The natural anthraquinone dye emodin: Eco/genotoxicological characterization for aquatic organisms.

Emodin is an anthraquinone secondary metabolite produced by several species of plants and fungi. Emodin is known for its pharmacological versatility, and, in the textile industry, for its good dyeing properties. However, its use in the textile industry can result in the formation and disposal of large volumes of wastewater. Emodin mutagenicity has been shown in bacteria and in human cells, but little is known about its possible toxic, genotoxic, or mutagenic effects in aquatic organisms. We have evaluated the eco/genotoxicity of emodin to aquatic organisms. Emodin was toxic to Daphnia similis (EC50 = 130 µg L-1) and zebrafish embryos (LC50 = 25 µg L-1). No toxicity was observed for Raphidocelis subcapitata, Ceriodaphnia dubia, or Parhyale hawaiensis. Additional biochemistry/molecular studies are needed to elucidate the toxic/mutagenic pathways of emodin in aquatic organisms. The PNEC value for emodin was 0.025 µg L-1. In addition to mutagenicity in the Salmonella/microsome assay, emodin was mutagenic in the micronucleus assay in the amphipod P. hawaiensis. Among the anthraquinone dyes tested to date, natural or synthetic, emodin was the most toxic to aquatic species.

Evaluation of precopulatory pairing behaviour and male fertility in a marine amphipod exposed to plastic additives.

Plastics contain a mixture of chemical additives that can leach into the environment and potentially cause harmful effects on reproduction and the endocrine system. Two of these chemicals, N-butyl benzenesulfonamide (NBBS) and triphenyl phosphate (TPHP), are among the top 30 organic chemicals detected in surface and groundwater and are currently placed on international watchlist for evaluation. Although bans have been placed on legacy pollutants such as diethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP), their persistence remains a concern. This study aimed to examine the impact of plastic additives, including NBBS, TPHP, DBP, and DEHP, on the reproductive behaviour and male fertility of the marine amphipod Echinogammarus marinus. Twenty precopulatory pairs of E. marinus were exposed to varying concentrations of the four test chemicals to assess their pairing behaviour. A high-throughput methodology was developed and optimised to record the contact time and re-pair time within 15 min and additional point observations for 96 h. The study found that low levels of NBBS, TPHP, and DEHP prolonged the contact and re-pairing time of amphipods and the proportion of pairs reduced drastically with re-pairing success ranging from 75% to 100% in the control group and 0%-85% in the exposed groups at 96 h. Sperm count declined by 40% and 60% in the 50 µg/l and 500 µg/l DBP groups, respectively, whereas TPHP resulted in significantly lower sperms in 50 µg/l exposed group. Animals exposed to NBBS and DEHP showed high interindividual variability in all exposed groups. Overall, this study provides evidence that plastic additives can disrupt the reproductive mechanisms and sperm counts of amphipods at environmentally relevant concentrations. Our research also demonstrated the usefulness of the precopulatory pairing mechanism as a sensitive endpoint in ecotoxicity assessments to proactively mitigate population-level effects in the aquatic environment.

Is natural better? An ecotoxicity study of anthraquinone dyes.

The concept of sustainability has gained prominence in recent years, enhancing the need to develop products that are less harmful to the environment. Dyes are used by various industrial sectors and have a lot of market value; they are used on a large scale mainly by the textile industry that uses large volumes of water and is one of the main contributors to the contamination of water bodies. Some natural compounds, especially anthraquinones are re-emerging as possible alternatives to synthetic dyes, some of which are known for their toxic and/or mutagenic effects. The BioColour project (https://biocolour.fi/) which is interested in promoting the development of new alternative molecules to synthetic dyes, provided us highly purified anthraquinone dyes dermocybin and dermorubin (>98% purity) extracted from a specie of fungus Cortinarius sanguineus. Dyes were tested for their acute and chronic toxicity using different aquatic organisms. Dermorubin was not toxic to any of the organisms tested for the highest test concentration of 1 mg L-1 and it was the most promising dye. Dermocybin was toxic to Daphnia similis (EC50 = 0.51 mg L-1), Ceriodaphnia dubia (IC10 = 0.13 mg L-1) and Danio rerio embryos (extrapolated LC50 = 2.44 mg L-1). A safety limit, i.e, predicted no-effect concentration (PNEC) of 0.0026 mg L-1 was derived based on the toxicity of dermocybin. The PNEC value can be used to provide hazard information for future application in commercial dyeing processes. Then, we compared the toxicity of dermocybin and dermorubin with ecotoxicity data available in the literature on other anthraquinone dyes of natural and synthetic origin. Some natural dyes can be as toxic as synthetic ones, or more toxic when chronic effects are considered. Despite natural dyes being used since centuries past, there are few ecotoxicological studies available. This study is designed to help develop a more comprehensive understanding of their toxicological properties.

