A larval medaka (Oryzias latipes) acute toxicity assay combined with solid-phase extraction to efficiently determine the toxicity of organic contaminants in river water.

Using a larval medaka (Oryzias latipes) acute toxicity assay combined with solid-phase extraction, we proposed a method for efficiently determining the fish toxicity of organic contaminants in river water. Organic toxicants were 10, 20, 50 and 100-fold concentrated from 4 L of the sample with adsorption cartridges. The lethal effect was observed by exposing every ten individuals of 48-72 h old larval medaka to 20 mL of each solution for 48h. The median lethal concentration rate (LCR50) was used as an indicator for the toxicity. With the developed toxicity test method, more than seven times difference was found in the LCR50 of the river water samples. LCR50 distribution profiles were compared with 125 samples in two typical rivers. The result revealed a lower toxicity level in the mainstream than in the confluences, and a lower toxicity level in Sagami River than in Ayase River. LCR50 proved unique as a toxicity indicator, which was impossible to speculate from the conventional water quality indicator of the dissolved organic carbon concentration. As an effective screening test for priority settings, the method can help us with an efficient planning for the environmental investigation and management.

Embryonic development assay with Daphnia magna: application to toxicity of aniline derivatives.

An assay system using Daphnia magna embryos was applied to investigate the adverse effects of aniline derivatives. The data were compared with our previous data for chlorophenols. This new assay provides useful information to evaluate the toxicity of chemicals and the differences in sensitivity between the life stages. The effects of 15 aniline derivatives on embryonic development of D. magna embryos were determined. At the start of exposure, 2-6-h old eggs (between stages 1 and 2, round in shape, diameter approx. 400 microm), were used. In control and solvent control groups, embryonic development from an egg to a free-swimming animal proceeded completely within 3 days with more than 90% hatchability. Median effective concentrations (EC50s) to reduce the numbers hatched were determined and gross morphological abnormalities of hatched animals recorded. Anilines induced no obvious morphological abnormalities and no developmental delay although premature deaths occurred. However, they affected the number of embryos hatching in a dose-dependent manner. In addition, this embryo assay was more sensitive to aniline derivatives (except for aniline) than acute juveniles immobilization assay. Ratios of 48-h EC50 (juvenile)/3-day EC50 (embryo) for eight anilines were greater than 5.0. Particularly, the ratios of 4-methyl-, 4-ethyl- and 3-methylaniline were 77, 23 and 11, respectively. EC50s for embryos and juveniles were poorly correlated (r = 0.41). This indicated that the sensitivities of the two life stages were different to the effects of anilines. EC50s were poorly correlated (r = -0.097) with the log Kow (1-octanol/water partition coefficient). These results were compared with previous results for phenols.

Synthesis of fluorine analogs of protoporphyrin potentially useful for diagnosis and therapy of cancer. IV. Synthesis of (trifluorovinyl)vinyl- and (1-chloro-2,2-difluorovinyl)vinyldeuteroporphyrins.

Trifluoro or chlorodifluoro analogs of protoporphyrin, the compounds in the title, were synthesized for use in the diagnosis and therapy of cancer. 3- Or 8-acetyldeuteroporphyrin dimethyl esters (2 and 3) were iodinated with iodine in the presence of potassium carbonate to the corresponding iodo compounds (5 and 6). The iodo compounds (5 and 6) were treated with bis(trifluorovinyl)zinc in the presence of tetrakis(triphenylphosphine)-palladium to give trifluorovinyl derivatives (7 and 8) in good yields. Reduction of the acetyl group of 7 and 8 with sodium borohydride afforded the corresponding hydroxyethyl derivatives (9 and 10). Compounds (9 and 10) were dehydrated with methanesulfonyl chloride and triethylamine to give (trifluorovinyl)vinyldeuteroporphyrin dimethyl esters (11 and 12). Treatment of 5 and 6 with bis(1-chloro-2,2-difluorovinyl)zinc in the presence of tetrakis(triphenylphosphine)palladium, followed by similar reactions as above gave (1-chloro-2,2-difluorovinyl)-vinyldeuteroporphyrin dimethyl esters (17 and 18).

