Enhancing bioremoval of textile dyes by eight fungal strains from media supplemented with gelatine wastes and sucrose

Eight Aspergillus strains were found to be successful in removing textile dyes from liquid media. These fungal strains were grown on medium containing: gelatine wastes and sucrose, as sources of nitrogen and carbon to test the possible speed up of the dyes removing while fungus biomass is building up in the media. The growth of fungal strains ranged from 10 to 110 mg biomass dry weight/100 ml medium. This growth induced high decolorization percentages, which ranged 33-95% within eight days. Two textile dyes Direct brown and Polar red were included in the study. The growth of the fungal strains as well as decolorization percentage of the dyes increased after 5, 6, and 8 days from incubation time with most tested strains. With Direct brown dye the strains number 2, 5, 31 and 37 recorded the highest percentage of decolorization (91, 92, 93 and 95 respectively) after incubation for 6 days. Fungal strains Aspergillus 5 and 31 gave the highest mycelium dry weight being 110 mg. Most of fungal strains induced 86 to 95 percentage of decolorization after 6 days incubation with Polar red dye. The possible toxicity of the remaining supernatant media after fungal biomass removal was tested by Ames test to assess the residual mutagenic agents remaining after dye removal, using three strains of Salmonella typhimurium (TA 1535, TA 1537, TA 1538). The results showed that the toxicity of the dyes, measured by Ames test could be removed by the dye absorption on the fungal biomass.     

Evaluation of laser dye mutagenicity using the ames/salmonella microsome test

Twenty-five laser dyes and four analogs were tested for mutagenicity in the Ames/Salmonella test. Seven dyes and two analogs gave positive mutagenic responses with bacterial strains TA1538 and TA98. Of two widely used families of laser dyes (coumarins and rhodamines), four coumarin samples, but none of the rhodamine samples, were mutagenic. All mutagenic compounds require enzyme activation for positive response except two terphenyl analogs, which are mutagenic with or without activation. Using high-performance liquid chromatography (HPLC), it was determined that five mutagenic dye samples had multiple components. The dyes themselves may not be the mutagenic agents in all cases (as with Nile Blue) but may contain impurities that are mutagenic. One dye, adicyanome-thylene (DCM) (≥95% pure), was mutagenic at doses below 0.5 μg/plate on strains TA1538 and TA98. DCM also induced reversions in strains TA96, TA97, TA100, TA102, and TA104, although less efficiently. This study indicates the need for further toxicological testing of these types of compounds. The mutagenic components of these dye mixtures, whether it is the dye or a contaminant, presents a possible hazard to those handling them. Therefore, practices and procedures for the safe handling of specific dyes should be reviewed in light of these findings.     

A promising Ames battery for mutagenicity characterization of new dyes

When testing new products, potential new products, or their impurities for genotoxicity in the Ames test, the quantity available for testing can be a limiting factor. This is the case for a dye repository of around 98,000 substances the Max Weaver Dye Library (MWDL). Mutagenicity data on dyes in the literature, although vast, in several cases is not reliable, compromising the performance of the in silico models. In this report, we propose a strategy for the generation of high‐quality mutagenicity data for dyes using a minimum amount of sample. We evaluated 15 dyes from different chemical classes selected from 150 representative dyes of the MWDL. The purity and molecular confirmation of each dye were determined, and the microplate agar protocol (MPA) was used. Dyes were tested at the limit of solubility in single and concentration‐response experiments using seven strains without and with metabolic activation except for anthraquinone dyes which were tested with eight strains. Six dyes were mutagenic. The most sensitive was YG1041, followed by TA97a > TA98 > TA100 = TA1538 > TA102. YG7108 as well as TA1537 did not detect any mutagenic response. We concluded that the MPA was successful in identifying the mutagenicity of dyes using less than 12.5 mg of sample. We propose that dyes should be tested in a tiered approach using YG1041 followed by TA97a, TA98, and TA100 in concentration‐response experiments. This work provides additional information on the dye mutagenicity database available in the literature.     

