Isao Aoyama

Toxicity evaluation of new antifouling compounds using suspension-cultured fish cells.

A simple, rapid toxicity test was developed using the suspension-cultured fish cell line CHSE-sp derived from chinook salmon Oncorhynchus tshawytscha embryos in order to assess the toxicity of new marine antifouling compounds. The compounds tested were copper pyrithione, Diuron, Irgarol 1051, KH101, Sea-Nine 211, and zinc pyrithione, all of which have been nominated in Japan as possible replacements for organotin compounds. The in vitro acute toxicity (24-h EC50) of the six compounds to these fish cells was evaluated using the dye Alamar Blue to determine cell viability, and then correlated with the results of in vivo chronic toxicities (28-day LC50) to juvenile rainbow trout Oncorhynchus mykiss. The suspension-cultured fish cells were found to be suitable for the screening of such chemicals before performing an in vivo test. The toxicities of the test compounds obtained from both tests, shown in decreasing order, were as follows: copper pyrithione > zinc pyrithione > KH101 > or = Sea-Nine 211 > Diuron > Irgarol 1051. The herbicides Diuron and Irgarol 1051 showed the least toxicity, while the pyrithiones had the greatest toxicity.

Antifouling herbicides in the coastal waters of western Japan

Residue analyses of some antifouling herbicides (Diuron, Irgarol 1051 and the latters degradation product M1, which is also known as GS26575), were conducted in waters collected along the coast of western Japan. In total, 142 water samples were collected from fishery harbours (99 sites), marinas (27 sites), and small ports (16 sites) around the Seto Inland Sea, the Kii Peninsula, and Lake Biwa, in August 1999. A urea-based herbicide, Diuron, was positively identified for the first time in Japanese aquatic environments. Diuron was detected in 121 samples (86%) up to a highest concentration of 3.05 microg/l, and was found in 86% of samples from fishery harbours, 89% from marinas, and 75% from ports. Four freshwater samples out of 11 collected at Lake Biwa contained Diuron. Neither Irgarol 1051 nor M1 was found in the lake waters, but both were found in many coastal waters. Irgarol 1051 was found in 84 samples (60%) at a highest concentration of 0.262 microg/l. The concentrations detected were of similar magnitude to those in our previous surveys, taken in 1997 and 1998. M1 was found in 40 samples (28%) up to a highest concentration of 0.080 microg/l. The concentrations detected were generally lower than those found in our previous surveys. The detection frequency among fishery harbours, marinas, and ports was 57-70% for Irgarol 1051 and 25-30% for M1. Ninety-five per cent of the coastal waters in which M1 was detected also contained Irgarol 1051, and 93% of the samples in which Irgarol 1051 was detected also contained Diuron. These results clearly suggest that commercial ship-bottom paints containing both Diuron and Irgarol 1051 are used extensively in the survey area.

Transformation of the new antifouling compound Irgarol 1051 by Phanerochaete chrysosporium

Irgarol 1051, 2-methylthio-4-tert-butylamino-6-cyclopropylamino-s-triazine, is a newly developed herbicidal additive for use in copper-based antifouling paints. It is intended to replace the antifouling agent tributyltin, which has been regulated internationally due to its severe impact on the aquatic ecosystem. However, there is no information in the open literature on the persistence and degradation of Irgarol, a fact that hinders the assessment of its ultimate impact on the environment. This study showed that the white rot fungus Phanerochaete chrysosporium was capable of biotransforming Irgarol 1051. It appears that the metabolism of Irgarol by the fungus proceeds mainly via partial N-dealkylation. Metabolic dealkylation occurs at the cyclopropylamino group resulting in metabolite M1, which has tentatively been identified as 2-methylthio-4-tert-butylamino-6-amino-s-triazine. M1 appeared to be a stable and/or terminal metabolite. No evidence of the heterocyclic ring cleavage of Irgarol 1051 was observed, thus implying a possibility of its degradation product(s) accumulating in the environment.

Algal growth inhibition by river water pollutants in the agricultural area around Lake Biwa, Japan.

