Environ Sci Pollut Res (2014) 21:8233–8241 DOI 10.1007/s11356-014-2806-y
RESEARCH ARTICLE
(Eco)toxicological effects of 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) in zebrafish (Danio rerio) and permanent fish cell cultures Krisztina Vincze & Martin Gehring & Thomas Braunbeck
Received: 27 October 2013 / Accepted: 17 March 2014 / Published online: 1 April 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) is a high-production volume chemical used in paper, ink, pesticide, and adhesive industries as a wetting and anti-foaming agent. The physicochemical properties and slow biodegradation rate of TMDD indicate a low bioaccumulation potential but a high prevalence in the environment. As a consequence, TMDD has been detected in several European rivers in the nanogram per liter and lower microgram per liter range; however, its environmental risk to aquatic organisms is considered low. Recent studies almost exclusively focused on acute effects by TMDD, little is known about cytotoxic and genotoxic effects, reproduction and developmental toxicity, endocrine disruption, and any kind of long-term toxicity and carcinogenicity so far. The present study aims to provide more specific baseline information on the ecotoxicological effects of TMDD in fish. For this end, cyto- and genotoxicity assays were carried out in vitro with the permanent fish cell line RTLW1; in addition, in vivo studies were conducted with the early
Responsible editor: Markus Hecker K. Vincze (*) : T. Braunbeck Aquatic Ecology and Toxicology Group, Center for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany e-mail: [email protected] T. Braunbeck e-mail: [email protected] M. Gehring Verband kommunaler Unternehmen (VKU), Invalidenstr. 91, 10115 Berlin, Germany e-mail: [email protected] Present Address: K. Vincze Institute for Evolution and Ecology, Animal Physiological Ecology, University of Tübingen, Konrad-Adenauer-Strasse 20, 72072 Tübingen, Germany
life stages of zebrafish (Danio rerio) in order to fill the data gaps in developmental toxicity and endocrine disruption. TMDD showed a cytotoxic and slight genotoxic potential in fish cell lines; moreover, various sublethal and lethal effects could be detected in developing zebrafish embryos. There was no evidence of endocrine-disrupting effects by TMDD; however, mortality following prolonged exposure to TMDD during fish sexual development test was clearly higher than mortality in the fish embryo test after 96-h exposure. Our results thus confirmed previous findings of laboratory screening tests, suggesting short-term toxic effects of TMDD in the intermediate, and long-term effects in the lower milligram per liter range. Keywords TMDD . Zebrafish . RTL-W1 fish cell line . Fish embryo test . Fish sexual development test . Comet assay . Neutral red retention assay . Micronucleus test
Introduction Surfactants are widely used for industrial applications as well as for personal care products and household formulations. Due their extensive usage and production, they usually end up in wastewater treatment plants and are removed mainly by biological and physical processes (Camacho-Muñoz et al. 2014). Still, surfactants can be found in considerable variety in waste and surface waters (Schröder and Ventura 2000). 2,4,7,9-Tetramethyl-5-decyne-4,7-diol (TMDD) is a nonionic surfactant (alcohol ethoxylate) with a structure of two hydrophilic moieties connected to hydrophobic residues; it is used as a coating and anti-foaming agent in pesticide, ink, paper, and adhesive industries. TMDD enhances the wetting of materials like plastic, wood, and paper through reducing the surface tension (Air Products and Chemicals Inc. 2001).