Combining different assays and chemical analysis to characterize the genotoxicity of waters impacted by textile discharges

Waters receiving textile discharges can exhibit genotoxic and mutagenic activity, which has been related to the presence of dyes and aromatic amines as synthesis precursors or byproducts. The aim of this study was to identify dyes and aromatic amines in water samples impacted by textile discharges, and to evaluate the genotoxic responses of these samples using the Salmonella/microsome assay in strains TA98 and YG1041, and the Fpg‐modified comet assay in the RTL‐W1 fish cell line. The genotoxicity of river samples downstream of the discharge was greater than the upstream samples in both of the Ames tests. The Fpg‐modified comet assay detected similar levels of DNA damage in the upstream and downstream samples. Mutagenicity was not detected with TA98, except for the Quilombo River samples, but when YG1041 was used as the tester strain mutagenicity was detected for all sites with a very different profile in upstream sites relative to the other sites. The mutagenic response strongly indicated that aromatic amines or dyes were contributing to the mutagenic activity downstream. The impact of textile discharges was also confirmed by chemical analysis, because the highest concentrations of azo dyes and aromatic amines were detected in the river downstream. This study shows the value of combining assays measuring complementary endpoints to better characterize the mutagenicity of environmental samples, with the advantage that this approach provides an indication of what classes of compounds are responsible for the effect. Environ. Mol. Mutagen. 57:559–571, 2016. © 2016 Wiley Periodicals, Inc.     

Reduction in the mutagenicity of synthetic dyes by successive treatment with activated sludge and the ligninolytic fungus, Irpex lacteus

Synthetic dyes are released in wastewater from textile manufacturing plants, and many of these dyes are genotoxic. In the present study, the mutagenicity of azo, anthraquinone, and triphenyl methane dyes was investigated before and after successive biodegradation with activated sludge and the ligninolytic fungus, Irpex lacteus. Two biodegradation systems were used to reduce the genotoxicity of dyes that were not efficiently inactivated by activated sludge alone. Mutagenicity was monitored with the Salmonella reversion assay conducted with the base-pair substitution detector strains, TA100 and YG1042, and the frame-shift detector strains, TA98 and YG1041, with and without rat liver S9. All dyes except for Congo Red (CR) were mutagenic with S9 activation. Assays conducted with the dyes indicated that only the azo dye Reactive Orange 16 (RO16) was mutagenic in both TA98 and TA100. Methyl Red and Disperse Blue 3 (DB3) were mutagenic in TA98, YG1041 and YG1042, while Reactive Black 5 was mutagenic in YG1041 and YG1042. Remazol Brilliant Blue R (RBBR), Crystal violet (CV) and Bromophenol Blue (BPB) were mutagenic only in TA98, but the toxicity of the latter two dyes complicated the evaluation of their mutagenicity. CR was not mutagenic in any of the tester strains. Biodegradation studies conducted with RO16 and DB3 indicated that the two-step biodegradation process reduced the mutagenic potential of RO16 and DB3 to a greater extent than activated sludge alone; the mutagenicity of the two dyes was reduced by 95.2% and 77.8%, respectively, by the two-step process. These data indicate that the combined biodegradation process may be useful for reducing the mutagenicity associated with wastewater from textile factories that contain recalcitrant dyes.     

Lack of genotoxicity of silver iodide in the SCE assay in vitro, in vivo, and in the Ames/microsome test

Silver iodide was evaluated for mutagenicity in the Ames/microsome test (strains TA 1535, TA 102, TA 97, and TA 98) and for the ability to induce Sister Chromatid Exchanges (SCE) in human cultured lymphocytes and in P388 lymphocytic leukemia cells cultured in the mouse peritoneal cavity. From the cytogenetic in vitro studies, it was observed that silver iodide, either in acetone solutions or as a suspension with polyacrilamide, scarcely causes a doubling effect on SCEs at nearly toxic concentrations (1 microg/ml). Such a doubling effect by silver iodide on SCEs in P388 leukemia cells in vivo was not achieved even after using 100 microg/g mouse body weight. In the Ames/microsome test actually a doubling effect on revertants was only isolately achieved with 30 microg/ml in TA 102 (S9-) and at 150 microg/ml in TA 97 (S9+) doses, which appear to be nearly toxic for bacteria.     