Proposal of a chronic toxicity test using the tropical epibenthic amphipod Parhyale hawaiensis.

Chronic toxicity tests with representative organisms are essential for ecological risk assessment. The circumtropical marine amphipod Parhyale hawaiensis is a promising test organism in ecotoxicology. This study aimed to develop a chronic toxicity protocol for liquid samples testing with P. hawaiensis using reproduction and growth as endpoints. In the proposed protocol, organisms (≤52 days old) are placed in 5 replicates each containing 100 mL of solution, 10 organisms, and 5 g of crushed coral for 42 days of exposure. The protocol was successfully developed but reproduction showed better performance than growth rate. NOECs based on reproduction were determined for zinc (0.10 mg Zn L-1) and 3,4-DCA (0.50 mg L-1), and they are of the same order of magnitude compared with the values of other amphipods. The developed test based on reproduction can be considered a promising tool for hazard characterizations although more tests with different substances are still needed.

Toxicity and mutagenicity studies of 6PPD-quinone in a marine invertebrate species and bacteria.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone), an oxidation product of the tire additive, 6PPD, has been associated with high mortality of salmonids (0.1 µg/L). The objective of this study was to determine the acute toxicity using neonates and mutagenicity (micronuclei in hemolymph of exposed adults) of 6PPD-quinone in the marine amphipod Parhyale hawaiensis. Also, we studied its mutagenicity in the Salmonella/microsome assay using five strains of Salmonella with and without metabolic system (rat liver S9, 5%). 6PPD-quinone did not present acute toxicity to P. hawaiensis from 31.25 to 500 µg/L. Micronuclei frequency increased after 96 h-exposure to 6PPD-quinone (250 and 500 µg/L) when compared to the negative control. 6PPD-quinone also showed a weak mutagenic effect for TA100 only in the presence of S9. We conclude that 6PPD-quinone is mutagenic to P. hawaiensis and weakly mutagenic to bacteria. Our work provides information for future risk assessment of the presence of 6PPD-quinone in the aquatic environment.

Genotoxic effects of silver nanoparticles on a tropical marine amphipod via feeding exposure.

Silver nanoparticles (AgNP) are widely used in several applications including as antifouling agents; therefore, they can end up in estuarine and marine environments. These nanoparticles tend to aggregate and to deposit in the sediment, where many organisms feed and reproduce. Parhyale hawaiensis is an epibenthic amphipod globally distributed in tropical zones, and has been considered a potential model for ecotoxicology. The aim of this study was to evaluate genotoxic effects of AgNP and Ag salt via feeding, as P. hawaiensis lives in the sediment where nanoparticles tend to accumulate. Organisms were cultivated in the laboratory, and adults were exposed to food containing both AgNP and Ag salt. We collected hemolymph after different times of exposure, and analysed the hemocytes for nuclear abnormalities (including micronuclei) and DNA damage using the standard alkaline comet assay. Conditions of both assays were developed/optimized to allow their successful application in marine invertebrates. Increased frequencies of micronuclei, nuclear buds and total abnormalities were detected in relation to concentration and time in organisms exposed to AgNP and Ag salt in comparison to the controls. No DNA damage was detected when the alkaline comet assay was applied. After 5 days of exposure, we observed higher micronuclei frequencies in Ag salt treatment in comparison with AgNP. After 13 days, micronuclei frequencies were similar for both silver forms. We believe that the Ag, in its ion form, is causing the mutagenic effect; therefore, more time would be needed for the release of the ion from AgNP, explaining the delayed mutagenic effect.

The amphipod Parhyale hawaiensis as a promising model in ecotoxicology.

