1-(2,4-dimethylphenylazo)-2-naphthol

PBT assessment

PBT assessment: overall result

PBT status:

the substance is not PBT / vPvB

Justification:

Classification of 1-(2,4-dimethylphenylazo)-2-naphthol for effects in the environment:

 

The chemical 1-(2,4-dimethylphenylazo)-2-naphthol (CAS no. 3118-97-6) is used in as a dye for plastics and inks. The aim was to assess whether the PBT criterion within Annex XIII was fulfilled for 1-(2,4-dimethylphenylazo)-2-naphthol. The PBT criterion was herein assessed based on experimental data in conjunction with standardized environmental fate models. Here follows a description of the PBT assessment.

 

 

Persistence assessment

The tested substance does not fulfils the P criterion within Annex XIII based on the assessment that here follows:

 

Biotic degradation

In an experimental key study from peer reviewed journal (Haiyan Xu et. al; 2010), biodegradation experiment of 1- (2, 4-dimethylphenylazo)-2-naphthol (CAS no. 3118-97-6) was performed. The test is carried at a temperature of 37ᵒCwith a duration period of 2 days under anaerobic conditions using bacteria as a test inoculum. The anaerobic bacterial species used in the study were obtained from the American Type Culture Collection (ATCC).The bacterial seed cultures of 1.5 ml were inoculated into flasks containing 100 ml BHI broth. Dye stock solutions were added to the medium at final concentrations of 10µg/ml, the cultures were incubated at 37ᵒC in an anaerobic chamber for 2 days without agitation. Three control consisted of sterile liquid medium, sterile liquid medium with bacteria, and one of sterile liquid medium with dyes were used in the study. All strains were preserved at-80ᵒC in 10 to 15% glycerol stocks and revived as needed. The strains, except for Lactobacillus species, which were routinely cultured on de Mann-Rogosa-Sharpe (MRS) broth or agar (Becton Dickinson & Company), were routinely cultured on Brain Heart Infusion (BIH) broth supplemented with vitamin K and hemin or on PRAS brucella blood agar plates supplemented with vitamin K and hemin at 37C under an atmosphere of 91% nitrogen, 4% hydrogen and 5% carbon dioxide. The bacterial strains were grown anaerobically at 37ᵒC by using BHI broth or MRS supplemented with various Sudan dye. A loopful for each strain was cultured in static conditions at 37ᵒC for 24 h in an Erlenmeyer flask containing 10 ml medium for use as a seed culture. Initial test subs. concentrations and inoculum conc. used for the study was 10 mg/l. Metabolites of the reduction of the test compound 1- (2, 4-dimethylphenylazo)-2-naphthol were isolated and identified by HPLC and Liquid chromatography/ electrospray ionization mass spectrometric(LC/ESI-MS). After extraction of samples and filtration, 40 µl of each sample was analyzed with a Helwet-Packard 1050 Series high performance liquid chromatography (HPLC) equipped with a with a variable wavelength detector (detection wavelengths were 250 and 500 nm), an auto sampler, and a Luna C18 column (150×3.0 mm, particle size, 5µm, Phenomenex) with a guard column (40 × 3.0 mm, Phenomenex). The mobile phase was composed of a linear gradient of acetonitrile: water containing 0.1% formic acid from 30:70 to 95:5 for 40 min. The peak area was used with a standard curve to calculate the concentration of Sudan dyes.

Identification of the metabolites was also performed using a similar procedure as detailed above with LC/ESI-MS. LC/ESI-MS data were acquired on a Thermo Finnigan Quantum Ultra mass spectrometer equipped with an Agilent 1100 Series HPLC system and a Prodigy ODS 2.0×250 mm 5µm 100 A HPLC column (Phenomenex). The mass spectrometer was operated in the positive-ion electrospray ionization (ESI) mode with an in-source collision-induced dissociation (CID) offset of 0 V. Other ESI conditions were spray voltage 4.0 kV, capillary temperature 350ᵒC, sheath gas 40 psi, ion sweep gas 0 and auxiliary gas 25. Standards and samples were extracted with ethyl acetate, which was dried and then redissolved in a starting buffer (acetonitrile: water containing 0.1% formic acid- 5:95). Much of the dried sample was insoluble, so samples were taken without including the precipitate. The starting buffer was held 20 min, ramped in 1 min to acetonitrile: water containing 0.1% formic acid-95:5 at 21 min and held until 40 min. Reduction of the1- (2, 4-dimethylphenylazo)-2-naphtholwas determined by measuring the disappearance of the absorbance at 500 nm immediately after extraction with ethyl acetate as well. The results are presented in percentage (%) obtained by the means from triplicate incubations.