In vitro genotoxicity of dyes present in colored smoke munitions

Genetic toxicology studies were conducted on organic dyes and mixtures used in colored smoke munitions. The dyes studied included Solvent Red 1; two different batches (Lot 1 and Lot 2) of Disperse Red 11; terephthalic acid; and a mixture of 25 parts Solvent Red 1, 5 parts Disperse Red 11, and 16 parts terephthalic acid. The dyes were evaluated for their ability to produce mutations in Salmonella bacterial strains and in Chinese hamster ovary (CHO) cells. The dyes were also tested in CHO cells to determine cytotoxicity and the induction of sister chromatid exchanges and chromosome aberration. None of the dyes were genotoxic in the standard Ames assay using bacterial strain TA1535 or TA100 with or without the addition of S-9 or in TA98 and TA1538 without S-9. With S-9, Disperse Red 11 (Lot 2) showed significant mutagenic activity in TA98 and TA1538 which increased as a function of S-9 concentration. However, the maximum level of mutagenic activity detected was low (3.8 revertants/micrograms). The azo dye Solvent Red 1 was also negative in a pre-incubation assay designed to reduce azo compounds to free amines. Solvent Red 1 was cytotoxic to mammalian cells, caused a significant increase in SCE, but was not mutagenic or clastogenic. Disperse Red 11 (Lot 1 and Lot 2) were not cytotoxic or clastogenic but produced an increase in cell cycle time and SCE frequency. Only Disperse Red 11 (Lot 2) increased mutations in the CHO/hypoxanthine-guanine phosphoribosyltransferase (HGPRT) assay. The mutagenic activity of the dye mixture was not significant, suggesting no synergistic interaction between the dyes. These studies demonstrated that none of the dyes was clastogenic and that a contaminant in Disperse Red 11 (Lot 2) may be responsible for the weak mutagenic activity in both mammalian and bacterial cell systems.     

Mutagenicity tests of fabric-finishing agents in salmonella typhimurium: Fiber-reactive wool dyes and cotton flame retardants

Thirty-nine fabric-finishing agents were tested for mutagenic activity in Salmonella typhimurium. Twenty-four fiber-reactive wool dyes and three acid dyes (not fiber-reactive) were screened by spot tests in strains TA100, TA98, TA1535, and TA1537. Among these dyes, seven bromoacrylamide dyes and one vinyl sulfone dye were mutagenic. Additionally, one of the three acid dyes was mutagenic in spot tests. The mutagenicity of the acid dye was due to an impurity or breakdown product rather than to the dye itself; the origin of the activities of the other dyes is unknown. No mutagenicity was observed among five chlorotriazine or four sulfonyl-ethane sulfonic acid dyes. Eight phosphorus-containing flame retardants (phosphonium, phosphine, phosphine oxide, and phosphonic acid derivatives) and methyl-N-methylolcarbamate, which is employed to obtain a flame-retardant finish on cotton, were tested for mutagenicity in strains TA100, TA98, TA1535, and TA1537, using quantitative incorporation assays. All were nonmutagenic. Two of three bromoalkyl-substituted triazine flame retardants were mutagenic in strains TA100 and TA1535. It is unknown whether this activity is due to impurities or to the parent compound. The flame retardants tested were either in actual commercial use or in experimental development for potential commercial processes. These results indicate the need for early testing of potential fabric-finishing agents and processes.     

Comparison of the results of a modified miniscreen and the standard bacterial reverse mutation assays

The bacterial reverse mutation assay (Ames test) provides a rapid assessment of the mutagenic potential of chemicals. The assay is widely used in the pharmaceutical industry for early assessment during candidate compound selection and for regulatory drug submissions. Early in development, many candidate compounds are available in only very small quantities. The use of the standard plate incorporation bacterial reverse mutation assay for screening, using only a single petri plate per concentration, requires the use of approximately 140 mg of test compound to test up to a stock concentration of 100 mg/ml (5000 microg/plate) in five strains of bacteria. A modification of the existing Ames Miniscreen assay has been developed using six-well cell-culture dishes that requires only 21 mg of compound to test a stock concentration of up to 100 mg/ml (2000 microg/well) in three strains of bacteria. The standard plate incorporation assay and the modified Miniscreen assays conducted on proprietary compounds without and with metabolic activation have yielded a high degree of concordance in findings.     