Near-shore marine/estuarine environments play an important role in the functioning of the marine ecosystem and are extremely vulnerable to the presence of chemical pollution. The ability to investigate the effects of pollution is limited by a lack of model organisms for which sufficient ecotoxicological information is available, and this is particularly true for tropical regions. The circumtropical marine amphipod Parhyale hawaiensis has become an important model organism in various disciplines, and here we summarize the scientific literature regarding the emergence of this model within ecotoxicology. P. hawaiensis is easily cultured in the laboratory and standardized ecotoxicity protocols have been developed and refined (e.g., miniaturized), and effects of toxicants on acute toxicity (Cd, Cu, Zn, Ag, ammonia, dyes, pesticides, environmental samples), genotoxicity as comet assay/micronuclei, and gene expression (Ag ion and Ag nanoparticles) and regeneration (pesticides) have been published. Methods for determination of internal concentrations of metals (Cu and Ag) and organic substances (synthetic dye) in hemolymph were successfully developed providing sources for the establishment of toxicokinetics models in aquatic amphipods. Protocols to evaluate reproduction and growth, for testing immune responses and DNA damage in germ cells are under way. The sensitivity of P. hawaiensis, measured as 50% lethal concentration (LC50), is in the same range as other amphipods. The combination of feasibility to culture P. hawaiensis in laboratory, the recent protocols for ecotoxicity evaluation and the rapidly expanding knowledge on its biology make it especially attractive as a model organism and promising tool for risk assessment evaluations in tropical environments.

Measuring concentrations of a dye in the hemolymph of a marine amphipod: Development of a protocol for exposure assessment.

The increasing pollution of aquatic environments due to old and emerging contaminants requires the development of integrative methods for exposure assessment. Internal concentrations are a reliable way to estimate total exposure of contaminants originated from different routes (water, sediment, and food). We developed a protocol to evaluate the concentration of a dye, C.I. Disperse Red 1, in the hemolymph of Parhyale hawaiensis, a marine amphipod. LOD and LOQ were satisfactory to detect the dye in all hemolymph samples. The concentration detected in the hemolymph varied related to exposure time and dye concentration (0.003 to 0.086 µg mL-1). Polynomial regression model was the best fit. The protocol was reliable to detect and quantify dye exposure in marine amphipods and can be considered for future assessments of estuarine and marine regions under the influence of dye processing plants. The method possibly can be easily adapted to other amphipods and other azo dyes.

Assessment of the compounds formed by oxidative reaction between p-toluenediamine and p-aminophenol in hair dyeing processes: Detection, mutagenic and toxicological properties.

Previous studies have demonstrated the presence of precursors and coupling agents in wastewater from hair dyeing processes. The complex reaction involved in the oxidation of these compounds can generate extremely hazardous sub-products, leading to an increase in the mutagenicity and toxicity of wastewater. Without proper treatment, this highly toxic wastewater may find its way into the drinking water treatment plant. The present work aimed to investigate the main products generated after the oxidation reaction involving p-toluenediamine (PTD) and p-aminophenol (PAP) - precursors that widely used in the composition of commercial permanent hair dyes, under experimental conditions close to the routine hair dyeing process (in the presence and absence of hydrogen peroxide in ammoniacal medium), using spectroscopic techniques. The study also investigated the mutagenicity and toxicity of the products formed in the hairdressing wash water and conducted detection analysis to determine the presence of the precursors and Bandrowski's Base Derivative (BBD) in samples of wastewater, surface and drinking water using HPLC-DAD and linear voltammetry techniques. Based on this investigation, we identified several PTD and PAP self-oxidation products and eleven sub-products derived from the reaction between PTD and PAP. Assays conducted using Salmonella typhimurium YG1041, with and without activation-induced rat liver metabolism (S9), indicated mutagenicity of the reaction products in concentrations above 10.0 µg µL-1. The concentrations of PTD, PAP, and several reactions and oxidation products of these precursors were detected in wastewater and water samples.

Occurrence and risk assessment of organophosphate esters in urban rivers from Piracicaba watershed (Brazil).

Organophosphate esters (OPEs) are substances globally used as flame retardants and plasticizers that have been detected in all environmental compartments. This study aimed to evaluate the occurrence and sources of ten OPEs in the Piracicaba River Basin (Brazil). Twelve sampling sites were selected in five rivers with different pollution sources; six sampling campaigns were performed encompassing dry and wet seasons. ΣOPEs ranged from 0.12 to 6.2 µg L-1; the levels in urban areas were higher than in rural and non-urban areas, but no overall tendency concerning the seasonal effect on OPEs concentrations was observed. Tris(2-butoxyethyl) phosphate (TBOEP), tris(2-chloroisopropyl) phosphate (TCIPP), and tris(1,3-dichloroisopropyl) phosphate (TDCIPP) were the most abundant and frequently detected compounds. Nine OPEs were detected at higher concentrations in a site affected by effluents from textile industries. An acute toxicity test using Daphnia similis was performed for tris(2-ethylhexyl) phosphate (TEHP) for the calculation of a preliminary predicted no effect concentration (PNEC). The risk quotient (RQ) approach was applied and risk to aquatic environment related to TEHP levels was observed in areas adjacent to textile industries, but more toxicity studies are required for the determination of a more reliable PNEC.