Among the tested bacterial strains, B. infantis, C. indolis, E. faecalis, L. rhamnosus and R. obeum were able to completely degrade (100%) the test substance 1- (2, 4-dimethylphenylazo)-2-naphthol whereas partial degradation of the 1- (2, 4-dimethylphenylazo)-2-naphthol occurred by the bacterial strains such as B. ovatus, B. distasonis, B. thetaiotaomicron, B. caccae, C. clostridioforme, E. faecium, F. russi and L. paracasei. Thus, the test substance 1 - (2, 4 -dimethylphenylazo)-2 -naphthol is readily biodegradable in water.The bacteria which are unable to degrade the 1 - (2, 4 -dimethylphenylazo)-2 -naphthol are B. vulgatus, B. uniformis, B. fragilis, B. longum, B. adolesecentis, B. catenulatum, B. angulatum, C. perfringens, C. butyricum, C. ramosum, C. difficle, C. leptum, E. aerofaciens, E. limosum, E. tenue, E. coli, F. nucleatum, L. bifidus, L. reuteri, L. ruminis, P. magnus and R. gnavus. The metabolite produced from 1 - (2, 4 -dimethylphenylazo)-2 -naphthol by E. faecalis was identified as 2,4 -dimethylaniline, based on an identical retention time of 16.55 min and ions at m/z122 [MH+] and 163 [MH++acetonitrile]. 1 -Amino-2 -naphthol from1 - (2, 4 -dimethylphenylazo)-2 -naphthol could not be detected in the extracted samples. No metabolites of 1 - (2, 4 -dimethylphenylazo)-2 -naphthol produced by E. coli was detected by LC/ESI-MS, indicating that the dyes were not degraded by the bacterium. Thus, based on percentage degradation of test chemical,1 - (2, 4 -dimethylphenylazo)-2 -naphthol is considered to be readily biodegradable in nature.

 

In an another study from peer reviewed journal (Haiyan Xu et. al; 2007), biodegradation experiment of 1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol (CAS no. 3118-97-6) was performed. The test is carried for 30 hr under anaerobic conditions using microorganisms as a test inoculum. Test microorganisms were obtained from human intestine. Test chemical conc. used for the study was 10 mg/l. Stock solutions of test chemical Sudan II was prepared by dissolving in 100% ethanol (1 mg/ml). Fresh diluted human fecal suspensions (6 ml; 10%, wt/vol) were transferred under anaerobic conditions into flasks containing 300 ml brain heart infusion broth to observe the effect of the microflora on decolorization of the 1 -[(2,4 -dimethylphenyl)diazenyl]-2 -naphthol. Samples (supernatants, cell extracts, and debris) were extracted with ethyl acetate to ensure that dye bound to bacterial cells could be released from the cells as well. Each residue was dissolved in 1 ml acetonitrile, and 40µl of each sample was analyzed by high-performance liquid chromatography with a Hewlett-Packard 1050 equipped with a variable-wavelength detector (the detection wavelengths used were 250 and 500 nm) and a reversed-phase Luna C18 column. The peak area was used to calculate the concentration of Sudan dye. Reduction of Sudan dyes was determined by monitoring the disappearance of the absorption peak for each dye at 500 nm and by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESIMS/ MS) when the cultures were extracted with ethyl acetate as well. To identify the Sudan dye metabolites, separate experiments were conducted in which the human intestinal microflora was incubated with Sudan II for 30 hr. Ethyl acetate extracts of incubation cultures were dried, and the residues were extracted with starting buffer (5% acetonitrile, 94.9% water, 0.1% formic acid). Much of the dried sample was insoluble, and soluble metabolites were analysed by LC/ESI-MS/MS. For analyses of Sudan dye metabolites, the starting buffer composition was held for 20 min, ramped quickly (in 1 min) to 95% acetonitrile–4.9% water–0.1% formic acid at 21 min, and held to 40 min. Product ion spectra, retention times (Rts), and UV data for metabolites were compared to those for authentic compounds for identification. The metabolites from Sudan dyes were detected by LC/ESI-MS/MS after 4 h incubation, and the amounts of the metabolites increased with the incubation time. No reduction of the tested Sudan dyes was observed in un-inoculated controls. The percentage degradation of test substance 1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol was determined to be 100% degradation after 30 hrs. The metabolite with an Rt of 18.7 min from1-[(2,4-dimethylphenyl)diazenyl]-2-naphtholwas identified as 2.3µg/ml 2,4-dimethylaniline (52.8%) on the basis of its Rt and product ion mass spectrum. Thus, based on percentage degradation,1 -[(2,4 -dimethylphenyl)diazenyl]-2 -naphthol was considered to be readily biodegradable in nature.

