to the extremely contaminated soil. This study shows that Hg accumulated primarily in the roots, but results also indicate uptake of gaseous Hg by the aerial plants of barley. Tissue-Hg concentrations of both exposed organisms were correlated with water-soluble and CaCl2-extractable Hg, and growth inhibition was in agreement with Hg fractionation.     

Fate of [14C]aldrin in crop rotation under outdoor conditions

[14C]Aldrin was applied to soils (about 3kg/ha) in outdoor boxes at various locations (Germany, England, and United States), and crops were cultivated (maize, wheat, sugar beets, and potatoes). In the following year, crop rotation experiments were carried out in the same soils without retreatment; in addition, wheat was grown in soils retreated with [14C]aldrin (3.5 kg/ha). After the harvest of both years, the distribution of aldrin and major metabolites (dieldrin; photodieldrin; hydrophilic metabolites including dihydrochlordene dicarboxylic acid; an unidentified nonpolar compound X; and unextractable metabolites) was determined in plants, soils, and leaching water. Two further conversion products, photoaldrin and aldrin-trans-diol, occurred in trace amounts only in a few samples. Metabolic pathways for aldrin under outdoor conditions are presented. The distribution of radioactive residues in soils and plants as well as their quantitative chemical composition are discussed, and comparisons are made between the different experimental sites, the crops, the first and second year, and retreated and nonretreated samples. The quantitative results are compared to those of field trials.     

Oviposition habitats of Psorophora columbiae in soybean fields of a Texas riceland agroecosystem

Soil samples were collected over a 21/2-year period from microhabitats in fields planted to soybeans in Chambers County, TX, to study the oviposition behavior of Psorophora columbiae. An analysis of egg collection data gathered from the processing of 1,098 soil samples suggested that, during wet years, Ps. columbiae eggs tend to be scattered throughout soybean fields. During dry years, a greater abundance of eggs occur in the low areas of these types of fields. The data also suggested that oviposition by Ps. columbiae is concentrated in soil near the base of soybean plants rather than in open furrows between the rows of plants.     

Plant-facilitated mobilization and translocation of weathered 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE) from an agricultural soil

Field experiments were conducted to determine the uptake and translocation of highly weathered 2,2-bis(p-chlorophenyl)1,1-dichloroethylene (p,p'-DDE) from an agricultural soil. In soil containing known amounts of p,p'-DDE, experimental plots containing zucchini, pumpkin, or spinach were constructed. At destructive harvest, three soil fractions were collected that differed in the degree of influence exerted by the plant roots. The bulk soil was vegetation-free, the near-root zone was within the area encompassed by the roots, and the rhizosphere remained physically attached to the roots at harvest. For each crop, statistically significant decreases were found in the concentration of p,p'-DDE in either the near-root zone or the rhizosphere relative to the bulk soil, suggesting plant-facilitated mobilization and/or degradation of the residue. Plant tissues were analyzed to determine the extent of contaminant removal from the soil and the magnitude of translocation through the shoot system. The concentration of p,p'-DDE in the roots of both zucchini and pumpkin was more than an order of magnitude larger than the bulk soil concentration, followed by significant translocation through the plant tissues to the fruit. The data indicate that certain plants may effectively accumulate residues of persistent organic pollutants in their tissues, suggesting phytoremediation as a possible treatment strategy.     

Toxicological characterization of 2,4,6-trinitrotoluene, its transformation products, and two nitramine explosives

The soil and groundwater of former ordnance plants and their dumping sites have often been highly contaminated with the explosive 2,4,6-trinitrotoluene (2,4,6-TNT) leading to a potential hazard for humans and the environment. Further hazards can arise from metabolites of transformation, by-products of the manufacturing process, or incomplete combustion. This work examines the toxicity of polar nitro compounds relative to their parent compound 2,4,6-TNT using four different ecotoxicological bioassays (algae growth inhibition test, daphnids immobilization test, luminescence inhibition test, and cell growth inhibition test), three genotoxicological assays (umu test, NM2009 test, and SOS Chromotest), and the Ames fluctuation test for detection of mutagenicity. For this study, substances typical for certain steps of degradation/transformation of 2,4,6-TNT were chosen for investigation. This work determines that the parent compounds 2,4,6-TNT and 1,3,5-trinitrobenzene are the most toxic substances followed by 3,5-dinitrophenol, 3,5-dinitroaniline and 4-amino-2-nitrotoluene. Less toxic are the direct degradation products of 2,4,6-TNT like 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2-amino-4,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene. A weak toxic potential was observed for 2,4,6-trinitrobenzoic acid, 2,4-diamino-6-nitrotoluene, 2,4-dinitrotoluene-5-sulfonic acid, and 2,6-diamino-4-nitrotoluene. Octahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine and hexahydro-1,3,5-trinitro-l,3,5-triazine show no hint of acute toxicity. Based on the results of this study, we recommend expanding future monitoring programs of not only the parent substances but also potential metabolites based on conditions at the contaminated sites and to use bioassays as tools for estimating the toxicological potential directly by testing environmental samples. Site-specific protocols should be developed. If hazardous substances are found in relevant concentrations, action should be taken to prevent potential risks for humans and the environment. Analyses can then be used to prioritise reliable estimates of risk.     

Genotoxicity of polar fractions from a herbicide-contaminated soil does not correspond to parent contaminants

Fractionation with biological and chemical analysis has been proposed to characterize the risk associated with complex samples. Thus, we have employed hot pressurized water for fractionating a herbicide-contaminated soil. Genotoxicity was found in polar and midpolarity fractions extracted from a historically herbicide-contaminated soil. However, the parent herbicides as well as organic solvent extracts (consisting of herbicides) were not genotoxic. Thus, polar organics formed by natural degradation of herbicides may be responsible for the genotoxic response. For correct assessment of the toxicity of complex matrices such as soils, both contaminated and uncontaminated samples need to be studied for their toxicological impact.     

