Evidence of coprostanol estrogenicity to the freshwater mussel Elliptio complanata

Coprostanol (5 beta (H)-cholestan-3 beta ol) is a reduced metabolite of cholesterol produced by micro-organisms found in the intestinal tract of mammals. This substance abounds in urban effluents and is accumulated by organisms living in the vicinity of municipal effluent outfalls. In an earlier study, freshwater mussels exposed to contaminated river water for 62 days accumulated large quantities of coprostanol (Cop) in their soft tissues (16 micrograms/g dry wt.). Moreover, these mussels were found to have elevated levels of vitellin in their hemolymphs, suggesting estrogenic effects. Although municipal wastewaters are known to contain other estrogenic compounds capable of inducing Vn synthesis in mussels, the estrogenic potential of coprostanol was singled out for examination. To this end, mussels were first injected with concentrations of coprostanol via the abductor muscle route, and allowed to stand in aerated water for 72 h at 15 degrees C. The levels of Vn in mussel hemolymph were assayed using the organic alkali-labile phosphate method. A competitive estradiol-binding assay was then devised to measure the ability of coprostanol to compete in the binding of fluorescein-labeled estradiol-albumin to cytosolic proteins. Coprostanol partially reversed the binding of labeled estradiol-albumin to cytosolic proteins with an EC50 of 1 mM. In addition, injections of coprostanol and estradiol-17 beta led to increased levels of vitellins in the hemolymph of treated mussels. Moreover, incubation of cop in gonad homogenate extracts in the presence of NADPH led to the formation of two compounds, as determined by high-performance thin-layer chromatography. One of these compounds appears to be the C17 oxidation product of coprostanol, whose polarity is similar to that of estradiol. The results present evidence that coprostanol is estrogenic to freshwater mussels.     

Preliminary ecotoxicological characterization of a new energetic substance, CL-20

A new energetic substance hexanitrohexaazaisowurtzitane (or CL-20) was tested for its toxicities to various ecological receptors. CL-20 (epsilon-polymorph) was amended to soil or deionized water to construct concentration gradients. Results of Microtox (15-min contact) and 96-h algae growth inhibition tests indicate that CL-20 showed no adverse effects on the bioluminescence of marine bacteria Vibrio fischeri and the cell density of freshwater green algae Selenastrum capricornutum respectively, up to its water solubility (ca. 3.6 mg l(-1)). CL-20 and its possible biotransformation products did not inhibit seed germination and early seedling (16-19 d) growth of alfalfa (Medicago sativa) and perennial ryegrass (Lolium perenne) up to 10,000 mg kg(-1) in a Sassafras sandy loam soil (SSL). Indigenous soil microorganisms in SSL and a garden soil were exposed to CL-20 for one or two weeks before dehydrogenase activity (DHA) or potential nitrification activity (PNA) were assayed. Results indicate that up to 10,000 mg kg(-1) soil of CL-20 had no statistically significant effects on microbial communities measured as DHA or on the ammonium oxidizing bacteria determined as PNA in both soils. Data indicates that CL-20 was not acutely toxic to the species or microbial communities tested and that further studies are required to address the potential long-term environmental impact of CL-20 and its possible degradation products.     

Chronic toxicity of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in soil determined using the earthworm (Eisenia andrei) reproduction test

The sublethal and chronic effects of the environmental contaminant and explosive octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in artificial soil were assessed using the earthworm (Eisenia andrei). Based on various reproduction parameters (total and hatched number of cocoons, number of juveniles and their biomass), fecundity was reduced at the different concentrations of HMX tested (from 280.0 +/- 12.3 to 2502.9 +/- 230.0 mg kg-1 dry soil) in spiked artificial soil (LOEC: 280.0 +/- 12.3 mg kg-1 dry soil). The growth of adult E. andrei was also reduced at the different concentrations tested, though no mortality occurred, even at the highest tested concentrations. The number of juveniles produced was correlated with the number of total and hatched cocoons, and the biomass of juveniles was correlated with the number of cocoons. Pooled results of these and earlier studies on explosives (TNT, RDX) using the E. andrei reproduction test confirm that effects of HMX on cocoon production are indicative of some reproductive consequences (number of juvenile and their biomass), whereas adult growth, in general, does not correlate strongly with change in reproduction capacity.     