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Environ Sci Pollut Res (2014) 21:8233–8241
Table 1 summarizes the basic physicochemical properties of TMDD. With an annual production and import volume of over one million pounds alone in the USA (US Environmental Protection Agency 2008), TMDD has become a highproduction volume (HPV) chemical; the production amount in Europe is so far unknown (Guedez et al. 2009). Generally, there is increasing evidence that TMDD may easily reach the environment through its various applications. TMDD has a low potential to bioaccumulate; however, its biodegradation rate is slow to negligible; therefore, it is judged to have high persistence in the environment (US Environmental Protection Agency 2008). As a consequence, TMDD has been detected in several European rivers like Lippe (Dsikowitzky et al. 2004a, b), Main, Nidda (Guedez et al. 2008), Rhine (Guedez et al. 2009; Schwarzbauer and Heim 2005; van Stee et al. 2002), Meuse, Scheldt (van Stee et al. 2002), Mulde (Heinzel 2006), Spree (Schwarzbauer and Ricking 2010), Ruhr (LANUV 2008) and in the industrial area of Kavala in Northern Greece (Grigoriadou et al. 2004) with concentrations in the nanogram per liter and lower microgram per liter range. In the river Rhine at Worms, Guedez et al. (2009) calculated an overall annual TMDD load of about 23 t/year, suggesting a fairly constant discharge, the most likely source being effluents from municipal wastewater treatment plants. Guedez and Püttmann (2011) also investigated the occurrence and fate of TMDD in four wastewater treatment plants and found only partial removal from municipal wastewater with elimination rates between 33 and 68 %. Recycled paper residues entering the sewage sludge represent a potential source of TMDD contamination in surface waters. Gehring et al. (2009)
analyzed six recycling toilet paper products of different origin; TMDD was reported in all samples with concentrations between 0.16 and 2.39 mg/kg. So far, little is known about the presence of TMDD in drinking water. However, TMDD was already detected in mineral water samples with concentrations up to 50 μg/L (Kleinschnitz and Schreier 1998). TMDD is considered to have only a minor impact on the aquatic environment (Deutscher Bundestag 2009), although risk assessment has been mainly based on acute toxicity. The 96-h LC50 values of acute fish tests with fathead minnow (Pimephales promelas) and common carp (Cyprinus carpio) are at 36 and 42 mg/L, respectively; acute tests with Daphnia magna gave 48-h EC50 values of 15 mg/L (Air Products and Chemicals Inc. 2001). With respect to effects other than acute toxicity, there is a fundamental lack of investigations into basic cytotoxic and genotoxic effects, reproduction and developmental toxicity, endocrine disruption, and any kind of long-term toxicity and carcinogenicity. Therefore, the present study was designed to provide baseline information on ecotoxicological effects of TMDD in fish. To this end, cyto- and genotoxicity assays (neutral red retention assay, comet assay, micronucleus test) were carried out in vitro with the permanent fish cell line RTLW1; in addition, prolonged in vivo studies focusing on teratogenic and endocrine potentials (fish embryo test (FET), fish sexual development test (FSDT), OECD 2011) were carried out with the zebrafish (Danio rerio).
Materials and methods Chemicals
Table 1 Physicochemical properties of TMDD (US Environmental Protection Agency 2008) Physicochemical features of 2,4,7,9-Tetramethyl-5-decyne-4,7-diol (TMDD)
All chemicals used were of the highest purity available. Unless stated otherwise, chemicals were purchased from Sigma-Aldrich (Deisenhofen, Germany).
Structure
In vitro toxicity screening Cell culture techniques
CAS number Melting point (measured) Boiling point (measured) Vapor pressure (measured) Water solubility (measured) Log octanol-water partition coefficient (Kow) (measured) Henry’s law constant (estimated by Estimation Program Interface Suite™) Half life (estimated by Estimation Program Interface Suite™)
126-86-2 54–55 °C 262–263 °C 0.62–0.7 kPa at 20 °C 1.7 g/L at 20 °C 2.8 8,58483 E-007 Atm-m3/mol Air—6,039 h Water—900 h Soil—900 h Sediment—3,600 h
The cell line RTL-W1 is a fibroblast-like permanent cell line derived from the liver of adult rainbow trout (Oncorhynchus mykiss), growing as a monolayer. It is a sensitive tool for assessing the toxic potentials of chemicals due to its high biotransformation capacity, i.e., the expression of cytochrome P450 enzymes (Lee et al. 1993). Cells were maintained in 75cm 2 plastic culture flasks (Nunc Nunclon Delta T-25, Langenselbold, Germany) in Leibowitz (L15) medium supplemented with 10 % fetal bovine serum (Biochrom, Berlin, Germany) and 1 % penicillin/streptomycin solution (Seiler et al. 2006). Cells were transferred into new flasks every 6– 8 days and were stored at 20 °C until further use.