Metabolism of 14C-labelled sucrose esters of stearic acid in rats.

Rats were dosed by oral gavage (250 mg/kg) with compounds containing sucrose esterified in four, six or eight positions with stearic acid. For each compound, rats excreted greater than 95% of the dose in the faeces. The extent of absorption and metabolism of radioactivity was inversely related to the degree of esterification. For rats dosed with sucrose esters labelled in the fatty acid moieties, the degree of absorption of radioactivity was highest for the tetraesterified compound (5.9% of the dose). At 120 hr after dosing with this derivative, the highest concentrations of radioactivity, aside from tissues of the gastrointestinal tract, were found in fat (183 micrograms-equivalents/g tissue), lymph nodes (117 micrograms-equivalents/g tissue) and the liver (88 micrograms-equivalents/g tissue); appreciable radioactivity appeared in the blood (3.9 micrograms-equivalents/g tissue) and collected lymph (5.0-7.6% of the dose). For rats dosed with esters labelled in the sucrose moiety, the amounts of radioactivity absorbed were lower than after dosing with the corresponding sucrose derivatives labelled in the fatty acid moieties; the absorbed radioactivity was greatest following administration of the tetraesterified compound (3.0%). Relatively little radioactivity was found in tissue samples collected from these rats. These results are consistent with limited hydrolysis of the sucrose esters, presumably to sucrose and fatty acids, prior to intestinal absorption.

Cytotoxicity of chlorophenols to goldfish GFS cells with the MTT and LDH assays.

The cytotoxicity of phenol and 14 chlorophenols (CPs) were examined in goldfish scale (GFS) cells, a fibroblastic cell line derived from goldfish scales. MTT reduction and intracellular LDH activities were examined as cytotoxicity indicators. Cell densities at inoculation were 1.0 x 10(5) cells/ml in the MTT assay and 0.5 x 10(5) or 0.1 x 10(5) cells/ml in the LDH assay. No change in the midpoint cytotoxicity concentration (IC(50)) was observed in either assay at the same order of cell density (0.5-1.0 x 10(5) cells/ml). However, at low chemical concentrations, the LDH assay was more sensitive than the MTT assay. In addition, for many CPs, the LDH assay at 0.1 x 10(5) cells/ml was more sensitive than the MTT and LDH assays at the higher cell density levels. The correlation coefficients (r) for all IC(50) determinations were greater than 0.97. Good correlations between these assays and the neutral red (NR) assay were also observed (r > 0.97). The 1-octanol/water partition coefficient (log P) gave good correlation coefficients (r > 0.90) with IC(50) values of both the MTT and LDH assays in simple linear regression analyses. The study demonstrates that both the MTT and LDH cytotoxicity assays with GFS cells, together with the NR assay, can be applied to screen the acute toxicities of chemicals.

Mass propagation of shoots of Stevia rebaudiana using a large scale bioreactor.

A procedure for the mass propagation of multiple shoots of Stevia rebaudiana is described. Isolated shoot primordia were used as the inoculum to obtain clusters of shoot primordia. Such clusters were grown in a 500 liter bioreactor to obtain shoots. A total of 64.6 Kg of shoots were propagated from 460 g of the inoculated shoot primordia. These shoots were easily acclimatized in soil.

Immunological properties of the primer-independent glucosyltransferase of Streptococcus mutans serotypes d and g.