Biotransformation of 2,4,6-trinitrotoluene (TNT) by enchytraeids (Enchytraeus albidus) in vivo and in vitro

2,4,6-Trinitrotoluene (TNT) is toxic to soil invertebrates, but little is known about its toxicokinetic behavior in soil. Tissue residue analysis was used to evaluate whether the presence of TNT and its reduced metabolites in soil invertebrates was due to uptake of these compounds from the soil into the organism, or due to microbial transformation of TNT associated with the organism followed by uptake. Adult white potworms (Enchytraeus albidus) were exposed to non-lethal concentrations of TNT in amended artificial soil for 21 d, or to TNT in solution for 20 h. Soil exposure studies confirmed earlier reports that TNT was transformed in enchytraeids in vivo to 2- and 4-aminodinitrotoluenes. However, enchytraeid exposure to TNT in solution led to the additional presence of 2,4-diaminonitrotoluene as well as 2- and 4- hydroxyamino-dinitrotoluenes and azoxy-compounds, suggesting that TNT can be metabolized in vivo in the absence of soil. Incubation of unexposed enchytraeid homogenates with TNT led to a protein-dependent appearance of these metabolites in vitro after > or =16 h incubation. Cellular fractionation studies indicated that most of this activity resided in the 8000 x g pellet, and was completely inhibited by broad-spectrum antibiotics. These studies demonstrate that enchytraeids can transform TNT in vivo and in vitro, at least in part, by bacteria associated with the host organism.     

Nonferrous metal (loid) s mediate bacterial diversity in an abandoned mine tailing impoundment

Environmental Science and Pollution Research - Migration and transformation of toxic metal (loid) s in tailing sites inevitably lead to ecological disturbances and serious threats to the...     

Ecotoxicity characterization of dinitrotoluenes and some of their reduced metabolites

In the present study, the toxic effects of 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT) and a selection of their respective metabolites were examined and compared to 2,4,6-trinitrotoluene (TNT) using the 15-min Microtox (Vibrio fischen) and 96-h freshwater green alga (Selenastrum capricomutum) growth inhibition tests. All of the compounds tested were less toxic than TNT. Using the Microtox assay, 2,6-DNT was more toxic than 2,4-DNT and the order of toxicity for 2,6-DNT and its metabolites was: 2,6-DNT > or = 2A-6NT > 2,6-DAT; whereas that for 2,4-DNT was: 4A-2NT > 2A-4NT > 2,4-DNT > 2,4-DAT. For the algal test, 2,4-DNT was more toxic than 2,6-DNT and the order of toxicity for 2,4-DNT and its metabolites was: 2,4-DNT > 2,4-DAT approximately equal to 4A-2NT = 2A-4NT. The order of toxicity for 2,6-DNT and its reduced metabolites using the algal test was very similar to the Microtox bioassay. These results demonstrate that the reduced metabolites of 2,6-DNT tested in this study were less toxic than that of the parent compound, but certain partially reduced metabolites of 2,4-DNT can be more toxic than the parent molecule. These data put into question the general hypothesis that reductive metabolism of nitro-aromatics is associated with a sequential detoxification process.     

Phytotoxicity of nitroaromatic energetic compounds freshly amended or weathered and aged in sandy loam soil

The toxicities of 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), 2,4-dinitrotoluene (2,4-DNT), and 2,6-dinitrotoluene (2,6-DNT) to terrestrial plants alfalfa (Medicago sativa L.), Japanese millet (Echinochloa crusgalli L.), and perennial ryegrass (Lolium perenne L.) were determined in Sassafras sandy loam soil using seedling emergence, fresh shoot, and dry mass measurement endpoints. A 13-week weathering and aging of energetic materials in soils, which included wetting and drying cycles, and exposure to sunlight of individual soil treatments, was incorporated into the study design to better reflect the soil exposure conditions in the field than toxicity determinations in freshly amended soils. Definitive toxicity tests showed that dinitrotoluenes were more phytotoxic for all plant species in freshly amended treatments based on EC20 values for dry shoot ranging from 3 to 24mgkg(-1) compared with values for TNB or TNT ranging from 43 to 62mgkg(-1). Weathering and aging of energetic materials (EMs) in soil significantly decreased the toxicity of TNT, TNB or 2,6-DNT to Japanese millet or ryegrass based on seedling emergence, but significantly increased the toxicity of all four EMs to all three plant species based on shoot growth. Exposure of the three plant species to relatively low concentrations of the four compounds initially stimulated plant growth before the onset of inhibition at greater concentrations (hormesis).     