Environ Sci Pollut Res (2014) 21:8233–8241
Neutral red retention assay The neutral red retention assay is a rapid in vitro assay assessing cell viability through the quantification of lysosomal retention of the dye neutral red (Borenfreund et al. 1988). Suspensions of RTL-W1 cells were exposed to TMDD concentrations along a 96-well plate (Renner, Dannstadt, Germany) in 1:2 serial dilutions with L15 medium in a range from 800 to 12.5 mg/L. Supplemented L15 medium served as a negative control, and 80 mg/L 3,5-dichlorophenol were used as positive control. The cytotoxic potential of TMDD was determined after cell incubation for 48 h at 20 °C as described by Babich and Borenfreund (1991). Neutral red retention was measured at a wavelength of 540 nm using a Spectra III multiwell plate reader (Tecan, Crailsheim, Germany). Dose–response curves expressing the viability of cells compared to controls were analyzed using SigmaPlot 11.0. (SYSTAT, Erkrath, Germany). The neutral red retention assay of TMDD was carried out in three independent replicates. Comet assay The alkaline comet assay or single cell gel electrophoresis is a sensitive technique for the detection of DNA single- and double-strand breaks, alkali-labile sites, and incomplete excision repair events (Schnurstein and Braunbeck 2001). A volume of 2 ml of RTL-W1 cell suspension in L15 medium with a density of 5×106 cells/ml was given into each well of a 6well plate (Renner) and incubated overnight at 20 °C. After rinsing the wells with phosphate-buffered saline (PBS), cells were exposed for 48 h to TMDD concentrations of 0, 20, 40, 60, 80, and 100 mg/L suspended in L15 medium. Pure medium served as a negative control, 5-min UV light radiation was used as a positive control. After exposure, cells were rinsed with PBS, detached from the wells by trypsin and processed as described in detail by Boettcher et al. (2011). The comet assay was performed according to the original protocol by Singh et al. (1998). DNA was stained with 20-μm ethidium bromide immediately before scoring. An Axioskop fluorescence microscope (Zeiss, Jena, Germany) equipped with a ×34 magnification and an excitation filter of 518 nm was used for evaluation. Exactly 100 randomly selected cells were scored with a high-sensitivity gray scales CCD camera (Optilas, Munich, Germany), and DNA tail moments (tail length × fluorescence intensity in the tails) were calculated with the image-analysis system Komet 5.5™ (Kinetic Images, Liverpool, UK). TMDD genotoxicity was assayed in three independent replicates. Micronucleus test Micronuclei are generated during failure of cell division through the lack of centromeres or inadequate function of
8235
microtubules (Al-Sabti and Metcalfe 1995), thus indicating potentials of genotoxicity. Six-well plates (Renner, Dannstadt, Germany) were supplied with 2 ml of RTL-W1 cell suspension at a density of 3×104 cell/ml. Glass cover slips (22× 22 mm; Langenbrinck, Emmendingen, Germany) were placed in each well to provide an optimal growth surface. After overnight incubation at 20 °C, cells were exposed to TMDD concentrations of 40, 80, and 160 mg/L diluted in L15 medium. Pure L15 medium served as a negative control, 4nitroquinoline-1-oxide (4-NQO) at a concentration of 190 μg/L was used as a positive control. After 48-h exposure, cells were rinsed with phosphate-buffered saline (PBS), and TMDD solutions were replaced by pure L15 medium for another 72 h in order to ensure cell division. Cell fixation was carried out according to Boettcher et al. (2010). After staining with acridine orange solution, 2,000 randomly selected cells per slide were scored with an Aristoplan microscope equipped with a ×340 lens and an excitation filter of 490 nm. Results were recorded as the percentage of cells containing micronuclei of the total number of cells inspected. Three independent replicates of the micronucleus assay with TMDD were conducted. In vivo toxicity screening Ethic statement In vivo toxicity tests with embryos were only conducted with non-protected stages of zebrafish embryos in order to comply with current EU animal welfare regulations (European Union 2010). The long-term experiments (fish sexual development test) were carried out in compliance with German animal ethics regulations under permission 35–9185.81/G-134/07 by local authorities at Karlsruhe (Germany). Maintenance and breeding of zebrafish The Aquatic Ecology and Toxicology Group at the Heidelberg University rears several stocks of zebrafish based on the West Aquarium strain. Fish were kept at 26±1 °C in 160-L tanks under flow-through conditions. The room was light-isolated, and an artificial dark/light cycle of 10:14 h was maintained. The animals were fed twice daily with dry flake food (TetraMin™ Hauptfutter, Tetra, Melle, Germany) and additionally with freshly hatched Artemia larvae the day before spawning. For egg production, mating groups were transferred into special 4-L aquaria equipped with a stainless steel grid on the bottom with a mesh size of 1.25 mm, which allowed the passage of eggs into a separate spawning tray, thus preventing predation by adult zebrafish. Green plastic wire material was used as spawning stimulus. Spawning took place in the early morning period after the onset of light. Eggs