四种医用高分子材料的Ames试验研究

本文报道用Ａｍｅｓ法检测４种医用高分子材料的致突变性。结果表明，经ＴＡ９７，ＴＡ９８，ＴＡ１００和ＴＡ１０２菌株测试的复合膜，胶原膜，铬复合缝线和复合缝线各剂量在点试验和平皿掺入试验中未诱发阳性反应。但是４个菌株所对应的突变剂均诱发了阳性突变。因此４种医用高分子材料在本实验条件下无诱发突变作用。     

Comparison of genotoxicity of textile dyestuffs in Salmonella mutagenicity assay, in vitro micronucleus assay, and single cell gel/comet assay.

The mutagenicity of textile dyes is an important consideration for the assurance of consumer protection and work safety. The mutagenicity testing of textile dyestuffs is crucial for accurately predicting health risks for consumers and workers exposed to dyes. Unfortunately, these data are often lacking. We studied the genotoxic activity of ten selected commercial textile dyestuffs, which are made up of mixtures of azo dyes and azo metal complex dyes as well as two anthraquinone dyestuffs. We used the Salmonella mutagenicity assay and cultured human keratinocytes (HaCaT cell line). In the S. typhimurium strain TA98, with and without S9, eight often dyestuffs investigated, and in strain TA 100, with and without S9, six often dyes caused frameshift mutations and base-pair substitutions in the dose range of 1-5000 microg/plate in a dose-related manner. All dyes, including those negative in the Salmonella mutagenicity assay, induced clastogenic effects in the in vitro micronucleus (MN) test in HaCaT cells as direct-acting mutagens in the concentration range of 5-150 microg/mL and with maximum MN frequencies between 1.1 and 7.2%, compared to negative controls that showed 0.2-0.4% MN cells. In the single cell gel/comet assay, all ten dyestuffs investigated caused DNA damage in HaCaT keratinocytes. The alkaline (pH >13) version used is capable of detecting DNA single strand breaks, alkali-labile sites, and DNA-DNA/DNA-protein cross-linking. Under the conditions of these screening tests, the textile dyes investigated are direct-acting genotoxic substances. The HaCaT cells testing protocol proposed has been shown to be an appropriate test system for evaluating mutagenicity of textile dyes on a base level.     

Comparative mutagenicity of nine brands of coffee to Salmonella typhimurium TA100, TA102, and TA104

Nine coffee preparations, four caffeinated instant brands, three caffeinated drip coffees, and two decaffeinated coffees, one of which was an instant brand, were evaluated for mutagenicity by the Ames assay using Salmonella typhimurium TA100, TA102, and TA104. All the coffees contained direct-acting mutagens, which reverted the three strains. The inclusion of a rat microsomal enzyme preparation reduced the mutagenic response of the three strains in the presence of some of the coffee samples. Both glyoxal and methylglyoxal, 1,2-dicarbonyls found in the coffees were mutagenic. The concentration of glyoxal, methyglyoxal, diacetyl, and guiacol were measured by gas chromatography/mass spectrometry. Caffeine, furfural, and 5-methylfurfural concentrations were determined by high performance liquid chromatography. Although lower concentrations of methyglyoxal were found in the drip caffeinated coffees, the mutagenic potency of these preparations was higher than the instant coffees on a weight basis especially when TA104 was the indicator organism. Our findings agree with those of other workers who have shown that carbonyl compounds, which were present in all the brands tested, are partially responsible for the mutagenic response of coffee but that additional mutagens are also present.     

