Time-course analysis of pulmonary inflammation induced by intratracheal instillation of nanosized crystalline silica particles in F344 rats.

Crystalline silica is an important cause of serious pulmonary diseases, and its toxic potential is known to be associated with its surface electrical properties. However, in vivo data clarifying the relevance of silica's toxic potential, especially its long-term effects, remain insufficient. To investigate the contribution of physico-chemical property including surface potential on the hazard of nanocrystalline silica, we performed single intratracheal instillation testing using five different crystalline silicas in a rat model and assessed time-course changes in pulmonary inflammation, lung burden, and thoracic lymph node loads. Silica-nanoparticles were prepared from two commercial products (Min-U-Sil5 [MS5] and SIO07PB [SPB]) using three different pretreatments: centrifugation (C), grinding (G), and surface dissolving (D). The five types of silica particles-MS5, MS5_C, SPB_C, SPB_G, and SPB_D-were intratracheally instilled into male F344 rats at doses of 0 mg/kg (purified water), 0.22 mg/kg (SPB), and 0.67, 2, or 6 mg/kg (MS5). Bronchoalveolar lavage, a lung burden analysis, and histopathological examination were performed at 3, 28, and 91 days after instillation. Granuloma formation was present in MS5 group at 91 days after instillation, although granuloma formation was suppressed in MS5_C group, which had a smaller particle size. SPB_C induced severe and progressive inflammation and kinetic lung overload, whereas SPB_G and SPB_D induced only slight and transient acute inflammation. Our results support that in vivo toxic potential of nanosilica by intratracheal instillation may involve with surface electrical properties leading to prolonged effect and may not be dependent not only on surface properties but also on other physico-chemical properties.

Effects of adsorbent carriers in modified ready biodegradability tests of quaternary ammonium salts.

Standard ready biodegradability tests are conducted at unrealistically high test concentration and therefore cannot properly evaluate toxic substances to microorganisms. The present study evaluated the effects of four adsorbent carriers in modified ready biodegradability tests of four quaternary ammonium salts (QASs) demonstrating microbial toxicity according to the Organization for Economic Co-operation and Development Test Guideline 301F and cautions for using carriers were found. In the tests with silica gel, the concentration of all QASs in the test solutions decreased due to adsorption. The percentages of biodegradation of octadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, and benzyldimethyloctadecylammonium chloride, which have a linear alkyl chain, were 89.9%, 80.6%, and 70.1% on day 28, respectively; benzethonium chloride, with a branched alkyl chain, did not undergo biodegradation. In the tests with activated carbon, although the concentrations of QASs greatly decreased, no QASs underwent biodegradation. In the tests with sea sand or quartz sand, QASs were not adsorbed on the carriers and were not biodegraded. Using an adsorbent carrier for toxic substances will increase biodegradation, but it has no effect on highly persistent substances with specific chemical structures, e.g., branched carbons, which is very important because ready biodegradability is not overestimated. Carriers having moderate adsorbability should be selected.

Effects of dose volume and delivery device on bronchoalveolar lavage parameters of intratracheally administered nano-sized TiO2 in rats.

The intratracheal (IT) test is useful for screening the pulmonary toxicity of inhaled materials, including nanomaterials. However, a standard procedure has not yet been authorized internationally, and the effects of different test parameters are unknown. To determine appropriate experimental conditions for the IT test, we intratracheally administered nano-sized TiO2 to male F344 rats at 3.0 mg/kg body weight by using two delivery devices (gavage needle or microaerosolizer) and dose volumes of 0.5-3.0 mL/kg (gavage needle) or 0.5-2.0 mL/kg (microaerosolizer). We evaluated the pulmonary deposition and interlobar distribution of TiO2 at both 30 min and 3 days after administration. In addition, the inflammatory components in bronchoalveolar lavage (BAL) fluid were measured 3 days after administration of TiO2. At dose volumes of 0.5-2.0 mL/kg, the BAL values were comparable regardless of the device used. In addition, pulmonary TiO2 burden and lobar concentration patterns were equivalent at all combinations of dose volume and delivery device. In conclusion, the acute pulmonary toxicity of nanomaterials can be assessed effectively by using an IT test in which the test agent is provided to rats at a dose volume of 0.5-2.0 mL/kg with either a gavage needle or microaerosolizer.