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were collected 30–60 min after spawning and used for in vivo toxicity tests. Fish embryo test (FET) The embryo test with the zebrafish is a simple and costeffective test, which can be used for toxicity screening and early risk assessment of chemicals (Braunbeck et al. 2005; Lammer et al. 2009; Nagel 2002). The zebrafish embryonic development is short (detailed by Kimmel et al. 1995) and easy to observe due to the transparency of eggs. In the current study, TMDD concentrations of 0, 20, 40, 80, and 320 mg/L were tested for up to 120 h. The FET was carried out in 24well plates (Renner). Artificial soft water according to ISO 7346/3 (ISO 1996) was used as a negative control, and 3.7mg/L 3,4-dichloroaniline (DCA) served as a positive control. The development of the embryos was observed trough a CKX41 microscope (Olympus, Hamburg, Germany) at 24-, 48-, 72-, 96-, and 120-h post fertilization (hpf). Lethal and sublethal endpoints were recorded at appropriate time periods. Lethal endpoints were egg coagulation, non-development of somites, non-detachment of the tail and lack of heartbeat, whereas sublethal endpoints comprised non-development of the eyes, lack of eye pigmentation, absence of spontaneous movement and blood circulation, formation of edemata, malformations of the spinal cord, tail, head, heart and yolk sac, and growth retardation (Braunbeck et al. 2005; Nagel 2002). Time of hatching was determined as well. LC50 and EC50 values were calculated after generating dose–response curves in SigmaPlot 11.0. Three independent replicates of FETs with TMDD were carried out. Fish sexual development test (FSDT) The zebrafish is a gonochoristic species, i.e., in the early development all individuals develop ovary-like gonadal organs; later, males differentiate to hermaphrodites and finally into phenotypic males (Segner et al. 2003). The time of sexual development is, therefore, thought to be a period very sensitive to endocrine disrupting substances (Brion et al. 2004). Based on this, the fish sexual development test (OECD TG 229) with juvenile zebrafish was developed for the detection of androgenic and estrogenic potentials of chemicals (Holbech et al. 2006; OECD 2011). Exposure to 0-, 1-, 10-, and 20-mg/L TMDD was initiated in two replicates with fertilized eggs incubated in 20-cm diameter Petri dishes at 27 °C with a photoperiod of 12:12 h. After becoming free-swimming, 40 larvae were transferred into 12-L aquaria equipped with a flow-through system. The entire test solutions were renewed once daily by mixing of defined aerated water volumes and TMDD stock solutions in the overflow system to ensure high water quality and to maintain constant TMDD concentrations.
Environ Sci Pollut Res (2014) 21:8233–8241
For detailed description and zebrafish larvae feeding regime, see Knörr and Braunbeck (2002). At 60-days post-hatch, fish were euthanized by anesthesia with a saturated solution of benzocaine (4-aminobenzoic acid ethyl ester) for histological investigations. Fish were fixed in Davidson’s fixative (OECD 2010). Following dehydration in a graded series of ethanol and paraffin embedding, longitudinally sections of 5-μm thickness were cut with an HN-40 microtome (Leica, Nussloch, Germany) through the gonadal region. Staining the sections with hematoxylin and eosin solution allowed an easy light microscopical examination of the gonads. The percentage of intersex fish as well as the sex ratio was recorded as additional endpoints. Finally, any form of gonad malformation was recorded. Statistical analysis In vitro test data was first investigated for normal distribution using Shapiro–Wilks test. Variability among the replicates was assessed through Steel–Dwass test for each treatment group. Data was combined, since no differences could be detected between the replicates. For the neutral red retention assay, comet assay, and micronucleus test significant differences between treatment groups and negative control were determined using ANOVA-on-ranks for non-parametric data sets followed by a post-hoc test according to Dunn’s method. For the fish sexual development test, mortality after 60 days was assessed through two-tailed Fisher’s exact test, where TMDD-treated groups and negative control were always compared pair wise. In the following step, significance levels were adjusted using Holm–Bonferroni method. Effects of TMDD on zebrafish sexual development were evaluated through Pearson’s method by analyzing the frequency of male, female, and hermaphrodite individuals. Statistical analysis was carried out using SAS JMP version 9.0 (SAS Institute GmbH, Böblingen, Germany).
Results In vitro toxicity of TMDD Cytotoxicity in RTL-W1 cells The viability of TMDD-treated cells is given as percentage relative to two internal negative controls. Neutral red retention assay data is presented as the combined results of three replicates (Fig. 1). After sigmoid regression, data distribution was in accordance with a typical sigmoid dose–response curve (R=0.99, 95 % confidence interval). The average NR50 value was 182 mg/L TMDD. There were significant differences between the negative control and cells exposed to ≥100 mg/ L (p=0.001) TMDD.