The mutagenic and SOS-inducing potential of the soluble organic fraction collected from diesel particulate emissions

Studies involving the Ames Salmonella mutagenicity test and the Bacillus subtilis comptest have demonstrated that the soluble organic fraction of diesel particulate is potentially mutagenic and DNA damaging. The soluble organic fraction was extracted from exhaust particulate samples collected from four different diesel engines operated at specified conditions. For each fraction collected, an increase in the concentration of the organic material resulted in a subsequent increase in the number of histidine prototrophs obtained when this material was added to the histidine auxotrophic strains that comprise the Ames Salmonella test. Specifically, the number of induced revertants, for strains TA98 and TA100, ranged from less than one revertant per microgram of sample to 29 revertants per microgram of sample. The ability of these organic fractions to induce bacterial SOS functions also was determined by exposing competent cultures of Bacillus subtilis strain RUB827 to increasing concentrations of these extracts. With varying efficiencies, these samples were positive in their ability to induce the SOS system of B subtilis. Significantly, the toxicity of these mutagenic and DNA damaging samples never resulted in more than 95% killing, even for the highest concentrations tested in the Salmonella and B subtilis assay.     

Use of rat/hamster S-9 mixture in the Ames mutagenicity assay

Based on the findings of Nagao et al [1978] that phenacetin is negative in the standard Ames test with Aroclor induced rat S-9 and positive with hamster S-9, the test was performed with a mixture of rat/hamster S-9. Phenacetin was mutagenic with the mixture. The activity of the mixture was compared to the rat S-9 alone with low concentrations of 2-aminoanthracene (a strong promutagen for Salmonella typhimurium TA 1535, TA 100, TA 1537, and TA 98), nitrosopiperidine (a weak promutagen), and 1,2 epoxybutane (a weak, direct-acting mutagen). Except for an increased mutagenic activation by the mixture with nitrosopiperidine the mixture was comparable to the rat S-9 alone, indicating that replacing rat S-9 with a rat/hamster S9 mixture in the standard Ames test could increase the sensitivity of the test without interfering with rat S-9 activity.     

Mutagenic potentials of dental cements as detected by the Salmonella/microsome test

The potential mutagenicity of a zinc phosphate (Poscal), a polycarboxylate (Aqualox) and glass ionomer cements with (Argion) and without (Meron) silver reinforcement were characterized by employing the Ames Salmonella/microsome test. The materials were eluted in dimethyl sulphoxide or physiologic saline and the aliquots were used either immediately or after an incubation period of 24h at 37 degrees C. Mutagenic effects of the materials were tested on Salmonella typhimurium strains TA 98, TA 100, TA 102 and TA 1535 using the standard plate incorporation assay, and in the presence or absence of S9 fraction from rat liver. Poscal and Aqualox elicited mutagenic effects on S. typhimurium TA 98 and TA 1535, whereas Meron exhibited mutagenic effects on S. typhimurium TA 98. No mutagenic effects were detected for Argion. The type of solvent, dose of the material and incubation as well as the interactions between these factors exhibited varying degrees of influences on the mutagenic activities of the cements (P<0.05 and P<0.1). We conclude that zinc phosphate, polycarboxylate, and glass ionomer cements may have possible mutagenic activities.     

Structure-mutagenicity relationships of benzidine analogues

The mutagenic activities of benzidine, its dihydrochloride salt, and 12 of their analogues were compared in the Ames test using strains TA100 and TA98 with and without rat liver S9 activation. With the exceptions of 4,4'-methylenebis(3-nitroaniline) in both strains and 3,3-dichlorobenzidine in TA98, little or no mutagenicity was observed in the series when tested without S9 activation. All compounds, except tetramethylbenzidine, exhibited some activity in TA100 with S9 activation; dichlorobenzidine and 4-aminobiphenyl were significantly more mutagenic than the other compounds. This was in contrast to the TA98 results where the bridged diphenyl compounds, with the exception of the nitroaniline derivative, were only slightly mutagenic compared to the more planar biphenyl series. Only the nitroaniline compound was mutagenic in both strains in the presence or absence of S9 activation. For benzidine and the 3,3'-disubstituted benzidines (the dimethoxy-, diamino-, and dichloro- compounds), an increase in mutagenicity correlated to a decrease in basicity of the parent anilines in both TA100 and TA98.     