Metabolism of14C-heptachlor in goldfish (Carassius auratus)

Goldfish injected with¹⁴C-heptachlor (38.2 g/44 g fish) eliminated about 18% of the dose in 10 days. Analyses of the fish at the end of 10 days revealed the presence of five compounds of which 91.2% was unchanged heptachlor and the remainder was heptachlor epoxide (5.4%), 1-hydroxychlordene (1.0%), 1-hydroxy-2,3-epoxychlordene (1.1%) and a conjugate (1.2%). The conjugate, on acid hydrolysis, yielded a product more polar than dihydroxyheptachlor, most likely a trihydroxy product. Analyses of feces and water showed the presence of only polar products.     

Metal Pollution (Cd,Pb, Zn,and As) in Agricultural Soils and Soybean,Glycine max,in Southern China

Crops produced on metal-polluted agricultural soils may lead to chronic toxicity to humans via the food chain. To assess metal pollution in agricultural soils and soybean in southern China, 30 soybean grain samples and 17 soybean-field soil samples were collected from 17 sites in southern China, and metal concentrations of samples were analyzed by graphite furnace atomic absorption spectrophotometer. The integrated pollution index was used to evaluate if the samples were contaminated by Cd, Pb, Zn and As. Results showed that Cd concentration of 12 samples, Pb concentration of 2 samples, Zn concentration of 2 samples, and As concentrations of 2 samples were above the maximum permissible levels in soils. The integrated pollution index indicated that 11 of 17 soil samples were polluted by metals. Metal concentrations in soybean grain samples ranged from 0.11 to 0.91 mg kg^?1 for Cd; 0.34 to 2.83 mg kg^?1 for Pb; 42 to 88 mg kg^?1 for Zn; and 0.26 to 5.07 mg kg^?1 for As, which means all 30 soybean grain samples were polluted by Pb, Pb/Cd, Cd/Pb/As or Pb/As. Taken together, our study provides evidence that metal pollution is an important concern in agricultural soils and soybeans in southern China.     

Humic Acid Toxicity in Biologically Treated Soil Contaminated with Polycyclic Aromatic Hydrocarbons and Pentachlorophenol

Contaminated soil from a land treatment unit at the Libby Groundwater Superfund Site in Libby, MT, was amended with ¹⁴C pyrene and incubated for 396 days to promote biodegradation and the formation of soil-associated bound residues. Humic and fulvic acids were extracted from the treated soil microcosms and analyzed for the presence of pyrene residues. Biologic activity promoted ¹⁴C association with the fulvic acid fraction, but humic acid–associated ¹⁴C did not increase with biologic activity. The Aboatox flash toxicity assay was used to assess the toxicity of humic and fulvic acid fractions. The fulvic acid gave no toxic response, but the humic acid showed significant toxicity. The observed toxicity was likely associated with pentachlorophenol, a known contaminant of the soil that was removed by solvent extraction of the humic acid and that correlated well with toxicity reduction.     

Exposure assessment of residents living near a wood treatment plant

We report the results of environmental sampling and modeling in a neighborhood adjacent to a wood processing plant. This plant used creosote and pentachlorophenol (PCP) to treat wood for over 70 years. Between 1999 and 2001, environmental samples were obtained to quantify the level of environmental contamination from the wood processing plant. Blood from 10 residents was measured for chlorinated dioxins and dibenzofurans. Soil sediment samples from drainage ditches and attic/dust samples from nearby residents' homes were tested for polychlorinated dioxins, furans, and polycyclic aromatic hydrocarbons (PAH). The dioxin congeners analysis of the 10 residents revealed elevated valued for octachlorodibenzo-p-dioxin and heptachlorodibenzo-p-dioxin compatible with PCP as the source. The levels of carcinogenic PAHs were higher than background levels and were similar to soil contamination on wood preserving sites. Wipe sampling in the kitchens of 11 homes revealed that 20 of the 33 samples were positive for octachlorinated dioxins with a mean value of 10.27 ng/m2. The soil, ditch samples, and positive wipe samples from the homes indicate a possible ongoing route of exposure to the contaminants in the homes of these residents. Modeled air exposure estimated for the wood processing waste chemicals indicate some air exposure to combustion products. The estimated air levels for benzo(a)pyrene and tetrachlorodibenzodiozin in this neighborhood exceeded the recommended levels for these compounds in some states. The quantitative data presented suggest a significant contamination of a neighborhood by wood processing waste chemicals. These findings suggest the need for more stringent regulations on waste discharges from wood treatment plants.     

Diet and carcinogenesis

Carcinogens naturally occurring in food Include metabolites of microorganisms which infect plants during harvesting and storage; products of the normal metabolism of plants and products of chemical changes occurring during processing, preservation, preparation or which are picked up from the environment, including contaminated water or soil. Some examples of these types of carcinogens are: fungal metabolites such as aflatoxins in peanuts stored under improper conditions; safrol from the oil of various plants; tannin in tea, grain and grapes; and polycyclic hydrocarbons including benzo (a) pyrene formed by smoking meat and fish. Epidemiological studies indicate that there is a high incidence of gastric cancer in the areas of the world where smoked fish are common in diets. Vitamin A may play a role in preventing the carcinogenic action of polycyclic hydrocarbons.