Environ Sci Pollut Res (2014) 21:8233–8241
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Genotoxicity in the micronucleus test For the micronucleus assay, data were recorded as the proportion of RTL-W1 cells containing micronuclei of a total of 2,000 cells counted. The combined results of three independent replicates are shown. Exposure to TMDD did not result in significantly higher rates of micronucleus formation, if compared to the negative control (Fig. 3). Only the positive control (190 μg/L 4-NQO) showed a prominent genotoxic effect (p
RESEARCH ARTICLE
(Eco)toxicological effects of 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) in zebrafish (Danio rerio) and permanent fish cell cultures Krisztina Vincze & Martin Gehring & Thomas Braunbeck
Received: 27 October 2013 / Accepted: 17 March 2014 / Published online: 1 April 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) is a high-production volume chemical used in paper, ink, pesticide, and adhesive industries as a wetting and anti-foaming agent. The physicochemical properties and slow biodegradation rate of TMDD indicate a low bioaccumulation potential but a high prevalence in the environment. As a consequence, TMDD has been detected in several European rivers in the nanogram per liter and lower microgram per liter range; however, its environmental risk to aquatic organisms is considered low. Recent studies almost exclusively focused on acute effects by TMDD, little is known about cytotoxic and genotoxic effects, reproduction and developmental toxicity, endocrine disruption, and any kind of long-term toxicity and carcinogenicity so far. The present study aims to provide more specific baseline information on the ecotoxicological effects of TMDD in fish. For this end, cyto- and genotoxicity assays were carried out in vitro with the permanent fish cell line RTLW1; in addition, in vivo studies were conducted with the early
Responsible editor: Markus Hecker K. Vincze (*) : T. Braunbeck Aquatic Ecology and Toxicology Group, Center for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany e-mail: [email protected] T. Braunbeck e-mail: [email protected] M. Gehring Verband kommunaler Unternehmen (VKU), Invalidenstr. 91, 10115 Berlin, Germany e-mail: [email protected] Present Address: K. Vincze Institute for Evolution and Ecology, Animal Physiological Ecology, University of Tübingen, Konrad-Adenauer-Strasse 20, 72072 Tübingen, Germany
life stages of zebrafish (Danio rerio) in order to fill the data gaps in developmental toxicity and endocrine disruption. TMDD showed a cytotoxic and slight genotoxic potential in fish cell lines; moreover, various sublethal and lethal effects could be detected in developing zebrafish embryos. There was no evidence of endocrine-disrupting effects by TMDD; however, mortality following prolonged exposure to TMDD during fish sexual development test was clearly higher than mortality in the fish embryo test after 96-h exposure. Our results thus confirmed previous findings of laboratory screening tests, suggesting short-term toxic effects of TMDD in the intermediate, and long-term effects in the lower milligram per liter range. Keywords TMDD . Zebrafish . RTL-W1 fish cell line . Fish embryo test . Fish sexual development test . Comet assay . Neutral red retention assay . Micronucleus test
Introduction Surfactants are widely used for industrial applications as well as for personal care products and household formulations. Due their extensive usage and production, they usually end up in wastewater treatment plants and are removed mainly by biological and physical processes (Camacho-Muñoz et al. 2014). Still, surfactants can be found in considerable variety in waste and surface waters (Schröder and Ventura 2000). 2,4,7,9-Tetramethyl-5-decyne-4,7-diol (TMDD) is a nonionic surfactant (alcohol ethoxylate) with a structure of two hydrophilic moieties connected to hydrophobic residues; it is used as a coating and anti-foaming agent in pesticide, ink, paper, and adhesive industries. TMDD enhances the wetting of materials like plastic, wood, and paper through reducing the surface tension (Air Products and Chemicals Inc. 2001).