Occurrence of pesticides in waters from the largest sugar cane plantation region in the world.

In this study, a multi-residue method was used to analyze 13 pesticides and 1 degradation product in surface and groundwater in the region with the largest sugar cane production in the world. The potential effects of individual pesticides and their mixtures, for aquatic life and human consumption, were evaluated. For the surface water, 2-hydroxy atrazine, diuron, carbendazim, tebuthiuron, and hexazinone were the most frequently detected (100, 94, 93, 92, and 91%, respectively). Imidacloprid (2579 ng L-1), carbendazim (1114 ng L-1), ametryn (1101 ng L-1), and tebuthiuron (1080 ng L-1) were found at the highest concentrations. For groundwater, tebuthiuron was the only quantified pesticide (107 ng L-1). Ametryn, atrazine, diuron, hexazinone, carbofuran, imidacloprid, malathion, carbendazim, and their mixtures presented risk for the aquatic life. No risk was observed for the pesticides analyzed in this work, alone or in their mixtures for human consumption.

Assessment of p-aminophenol oxidation by simulating the process of hair dyeing and occurrence in hair salon wastewater and drinking water from treatment plant.

This work reports the study of oxidation reaction of p-aminophenol (PAP) in ammoniacal medium in dissolved atmospheric oxygen and hydrogen peroxide, simulating the process of hair dyeing with permanent dyes. The products formed, which included semi-quinoneimine radical, quinoneimine, dimers, trimers and tetramers, were identified by mass spectrometry, infrared spectroscopy, UV-vis spectrophotometry, and nuclear magnetic resonance of hydrogen. The process was found to involve an autoxidation mechanism. The mutagenicity of the products was carried out by Salmonella Typhimurium YG1041 assay, and the results indicated no mutagenic properties. The presence of PAP and its oxidative products in samples of wastewater collected from hairdressing salon effluent (WW), raw river water (RRW), and water inlet and outlet of drinking water treatment plant (DWTP) was analyzed by HPLC-DAD. PAP was detected in the collected samples of WW, water samples from DWTP (before and after treatment), at concentrations of 2.1 ± 0.5 mg L-1, 1.9 ± 0.3 × 10-3 mg L-1 and 1.3 ± 0.2 × 10-3 mg L-1, respectively. The reaction products, including dimers, trimers and tetramers were identified only in the WW sample; this shows that both the precursor in the sample and its derivatives were released into the wastewater.

Assessment of the autoxidation mechanism of p-toluenediamine by air and hydrogen peroxide and determination of mutagenic environmental contaminant in beauty salon effluent.

The present work investigated the autoxidation reaction of p-toluenediamine (PTD) - a precursor - widely used in permanent hair dyeing formulation, under experimental conditions close to the hair dyeing process (oxygen and/or peroxide in ammoniacal medium), by chromatographic and spectroscopic techniques. In additional, evaluated the mutagenicity of the PTD oxidation products and the presence of PTD and this products in wastewater from beauty salon, as well as in surface water and drinking water using HPLC coupled to a diode array detector and linear scan voltammetry. Through this study, it was possible the identification of semi-quinonediimine, quinonediimine, dimers (derived from toluenediamine), and trimer radical identified as Bandrowski's Base derivative (BBD) formed during autoxidation of PTD. Salmonella Typhimurium YG1041 assay with and without metabolic activation induced rat-liver (S9) indicated mutagenic activity for BBD. Levels of PTD were determined by the standard addition method in samples collected from the wastewater of a beauty salon, as well as from the water before and after treatment in a drinking water treatment plant (DWTP) reached concentrations of 2.08 ±â€¯0.21, 2.36 ±â€¯0.10 × 10-3, and 1.77 ±â€¯0.13 × 10-3 mg L-1, respectively. In addition, linear sweep voltammetry was used to monitor the BBD found at the concentration of 1.59 ±â€¯0.35 mg L-1 in wastewater collected from the beauty salon.

Critical issues and alternatives for the establishment of chemical water quality criteria for livestock.