An ecotoxicological study of river water discharged from the agricultural area around Lake Biwa was performed by using algal bioassays to guide chemical analysis. Water samples were collected once a week, at least, for 1 year starting in April 1997 and continuing until April 1998. The toxicities of the dissolved and particulate-adsorbed extracts of water samples were evaluated by the algal growth inhibition test and concentrations of individual pesticides were determined. Most of the river water that was collected during the periods when pesticides were applied to the paddy fields caused algal growth inhibition. Some extracts were found to contain herbicides (molinate, mefenacet, simetryn, or esprocarb) as major compounds. According to chemical assay and bioassay, simetryn was identified as the most toxic compound that caused algal growth inhibition.

Chemical stabilization of medical waste fly ash using chelating agent and phosphates: heavy metals and ecotoxicity evaluation.

Chemical stabilization of heavy metals in medical waste fly ash has been carried out using the following compounds: a chelating agent (Ashnite S803), a commercial acidic phosphoric acid solution (Ashnite R303) as well as basic one (Ashnite R201). In order to predict the leachability of heavy metals, Japanese Leaching Test (JLT-13) procedure was applied to the stabilized fly ash products. An ecotoxicity assessment of the stabilized fly ash products leachate and the unstabilized fly ash leachate was conducted using a battery of bioassays based on lettuce root elongation inhibition, Daphnia magna mortality and Vibrio fischeri photoinhibition. The results showed that the three stabilizing agents were able to significantly decrease (ANOVA, P < 0.05) the concentration of heavy metals in the leachates. Although the leachate from both stabilized and unstabilized fly ash were very toxic to lettuce and daphnids, the incorporation of these stabilizing agents diminished significantly (ANOVA, P < 0.05) the toxicity of the leachates towards the three tested organisms. Pearson correlation analysis was used to analyze the strength of the relationship between chemical elements concentration in the leachate and bioassays results. Most of the heavy metals in the leachate were significantly correlated (ANOVA, P < 0.05) with the toxicity values of the bioassays. However, the correlation was not found between the concentration of dissolved organic carbon (DOC) and the toxicity effect of the leachate to the tested organisms.

Application of short-term bioassay guided chemical analysis for water quality of agricultural land run-off

The effect of agricultural land run-off on the water quality of Lake Kojima, Japan, was investigated using a short-term bioassay-guided chemical analysis. Water samples were collected for 1 year starting from June 1995 to June 1996. Toxicity of the dissolved and adsorbed extracts in the water samples was evaluated using the Daphnia immobilization test and the concentrations of pesticides and putative toxic substance in the extracts were determined by high performance liquid chromatography. Most of the dissolved extracts caused immobilization of the test Daphnia magna at low concentrations during the period of paddy pesticide application. Some extracts were found to contain pesticides such as dymron, mefenacet and flutolanil, but their concentrations were too low to have a toxic effect on the daphnia. An unknown toxic compound, Peak C, was isolated from some river water samples, but it produced only a relatively weak toxicity to Daphnia. To better understand the impact of agricultural run-off on a receiving water body, the relationship between the observed toxicity and the concentrations of pesticides and Peak C in the water samples was studied both temporally and spatially.

Ecotoxicity assessment of the aquatic environment around Lake Kojima, Japan

To reduce the impact of chemical substances on the aquatic ecosystem, it is essential to understand their ecotoxicological properties in the natural aquatic environment. Consequently, we conducted an ecotoxicological study on the aquatic environment around Lake Kojima, a man-made lake located in the southwest of Japan. Lake Kojima receives its chemical inputs mainly from two rivers that flow through various agricultural and industrial areas. For ecotoxicity screening, surface water and sediment samples were collected 4 times in 1993 from 16 preselected sites. Then, the solutes in the filtered surface water were concentrated by ODS resin, and the organic chemicals in the suspended solids (SS) and sediments were extracted by acetone. A battery of five ecotoxicity tests (agar plate test using bacteria and yeast, algal growth inhibition test, Daphnia magna immobilization test, and root elongation test using lettuce seeds) was used to assess these extracts. The results show that the surface water extracts had a lethal effect on D. magna, the SS extracts suppressed algal growth, and the sediment extracts were inhibitory to the growth of yeast. A significant inhibitory effect by the sediment extracts from 4 lake sites and 3 river sites was detected by these ecotoxicity tests. Attempts also were made to identify the putative ecotoxic chemicals in the collected samples. Elementary sulfur was identified as one of the major toxicants in the sediment extracts that were inhibitory to the yeast growth. Moreover, samples of surface water around Lake Kojima, collected weekly from June to September in 1994, were found to contain three pesticides and were toxic to D. magna. But the concentration of the pesticides detected was too low to cause daphnia immobilization. It is believed that the toxicity of the water extracts was mainly due to the combined toxic effect of natural and man-made components.