8234
Environ Sci Pollut Res (2014) 21:8233–8241
Table 1 summarizes the basic physicochemical properties of TMDD. With an annual production and import volume of over one million pounds alone in the USA (US Environmental Protection Agency 2008), TMDD has become a highproduction volume (HPV) chemical; the production amount in Europe is so far unknown (Guedez et al. 2009). Generally, there is increasing evidence that TMDD may easily reach the environment through its various applications. TMDD has a low potential to bioaccumulate; however, its biodegradation rate is slow to negligible; therefore, it is judged to have high persistence in the environment (US Environmental Protection Agency 2008). As a consequence, TMDD has been detected in several European rivers like Lippe (Dsikowitzky et al. 2004a, b), Main, Nidda (Guedez et al. 2008), Rhine (Guedez et al. 2009; Schwarzbauer and Heim 2005; van Stee et al. 2002), Meuse, Scheldt (van Stee et al. 2002), Mulde (Heinzel 2006), Spree (Schwarzbauer and Ricking 2010), Ruhr (LANUV 2008) and in the industrial area of Kavala in Northern Greece (Grigoriadou et al. 2004) with concentrations in the nanogram per liter and lower microgram per liter range. In the river Rhine at Worms, Guedez et al. (2009) calculated an overall annual TMDD load of about 23 t/year, suggesting a fairly constant discharge, the most likely source being effluents from municipal wastewater treatment plants. Guedez and Püttmann (2011) also investigated the occurrence and fate of TMDD in four wastewater treatment plants and found only partial removal from municipal wastewater with elimination rates between 33 and 68 %. Recycled paper residues entering the sewage sludge represent a potential source of TMDD contamination in surface waters. Gehring et al. (2009)
analyzed six recycling toilet paper products of different origin; TMDD was reported in all samples with concentrations between 0.16 and 2.39 mg/kg. So far, little is known about the presence of TMDD in drinking water. However, TMDD was already detected in mineral water samples with concentrations up to 50 μg/L (Kleinschnitz and Schreier 1998). TMDD is considered to have only a minor impact on the aquatic environment (Deutscher Bundestag 2009), although risk assessment has been mainly based on acute toxicity. The 96-h LC50 values of acute fish tests with fathead minnow (Pimephales promelas) and common carp (Cyprinus carpio) are at 36 and 42 mg/L, respectively; acute tests with Daphnia magna gave 48-h EC50 values of 15 mg/L (Air Products and Chemicals Inc. 2001). With respect to effects other than acute toxicity, there is a fundamental lack of investigations into basic cytotoxic and genotoxic effects, reproduction and developmental toxicity, endocrine disruption, and any kind of long-term toxicity and carcinogenicity. Therefore, the present study was designed to provide baseline information on ecotoxicological effects of TMDD in fish. To this end, cyto- and genotoxicity assays (neutral red retention assay, comet assay, micronucleus test) were carried out in vitro with the permanent fish cell line RTLW1; in addition, prolonged in vivo studies focusing on teratogenic and endocrine potentials (fish embryo test (FET), fish sexual development test (FSDT), OECD 2011) were carried out with the zebrafish (Danio rerio).
Materials and methods Chemicals
Table 1 Physicochemical properties of TMDD (US Environmental Protection Agency 2008) Physicochemical features of 2,4,7,9-Tetramethyl-5-decyne-4,7-diol (TMDD)
All chemicals used were of the highest purity available. Unless stated otherwise, chemicals were purchased from Sigma-Aldrich (Deisenhofen, Germany).
Structure
In vitro toxicity screening Cell culture techniques
CAS number Melting point (measured) Boiling point (measured) Vapor pressure (measured) Water solubility (measured) Log octanol-water partition coefficient (Kow) (measured) Henry’s law constant (estimated by Estimation Program Interface Suite™) Half life (estimated by Estimation Program Interface Suite™)
126-86-2 54–55 °C 262–263 °C 0.62–0.7 kPa at 20 °C 1.7 g/L at 20 °C 2.8 8,58483 E-007 Atm-m3/mol Air—6,039 h Water—900 h Soil—900 h Sediment—3,600 h
The cell line RTL-W1 is a fibroblast-like permanent cell line derived from the liver of adult rainbow trout (Oncorhynchus mykiss), growing as a monolayer. It is a sensitive tool for assessing the toxic potentials of chemicals due to its high biotransformation capacity, i.e., the expression of cytochrome P450 enzymes (Lee et al. 1993). Cells were maintained in 75cm 2 plastic culture flasks (Nunc Nunclon Delta T-25, Langenselbold, Germany) in Leibowitz (L15) medium supplemented with 10 % fetal bovine serum (Biochrom, Berlin, Germany) and 1 % penicillin/streptomycin solution (Seiler et al. 2006). Cells were transferred into new flasks every 6– 8 days and were stored at 20 °C until further use.