Streptococcus mutans serotype g secretes at least three kinds of glucosyltransferase with different enzymological and immunological properties. One of them is a primer-independent enzyme and seems to be the source of primer for the others, both of which are primer-dependent enzymes. Recently, we purified the primer-independent enzyme, the third glucosyltransferase in this group from S. mutans strain AHT-k serotype g. In the present study, we examined the specificity of the antiserum against the primer-independent glucosyltransferase using extracellular culture-conditioned fluids of many strains of the various serotypes of S. mutans. The antiserum cross-reacted with the extracellular culture fluids from strains of serotypes d and a, in addition to serotype g, but not with those of other serotypes, indicating that the primer-independent glucosyltransferase is secreted by the S. sobrinus and S. cricetus, but not by S. mutans and S. rattus. The antiserum did not completely inhibit the activity of the enzyme, even at more than twofold antibody excess, determined by indirect precipitation with immobilized staphylococcal protein A.

Studies on the metabolic fate of sucrose esters in rats.

The metabolism in rats of sucrose esters of stearic acid and palmitic acid was studied in vivo and in vitro using esters labelled with 14C at the sucrose of fatty-acid moiety. In excretion studies, the ratio of expired radioactivity to absorbed radioactivity after oral administration of the sucrose esters labelled at the sucrose moiety was similar to that after the administration of [14C]sucrose. A similar correlation between the ester labelled at the fatty-acid moiety and the free [14C]fatty acid was also observed. No intact sucrose ester was detected in the urine. Studies in vitro using everted intestinal sacs showed that there was virtually no transport of 14C-labelled sucrose esters from the mucosal to the serosal solution through the intestinal tissues, and that the enzymes in the intestinal mucosa played a more important role in the hydrolysis of sucrose esters than did those in the digestive fluid. In studies of intestinal absorption through the mesenteric lymphatic system, during the 24 hr after ingestion 1.8% of the administered radioactivity was recovered in the lymph after dosing with [U-14C]sucrose monostearate whereas 20% was recovered in the lymph after dosing with sucrose [1-14C]monostearate. This difference in levels of recovery of administered radioactivity indicated that sucrose monostearate was absorbed only after hydrolysis. No intact ester was detected in the lymph or in the portal or femoral blood. The results of all of these experiments show that the sucrose esters are hydrolysed to sucrose and fatty acids prior to intestinal absorption.

Relationship between molar refraction and n-octanol/water partition coefficient.

The n-octanol/water partition coefficient (Pow) for organic chemicals which do not form the hydrogen bond was considered on the basis of London's dispersion force, because the dispersion force is the main component of van der Waals-type attractive forces between solute and solvent molecules. The dispersion force is a function of both the ionization potential and the molar refraction. As the variation of the ionization potential is small in comparison with that of the molar refraction, the latter is the most important factor to determine the value of Pow. Thus the relationship between Pow and the molar refraction can be established. The relationship was confirmed for some halogenated alkanes, alkenes and aromatics, and polycyclic aromatic hydrocarbons by analysis of observed values of Pow and calculated values of the molar refraction. Estimation of Pow from the molar refraction is advantageous for the preliminary hazard assessment of new chemicals, because direct measurement of Pow is laborious and time consuming for lack of information about analytical methods.

Non-steady-state equilibrium model for the preliminary prediction of the fate of chemicals in the environment.

Prediction of the fate of chemicals in the environment is essential to the assessment of the potential hazard of chemicals. The fate of a chemical in the environment depends mainly upon (1) transfer processes between environmental compartments due to physicochemical properties, (2) transformation processes in each compartment, and (3) characteristics of the environment. Many equilibrium or kinetic models have been proposed to predict the fate of chemicals in the environment. Though kinetic models are useful in predicting the concentration-time profile of a chemical at the non-steady state, they are rather complicated. In this work is proposed a simple and useful equilibrium model for prediction of the mass and concentration distribution fraction, mean residence time, and concentration-time profile of a chemical released into the environment, an advantage of our model. For the validation of this model, the results were compared with data from Neely's pond experiment and field data monitored by the Japanese Environment Agency. The results show that the equilibrium model is valuable for preliminary prediction of the fate of chemicals that are priority chemicals for hazard assessment in the environment.