Effect of test concentration in the ready biodegradability test for chemical substances: Improvement of OECD test guideline 301C.

In Japan, understanding the environmental persistence of chemicals is very important for risk assessment, and ready biodegradability tests are mainly conducted according to the Organisation for Economic Co-operation and Development test guideline 301C. However, the highest test concentration specified in test guideline 301C, 100 mg/L, may cause microbial toxicity and incomplete biodegradation. The authors performed test guideline 301C tests at test concentrations of 30 mg/L for 13 substances that were readily biodegradable in ready biodegradability tests but not in test guideline 301C tests. Of the 5 substances with potential to cause microbial toxicity at 100 mg/L, the percentage of biodegradation of sodium dimethyldithiocarbamate, 4-chloro-3-cresol (CC), thymol (THY), and p-tert-butyl-α-methylbenzenepropionaldehyde measured by biochemical oxygen demand (BOD) increased in the test guideline 301C test at 30 mg/L, suggesting a reduction in toxicity effects. Furthermore, CC and THY met the criteria for ready biodegradability, which are more than 60% of biodegradation by BOD and a 10-d window. Of the 8 substances with a low potential for causing microbial toxicity at 100 mg/L, the percentage of biodegradation of only 2-(diethylamino)ethanol increased in the test guideline 301C test at 30 mg/L. Employing a lower test concentration in the standard test guideline 301C test will contribute to improvement of consistency between results of a test guideline 301C test and other ready biodegradability tests.

Comparison of biodegradation performance of OECD test guideline 301C with that of other ready biodegradability tests.

The Organisation for Economic Co-operatoin and development (OECD) Guidelines for the Testing of Chemicals list 7 types of tests for determining the ready biodegradability of chemical compounds (301A-F and 310). The present study compares the biodegradation performance of test guideline 301C, which is applied in Japan's Chemical Substances Control Law, with the performance of the other 6 ready biodegradability tests (RBTs) listed in the guidelines. Test guideline 301C specifies use of activated sludge precultured with synthetic sewage containing glucose and peptone (301C sludge) as a test inoculum; in the other RBTs, however, activated sludge from wastewater treatment plants (WWTP sludge) is frequently employed. Analysis based on percentage of biodegradation and pass levels revealed that the biodegradation intensity of test guideline 301C is relatively weak compared with the intensities of RBTs using WWTP sludge, and the following chemical compounds are probably not biodegraded under test guideline 301C conditions: phosphorus compounds; secondary, tertiary, and quaternary amines; and branched quaternary carbon compounds. The relatively weak biodegradation intensity of test guideline 301C may be related to the markedly different activities of the 301C and WWTP sludges. These findings will be valuable for evaluating RBT data in relation to Japan's Chemical Substances Control Law.

Real-time PCR for rapidly detecting aniline-degrading bacteria in activated sludge.

We developed a detection method that uses quantitative real-time PCR (qPCR) and the TaqMan system to easily and rapidly assess the population of aniline-degrading bacteria in activated sludge prior to conducting a biodegradability test on a chemical compound. A primer and probe set for qPCR was designed by a multiple alignment of conserved amino acid sequences encoding the large (α) subunit of aniline dioxygenase. PCR amplification tests showed that the designed primer and probe set targeted aniline-degrading strains such as Acidovorax sp., Gordonia sp., Rhodococcus sp., and Pseudomonas putida, thereby suggesting that the developed method can detect a wide variety of aniline-degrading bacteria. There was a strong correlation between the relative copy number of the α-aniline dioxygenase gene in activated sludge obtained with the developed qPCR method and the number of aniline-degrading bacteria measured by the Most Probable Number method, which is the conventional method, and a good correlation with the lag time of the BOD curve for aniline degradation produced by the biodegradability test in activated sludge samples collected from eight different wastewater treatment plants in Japan. The developed method will be valuable for the rapid and accurate evaluation of the activity of inocula prior to conducting a ready biodegradability test.