Mutagenicity and effect on gap-junctional intercellular communication of 4,4'-methylenebis(2-chloroaniline) and its oxidized metabolites

Oxidized metabolites of 4,4'-methylenebis(2-chloroaniline) (MBOCA) were tested for direct mutagenicity in a Salmonella typhimurium assay and for effects on gap-junctional communication of WB-F344 rat liver cells. The mutagenicities of the N-hydroxy, mononitroso and o-hydroxy (ring) metabolites of MBOCA were assayed without adding activating enzyme systems, using the frame-shift sensitive strain TA98 and the base pair substitution sensitive strain TA100. The mutagenicity of the hydroxylamine was demonstrated by a linear increase in the formation of mutant colonies in both strains, with a formation of two revertants/nmol by TA98 and 21 revertants/nmol by TA100. The mononitroso metabolite showed a slight positive effect on TA100, but effects were masked by its cytotoxicity towards this strain. This metabolite was neither mutagenic nor cytotoxic to TA98. The o-hydroxy and the dinitroso metabolites were negative for mutagenicity at concentrations up to 50 and 500 micrograms/plate, respectively. The effects of parent MBOCA and N-hydroxy, mononitroso and o-hydroxy metabolites on cell-cell communication were determined by a scrape loading/fluorescent dye transfer technique. Cytotoxicity was assessed by determination of colony-forming efficiency and lactate dehydrogenase release. MBOCA itself caused an inhibition of dye transfer at concentrations of 7.5, 11.3 and 15 nmol/ml, whereas measures of cytotoxicity were not seen until 15 and 30 nmol/ml for LDH release and plating efficiency, respectively. None of the oxidized metabolites were active in inhibiting dye transfer at non-cytotoxic concentrations.     

Identification of mutagens in freshwater sediments by the Ames-fluctuation assay using nitroreductase and acetyltransferase overproducing test strains

Extracts of sediments from an area of concern in the Elbe river basins (Spittelwasser creek) were analyzed with the Ames-fluctuation test and in parallel with gas chromatography/mass spectrometry for compound identification. The standard test strains TA 98 and TA 100 showed mutagenicity mainly in medium-polar fractions of the sediment extracts. PAHs contribute to the overall mutagenic potential of the sample. Especially, cyclopenta[c,d]pyrene that was previously not defined as a priority hazardous substance has to be considered as well. The addition of metabolically competent test strains, which overexpress nitroreductase and acetyltransferase (e.g., YG1041 and YG1042) to the test battery, increased significantly the sensitivity of the Ames test for medium polar to polar genotoxins. The increased mutagenicity that was found in these bacterial strains indicates the presence of nitroarenes and/or aromatic amines. In fact, a number of heterocyclic and nitrogen-substituted aromatic compounds were identified in the sediments of the Spittelwasser creek of which methyl parathion, 1-naphthylamine, and N-phenyl-2-naphthylamine are mutagenic.     

Testing of sunset yellow and orange II for genotoxicity in different laboratory animal species

The azo dyes Sunset Yellow and Orange II were gavaged to rodent species to check bile, urine, and fecal extracts for possible mutagenic activity in the Ames test or in bone marrow cells for clastogenicity using cytogenetic test systems. After oral application the dyes showed a negative response in bile, excrements, and bone marrow. When an exogenous metabolic activation was performed, increased revertant numbers using Salmonella strain TA100 were obtained only in fecal extracts of Sunset Yellow-treated animals. It is concluded that no genotoxic harm is to be expected from the ingestion of Sunset-Yellow or Orange II.     

Mutagenic potential of bisphenol A diglycidyl ether (BADGE) and its hydrolysis-derived products in the Ames Salmonella assay.

The mutagenicity of bisphenol A diglycidyl ether (BADGE), its first and second hydrolysis products (the diol epoxide and bis-diol of BADGE, respectively) and the bis-chlorohydrin of BADGE were investigated using the Ames Salmonella assay with strains TA98, TA100, TA1535 and TA1537. The assays were performed in the absence and presence of various concentrations of rat liver S9 fraction. The results obtained confirm the mutagenic power of BADGE in strains TA100 and TA1535 and show a positive response to the diol epoxide of BADGE in these strains, although the latter compound was approximately 10 times less potent than the former. A lack of mutagenic activity of the bis-diol of BADGE and the chlorohydrin under study is also shown. These findings suggest that BADGE and, to a much lesser extent, the diol epoxide of BADGE may constitute a genotoxic hazard, but not the bis-diol or bis-chlorohydrin of BADGE.