Environ Sci Pollut Res (2014) 21:8233–8241
Neutral red retention assay The neutral red retention assay is a rapid in vitro assay assessing cell viability through the quantification of lysosomal retention of the dye neutral red (Borenfreund et al. 1988). Suspensions of RTL-W1 cells were exposed to TMDD concentrations along a 96-well plate (Renner, Dannstadt, Germany) in 1:2 serial dilutions with L15 medium in a range from 800 to 12.5 mg/L. Supplemented L15 medium served as a negative control, and 80 mg/L 3,5-dichlorophenol were used as positive control. The cytotoxic potential of TMDD was determined after cell incubation for 48 h at 20 °C as described by Babich and Borenfreund (1991). Neutral red retention was measured at a wavelength of 540 nm using a Spectra III multiwell plate reader (Tecan, Crailsheim, Germany). Dose–response curves expressing the viability of cells compared to controls were analyzed using SigmaPlot 11.0. (SYSTAT, Erkrath, Germany). The neutral red retention assay of TMDD was carried out in three independent replicates. Comet assay The alkaline comet assay or single cell gel electrophoresis is a sensitive technique for the detection of DNA single- and double-strand breaks, alkali-labile sites, and incomplete excision repair events (Schnurstein and Braunbeck 2001). A volume of 2 ml of RTL-W1 cell suspension in L15 medium with a density of 5×106 cells/ml was given into each well of a 6well plate (Renner) and incubated overnight at 20 °C. After rinsing the wells with phosphate-buffered saline (PBS), cells were exposed for 48 h to TMDD concentrations of 0, 20, 40, 60, 80, and 100 mg/L suspended in L15 medium. Pure medium served as a negative control, 5-min UV light radiation was used as a positive control. After exposure, cells were rinsed with PBS, detached from the wells by trypsin and processed as described in detail by Boettcher et al. (2011). The comet assay was performed according to the original protocol by Singh et al. (1998). DNA was stained with 20-μm ethidium bromide immediately before scoring. An Axioskop fluorescence microscope (Zeiss, Jena, Germany) equipped with a ×34 magnification and an excitation filter of 518 nm was used for evaluation. Exactly 100 randomly selected cells were scored with a high-sensitivity gray scales CCD camera (Optilas, Munich, Germany), and DNA tail moments (tail length × fluorescence intensity in the tails) were calculated with the image-analysis system Komet 5.5™ (Kinetic Images, Liverpool, UK). TMDD genotoxicity was assayed in three independent replicates. Micronucleus test Micronuclei are generated during failure of cell division through the lack of centromeres or inadequate function of
8235
microtubules (Al-Sabti and Metcalfe 1995), thus indicating potentials of genotoxicity. Six-well plates (Renner, Dannstadt, Germany) were supplied with 2 ml of RTL-W1 cell suspension at a density of 3×104 cell/ml. Glass cover slips (22× 22 mm; Langenbrinck, Emmendingen, Germany) were placed in each well to provide an optimal growth surface. After overnight incubation at 20 °C, cells were exposed to TMDD concentrations of 40, 80, and 160 mg/L diluted in L15 medium. Pure L15 medium served as a negative control, 4nitroquinoline-1-oxide (4-NQO) at a concentration of 190 μg/L was used as a positive control. After 48-h exposure, cells were rinsed with phosphate-buffered saline (PBS), and TMDD solutions were replaced by pure L15 medium for another 72 h in order to ensure cell division. Cell fixation was carried out according to Boettcher et al. (2010). After staining with acridine orange solution, 2,000 randomly selected cells per slide were scored with an Aristoplan microscope equipped with a ×340 lens and an excitation filter of 490 nm. Results were recorded as the percentage of cells containing micronuclei of the total number of cells inspected. Three independent replicates of the micronucleus assay with TMDD were conducted. In vivo toxicity screening Ethic statement In vivo toxicity tests with embryos were only conducted with non-protected stages of zebrafish embryos in order to comply with current EU animal welfare regulations (European Union 2010). The long-term experiments (fish sexual development test) were carried out in compliance with German animal ethics regulations under permission 35–9185.81/G-134/07 by local authorities at Karlsruhe (Germany). Maintenance and breeding of zebrafish The Aquatic Ecology and Toxicology Group at the Heidelberg University rears several stocks of zebrafish based on the West Aquarium strain. Fish were kept at 26±1 °C in 160-L tanks under flow-through conditions. The room was light-isolated, and an artificial dark/light cycle of 10:14 h was maintained. The animals were fed twice daily with dry flake food (TetraMin™ Hauptfutter, Tetra, Melle, Germany) and additionally with freshly hatched Artemia larvae the day before spawning. For egg production, mating groups were transferred into special 4-L aquaria equipped with a stainless steel grid on the bottom with a mesh size of 1.25 mm, which allowed the passage of eggs into a separate spawning tray, thus preventing predation by adult zebrafish. Green plastic wire material was used as spawning stimulus. Spawning took place in the early morning period after the onset of light. Eggs