Good water quality for livestock is essential for animal health, supply of safe food and food production economy. Few countries have established water quality criteria for livestock for chemical contaminants. For those that have them, the values are quite variable among each other for the same substance due to differences in the approach for the acceptable daily intakes and algorithms for the calculation. In several countries, including Brazil, these standards are based on international or other countries' data, which differ among protected species, exposure scenarios, and levels of protection. The objective of this work was to discuss critical issues to establish chemical water quality criteria for livestock in Brazil. A discussion about the difficulties involved and the alternatives when sufficient data are not available is presented. Using the Canadian framework for reference, we provide recommendations on a revised framework and alternatives regarding the use of chronic studies as mandatory, defaulting to human health drinking water quality guidelines/criteria for livestock, the use of additional safety factors, and alternatives in the absence of toxicological data.

Genotoxicity of textile dye C.I. Disperse Blue 291 in mouse bone marrow.

Color Index (C.I.) Disperse Blue 291 (DB291) is an azo dye used by the textile industry. After yarn dyeing, wastewater containing the dye, released into the aquatic environment, may pollute drinking water sources. We investigated the mutagenicity and genotoxicity of DB291 in male Swiss mice, following oral administration. Micronucleated cells, primary DNA damage (comet assay) in blood, liver, and kidney cells, and BAX, BCL2, SMAD4 and TNFA gene expression in leukocytes were evaluated. An increased frequency of micronucleated polychromatic erythrocytes (MNPCEs) was observed in animals treated with 50 mg/kg bw; no other genetic alteration was detected. Neither primary DNA damage nor changes in gene expression were observed.

In Vivo genotoxicity of a commercial C.I. Disperse Red 1 dye.

Color Index (C.I.) Disperse Red 1 (DR1) is a widely used textile azo dye found in rivers. As it may not be completely removed by conventional treatments, humans can be exposed through drinking water. Studies have supported in vitro toxicity and mutagenicity of commercial DR1. This study aimed to investigate the mutagenic and toxicogenomic effects of commercial DR1 in multiple tissues/organs of Swiss male mice. For that, animals were orally exposed to the dye (by gavage), at single doses of 0.0005, 0.005, 0.5, 50, or 500 mg/kg bw. The two lowest doses were equivalent to the ones found in two Brazilian rivers receiving influx of textile discharges. Cytotoxicity, micronucleated cell frequencies (for all doses tested), primary DNA damage (comet assay), and gene expression profiling of (0.0005 and 0.005 mg/kg of bw) were investigated 24 hr after animal exposure to commercial DR1. Data showed increased frequencies of micronucleated polychromatic erythrocytes in bone marrow cells after treatment with 0.5 and 50 mg/kg bw. At 0.005 mg/kg bw, commercial DR1 induced an increase of primary DNA damage in liver, but not in kidney cells. Additionally, upregulation of genes involved in the inflammatory process (IL1B) (0.0005 and 0.005 mg/kg bw) and cell-cycle control (CDKN1A) in liver cells, and apoptosis (BCL2 and BAX) in leukocytes (0.005 mg/kg bw) were also detected. In conclusion, the commercial DR1 was genotoxic (chromosome aberrations and primary DNA damage) and modulated gene expression in mice, and such effects were dependent on the doses and tissues analyzed. Environ. Mol. Mutagen. 59:822-828, 2018. © 2018 Wiley Periodicals, Inc.

Development of an acute toxicity test with the tropical marine amphipod Parhyale hawaiensis.

There is a lack of suitable tropical marine species for ecotoxicity tests. An attractive model organism for ecotoxicology is the marine amphipod Parhyale hawaiensis, which is already a model for genetic and developmental studies. This species is widespread, can tolerate changes in salinity, is easy to handle and is representative of circumtropical regions. The aim of this work was to describe standardized procedures for laboratory husbandry, define conditions for acute toxicity tests, and to provide acute toxicity test results for some reference toxicants. Culturing conditions for the organism in the laboratory were established in reconstituted seawater (30 ± 2 salinity), 24 ± 2 °C, photoperiod 12/12 h light/dark. Acute toxicity test procedures were developed for 96 h-exposure time, and organisms at ages <7 days. The miniaturized version of the test, based on 96-well microplates and 200 µL of exposure media provided consistent results compared to larger exposure volumes (80-mL vials protocol). Acute toxicity of Ag, Cd, Cu, Zn and ammonia determined for P. hawaiensis were consistent to previous results for other marine amphipods. We conclude that P. hawaiensis can be successfully cultured in standardized conditions and be effectively used in acute toxicity testing. Further development and use of this model will enable standardized and reproducible ecotoxicology investigations in understudied and vulnerable tropical marine ecosystems.