Toxicity of tetramethylammonium hydroxide to aquatic organisms and its synergistic action with potassium iodide

The aquatic ecotoxicity of chemicals involved in the manufacturing process of thin film transistor liquid crystal displays was assessed with a battery of four selected acute toxicity bioassays. We focused on tetramethylammonium hydroxide (TMAH, CAS No. 75-59-2), a widely utilized etchant. The toxicity of TMAH was low when tested in the 72 h-algal growth inhibition test (Pseudokirchneriellia subcapitata, EC50=360 mg L(-1)) and the Microtox® test (Vibrio fischeri, IC50=6.4 g L(-1)). In contrast, the 24h-microcrustacean immobilization and the 96 h-fish mortality tests showed relatively higher toxicity (Daphnia magna, EC50=32 mg L(-1) and Oryzias latipes, LC50=154 mg L(-1)). Isobologram and mixture toxicity index analyses revealed apparent synergism of the mixture of TMAH and potassium iodide when examined with the D. magna immobilization test. The synergistic action was unique to iodide over other halide salts i.e. fluoride, chloride and bromide. Quaternary ammonium ions with longer alkyl chains such as tetraethylammonium and tetrabutylammonium were more toxic than TMAH in the D. magna immobilization test.

Effect-directed investigation and interactive effect of organic toxicants in landfill leachates combining Microtox test with RP-HPLC fractionation and GC/MS analysis.

Landfill leachates contain a large amount of unknown harmful compounds derived from domestic and industrial sources. A toxicity effect-directed approach was used to identify biologically active compounds in three landfill leachate samples (S1–S3) by combining the Microtox test with reverse-phase high performance liquid chromatography (RP-HPLC) fractionation and gas chromatography/mass spectrometry (GC/MS) analysis. Organic toxicants were recovered from coarse fractions only in S1 and in S2. Fine fractionation exhibited a somewhat different toxicity pattern in S1 and S2. GC/MS analysis positively identified Bisphenol A (BPA) and 4-t-butylphenol (4-t-BP) in both samples, N-ethyltoluenesulfoneamide (NETSA) was detected only in S1. However, their concentrations were not high enough to be responsible for the observed toxicity in original samples. A synergistic effect among detected organic compounds (BPA, NETSA, and 4-t-BP) was demonstrated. Each compound present at 1/7 of its individual EC50, might lead to undesirable mixture toxicity, which indicated that interactive effects may, to a certain extent, play a role in landfill leachates with complex matrices. The results from further hydrophobicity analysis and estrogen receptor (ER) competitive binding assays of fraction 13 of both samples gave evidence that some possible toxicants that failed to be identified by GC/MS might be endocrine disrupting chemical(s) (EDC) with a log Kow range of 3.5–3.7 in both samples.

Experimental study on the concentration process of trace element through a food chain from the viewpoint of nutrition ecology

Abstract This paper describes the importance of a food chain to estimate the concentration of heavy metals from the viewpoint of nutrition ecology. The experiments were performed to investigate the effects of the following experimental conditions on the uptake of heavy metals by a predator, using 137 Cs as a tracer; ration size per day, feeding interval and the change in weight of the predator fish. Top minnows ( Oryzias latipes ) were used as preys and Golden astro ( Astronotus ocellatus ) as predators. The preys were raised without feeding in an aquarium and the predators were fed top minnows. The set of results showed that (1) the concentration of a metal in predator fish increased with the ration size, (2) the feeding interval had no effect on the uptake of the metal by fish under the experimental conditions performed, and (3) the concentration in the growing fish was suppressed increasing in value with the increase of its weight. They suggest the importance of generalization of the experimental food conditions when the study on the concentration process of heavy metals through a food chain is performed. The paper also deals with the compartment model applied to the concentration process of the metal by a fish. The theoretical results depicted well the experimental ones.