Fluctuations of the red tide flagellates Chattonella spp. (Raphidophyceae) and the algicidal bacterium Cytophaga sp. in the Seto Inland Sea, Japan

A marine algicidal gliding bacterium Cytophaga sp. strain J18/M01 was isolated in 1990 from a station in northern Harima-Nada, the Seto Inland Sea, Japan, using the harmful red tide alga Chattonella antiqua (Hada) Ono as a susceptible organism. The bacterium can prey upon various species of microalgae. Temporal fluctuations of this bacterium and Chattonella spp. [C. antiqua and C. marina (Subrahmanyan) Hara et Chihara] were investigated weekly at the above station in the summer of 1997 and 1998, using immunofluorescence assay employing highly specific polyclonal antibodies for the bacterium. In the summer of 1997, the cell density of Chattonella spp. showed a maximum value (70 cells ml⁻¹) on 8 July, and decreased thereafter. The bacterium Cytophaga sp. J18/M01 was commonly detected around a few hundreds of cells per milliliter or less. The number of Cytophaga sp. J18/M01 increased after the peak of Chattonella spp., and the maximum cell number of the bacterium was 1350 ml⁻¹. This algicidal bacterium also followed the changes of total amounts of microalgal biomass (chlorophyll a+pheophytin) when Chattonella spp. were absent. In the summer of 1998, Chattonella spp. were relatively less abundant (maximum 21 cells ml⁻¹), and the algicidal bacterium Cytophaga sp. J18/M01 showed a close relationship with the change of total microalgal biomass. The present study suggests that the algicidal bacterium Cytophaga sp. J18/M01 preyed upon, not only harmful red tide microalgae, but also other common microalgae such as diatoms, and the bacterium presumably plays an important role in regulating microalgal biomass in natural marine environments. Received: 20 April 2000 / Accepted: 1 December 2000     

Assessment of the Application of an Ecotoxicological Procedure to Screen Illicit Toxic Discharges in Domestic Septic Tank Sludge

ABSTRACT

An innovative screening procedure has been developed to detect illicit toxic discharges in domestic septic tank sludge hauled to the Montreal Urban Community waste-water treatment plant. This new means of control is based on an integrative approach, using bioassays and chemical analyses. Conservative criteria are applied to detect abnormal toxicity with great reliability while avoiding false positive results. The complementary data obtained from toxicity tests and chemical analyses support the use of this efficient and easy-to-apply procedure.

This study assesses the control procedure in which 231 samples were analyzed over a 30-month period. Data clearly demonstrate the deterrent power of an efficient control procedure combined with a public awareness campaign among the carriers. In the first 15 months of application, between January 1996 and March 1997, approximately 30% of the 123 samples analyzed showed abnormal toxicity. Between April 1997 and June 1998, that is, after a public hearing presentation of this procedure, this proportion dropped significantly to approximately 9% based on 108 analyzed samples.

The results of a 30-month application of this new control procedure show the superior efficiency of the ecotoxicological approach compared with the previously used chemical control procedure. To be able to apply it effectively and, if necessary, to apply the appropriate coercive measures, ecotoxicological criteria should be included in regulatory guidelines.     

Assessment of the application of an ecotoxicological procedure to screen illicit toxic discharges in domestic septic tank sludge

An innovative screening procedure has been developed to detect illicit toxic discharges in domestic septic tank sludge hauled to the Montreal Urban Community waste-water treatment plant. This new means of control is based on an integrative approach, using bioassays and chemical analyses. Conservative criteria are applied to detect abnormal toxicity with great reliability while avoiding false positive results. The complementary data obtained from toxicity tests and chemical analyses support the use of this efficient and easy-to-apply procedure. This study assesses the control procedure in which 231 samples were analyzed over a 30-month period. Data clearly demonstrate the deterrent power of an efficient control procedure combined with a public awareness campaign among the carriers. In the first 15 months of application, between January 1996 and March 1997, approximately 30% of the 123 samples analyzed showed abnormal toxicity. Between April 1997 and June 1998, that is, after a public hearing presentation of this procedure, this proportion dropped significantly to approximately 9% based on 108 analyzed samples. The results of a 30-month application of this new control procedure show the superior efficiency of the ecotoxicological approach compared with the previously used chemical control procedure. To be able to apply it effectively and, if necessary, to apply the appropriate coercive measures, ecotoxicological criteria should be included in regulatory guidelines.