Quantifying the contribution of dyes to the mutagenicity of waters under the influence of textile activities.

The combination of chemical analyses and bioassays allows the identification of potentially mutagenic compounds in different types of samples. Dyes can be considered as emergent contaminants and were detected in waters, under the influence of textile activities. The objective of this study was to evaluate the contribution of 9 azo dyes to the mutagenicity of representative environmental samples. Samples were collected along one year in the largest conglomerate of textile industries of Brazil. We analyzed water samples from an important water body, Piracicaba River, upstream and downstream two main discharges, the effluent of a wastewater treatment plant (WWTP) and the tributary Quilombo River, which receives untreated effluent from local industries. Samples were analyzed using a LC-MS/MS and tested for mutagenicity in the Salmonella/microsome microsuspension assay with TA98 and YG1041. Six dyes were detected in the collected samples, Disperse Blue 291, Disperse Blue 373, Disperse Orange 30, Disperse Red 1, Disperse Violet 93, and Disperse Yellow 3. The most sensitive condition for the detection of the mutagenicity was the strain YG1041 with S9. The concentration of dyes and mutagenicity levels varied along time and the dry season represented the worst condition. Disperse Blue 373 and Disperse Violet 93 were the major contributors to the mutagenicity. We conclude that dyes are contributing for the mutagenicity of Piracicaba River water; and both discharges, WWTP effluent and Quilombo River, increase the mutagenicity of Piracicaba River waters in about 10-fold. The combination of chemical analysis and bioassays were key in the identification the main drivers of the water mutagenicity and allows the selection of priority compounds to be included in monitoring programs as well for the enforcing actions required to protect the water quality for multiple uses.

In Vitro Genotoxicity Testing: Significance and Use in Environmental Monitoring.

There is ongoing concern about the consequences of mutations in humans and biota arising from environmental exposures to industrial and other chemicals. Genetic toxicity tests have been used to analyze chemicals, foods, drugs, and environmental matrices such as air, water, soil, and wastewaters. This is because the mutagenicity of a substance is highly correlated with its carcinogenicity. However, no less important are the germ cell mutations, because the adverse outcome is related not only to an individual but also to population levels. For environmental analysis the most common choices are in vitro assays, and among them the most widely used is the Ames test (Salmonella/microsome assay). There are several protocols and methodological approaches to be applied when environmental samples are tested and these are discussed in this chapter, along with the meaning and relevance of the obtained responses. Two case studies illustrate the utility of in vitro mutagenicity tests such as the Ames test. It is clear that, although it is not possible to use the outcome of the test directly in risk assessment, the application of the assays provides a great opportunity to monitor the exposure of humans and biota to mutagenic substances for the purpose of reducing or quantifying that exposure.

Occurrence and risk assessment of an azo dye - The case of Disperse Red 1.

Water quality criteria to protect aquatic life are not available for most disperse dyes which are often used as commercial mixtures in textile coloration. In this study, the acute and chronic toxicity of the commercial dye Disperse Red 1 (DR1) to eight aquatic organisms from four trophic levels was evaluated. A safety threshold, i.e. Predicted No-Effect Concentration (PNEC), was derived based on the toxicity information of the commercial product and the purified dye. This approach was possible because the toxicity of DR1 was accounting for most of the toxicity of the commercial mixture. A long-term PNEC of 60 ng L(-1) was proposed, based on the most sensitive chronic endpoint for Daphnia similis. A short-term PNEC of 1800 ng L(-1) was proposed based on the most sensitive acute endpoint also for Daphnia similis. Both key studies have been evaluated with the new "Criteria for Reporting and Evaluating ecotoxicity Data" (CRED) methodology, applying more objective criteria to assess the quality of toxicity tests, resulting in two reliable and relevant endpoints with only minor restrictions. HPLC-MS/MS was used to quantify the occurrence of DR1 in river waters of three sites, influenced by textile industry discharges, resulting in a concentration range of 50-500 ng L(-1). The risk quotients for DR1 obtained in this work suggest that this dye can pose a potential risk to freshwater biota. To reduce uncertainty of the derived PNEC, a fish partial or full lifecycle study should be performed.