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were collected 30–60 min after spawning and used for in vivo toxicity tests. Fish embryo test (FET) The embryo test with the zebrafish is a simple and costeffective test, which can be used for toxicity screening and early risk assessment of chemicals (Braunbeck et al. 2005; Lammer et al. 2009; Nagel 2002). The zebrafish embryonic development is short (detailed by Kimmel et al. 1995) and easy to observe due to the transparency of eggs. In the current study, TMDD concentrations of 0, 20, 40, 80, and 320 mg/L were tested for up to 120 h. The FET was carried out in 24well plates (Renner). Artificial soft water according to ISO 7346/3 (ISO 1996) was used as a negative control, and 3.7mg/L 3,4-dichloroaniline (DCA) served as a positive control. The development of the embryos was observed trough a CKX41 microscope (Olympus, Hamburg, Germany) at 24-, 48-, 72-, 96-, and 120-h post fertilization (hpf). Lethal and sublethal endpoints were recorded at appropriate time periods. Lethal endpoints were egg coagulation, non-development of somites, non-detachment of the tail and lack of heartbeat, whereas sublethal endpoints comprised non-development of the eyes, lack of eye pigmentation, absence of spontaneous movement and blood circulation, formation of edemata, malformations of the spinal cord, tail, head, heart and yolk sac, and growth retardation (Braunbeck et al. 2005; Nagel 2002). Time of hatching was determined as well. LC50 and EC50 values were calculated after generating dose–response curves in SigmaPlot 11.0. Three independent replicates of FETs with TMDD were carried out. Fish sexual development test (FSDT) The zebrafish is a gonochoristic species, i.e., in the early development all individuals develop ovary-like gonadal organs; later, males differentiate to hermaphrodites and finally into phenotypic males (Segner et al. 2003). The time of sexual development is, therefore, thought to be a period very sensitive to endocrine disrupting substances (Brion et al. 2004). Based on this, the fish sexual development test (OECD TG 229) with juvenile zebrafish was developed for the detection of androgenic and estrogenic potentials of chemicals (Holbech et al. 2006; OECD 2011). Exposure to 0-, 1-, 10-, and 20-mg/L TMDD was initiated in two replicates with fertilized eggs incubated in 20-cm diameter Petri dishes at 27 °C with a photoperiod of 12:12 h. After becoming free-swimming, 40 larvae were transferred into 12-L aquaria equipped with a flow-through system. The entire test solutions were renewed once daily by mixing of defined aerated water volumes and TMDD stock solutions in the overflow system to ensure high water quality and to maintain constant TMDD concentrations.
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For detailed description and zebrafish larvae feeding regime, see Knörr and Braunbeck (2002). At 60-days post-hatch, fish were euthanized by anesthesia with a saturated solution of benzocaine (4-aminobenzoic acid ethyl ester) for histological investigations. Fish were fixed in Davidson’s fixative (OECD 2010). Following dehydration in a graded series of ethanol and paraffin embedding, longitudinally sections of 5-μm thickness were cut with an HN-40 microtome (Leica, Nussloch, Germany) through the gonadal region. Staining the sections with hematoxylin and eosin solution allowed an easy light microscopical examination of the gonads. The percentage of intersex fish as well as the sex ratio was recorded as additional endpoints. Finally, any form of gonad malformation was recorded. Statistical analysis In vitro test data was first investigated for normal distribution using Shapiro–Wilks test. Variability among the replicates was assessed through Steel–Dwass test for each treatment group. Data was combined, since no differences could be detected between the replicates. For the neutral red retention assay, comet assay, and micronucleus test significant differences between treatment groups and negative control were determined using ANOVA-on-ranks for non-parametric data sets followed by a post-hoc test according to Dunn’s method. For the fish sexual development test, mortality after 60 days was assessed through two-tailed Fisher’s exact test, where TMDD-treated groups and negative control were always compared pair wise. In the following step, significance levels were adjusted using Holm–Bonferroni method. Effects of TMDD on zebrafish sexual development were evaluated through Pearson’s method by analyzing the frequency of male, female, and hermaphrodite individuals. Statistical analysis was carried out using SAS JMP version 9.0 (SAS Institute GmbH, Böblingen, Germany).
Results In vitro toxicity of TMDD Cytotoxicity in RTL-W1 cells The viability of TMDD-treated cells is given as percentage relative to two internal negative controls. Neutral red retention assay data is presented as the combined results of three replicates (Fig. 1). After sigmoid regression, data distribution was in accordance with a typical sigmoid dose–response curve (R=0.99, 95 % confidence interval). The average NR50 value was 182 mg/L TMDD. There were significant differences between the negative control and cells exposed to ≥100 mg/ L (p=0.001) TMDD.
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Genotoxicity in the micronucleus test For the micronucleus assay, data were recorded as the proportion of RTL-W1 cells containing micronuclei of a total of 2,000 cells counted. The combined results of three independent replicates are shown. Exposure to TMDD did not result in significantly higher rates of micronucleus formation, if compared to the negative control (Fig. 3). Only the positive control (190 μg/L 4-NQO) showed a prominent genotoxic effect (p