 

In an additional supporting study from authoritative database (SRC PhysProp Database, 2017), biodegradation experiment was carried out for 30 days for determining the percentage degradation of the test substance 1- (2, 4-dimethylphenylazo)-2-naphthol (CAS no. 3118-97-6). The test is carried under aerobic condition at a temperature of 27°C and pH 7. Sewage was used as a test inoculum for the study. Initial cell/ biomass concentration were1350, 1450, 1500, 1450, 1390 and 1500 mg/l, respectively. Conc. of test chemical used for the study were40, 60, 80, 100, 120 and 140 mg/l, respectively. Analytical methods used for the study were spectrophotometer and TLC. The percentage degradation of test substance 1- (2, 4-dimethylphenylazo)-2-naphthol was determined to be 100% in 30 days. Thus, based on percentage degradation, 1- (2, 4-dimethylphenylazo)-2-naphthol is considered to be readily biodegradable in nature.

 

Another biodegradation study was carried out for 60 days for determining the percentage degradation of the test substance 1- (2, 4-dimethylphenylazo)-2-naphthol (CAS no. 3118-97-6)(SRC PhysProp Database, 2017). The test is carried under aerobic condition at a temperature of 27°C and pH 7. Sewage was used as a test inoculum for the study. Initial cell/ biomass concentration were 1720; 1050 mg/l and1540; 920mg/l, respectively. Conc. of test chemical used for the study was 20mg/l, respectively. Analytical methods used for the study were spectrophotometer and TLC. The percentage degradation of test substance 1 - (2, 4 -dimethylphenylazo)-2 -naphthol was determined to be 100% after 60 days. Thus, based on percentage degradation, 1 - (2, 4 -dimethylphenylazo)-2 -naphthol is considered to be inherently biodegradable in nature.

 

Environmental fate

According to the fugacity model levels III, the most likely environmental fate for this test chemical is soil (i.e.estimated to 62.1%). In soil, 1-(2,4-dimethylphenylazo)-2-naphthol was expected to have negligible to slow mobility based upon a LogKOC in the range 3.6 – 5.14. Thehalf-life in soil (120 days estimated by EPI suite) indicates that the chemical is persistent in soil and the exposure risk to soil dwelling animals is moderate to low.

 

If released in to the environment, 33.6% and 4.24% of the chemical will partition into sediment and water according to the Mackay fugacity model level III in EPI suite version 4.1 (2017). However, the half-life (60 days in water and 541.66 days in sediment as estimated by EPI suite) indicates that the substance is persistent in both the compartments.

 

Although the half-life values in water and sediment indicate the chemical to be Very persistent (vP), since the substance is mainly partitioned in soil where it is non persistent and experimental biodegradation data is available for the substance, it has been concluded that 1-(2,4-dimethylphenylazo)-2-naphthol is not likely to be persistent in nature.

 

 

Bioaccumulation assessment

The tested substance fulfils the B criterion within Annex XIII based on the assessment that here follows:

 

Theestimated BCF value from authoritative database was determined to be in the range 1215-11749 and theoctanol water partition coefficient of the test chemical is 6.60 which is more than the threshold of 4.5. If this chemical is released into the aquatic environment, there should be a moderate to high risk for the chemical to bioaccumulate in fish and food chains.

 

Although the BCF value indicate the chemical to be Very bioaccumulative (vB), since only predicted data is available for the substance, it has been concluded that 1-(2,4-dimethylphenylazo)-2-naphthol is bioaccumulative infish and food chains.

 

Toxicity assessment

The tested substance does not fulfil the T criterion within Annex XIII based on the assessment that here follows:

 

Mammals

The tested chemical is regarded to be not classified for carcinogenicity, mutagenicity and reprotoxicity, Further, there is no evidence of chronic toxicity, as identified by the classifications STOT (repeated exposure), category 1(oral, dermal, inhalation of gases/vapours, inhalation of dust/mist/fume) or category 2 (oral, dermal, inhalation of gases/vapours, inhalation of dust/mist/fume).

 

Aquatic organisms

As per Column 2 (Annex VIII) of the REACH regulation, a study does not need to be conducted if there are mitigating factors indicating that aquatic toxicity is unlikely to occur for instance if the substance is highly insoluble in water. The test substance was determined to be highly insoluble in water (solubility= 0.05445 mg/L). The chemical was therefore not considered as hazardous to aquatic environments as per the criteria set out in Annex XIII.

 

 

Conclusion

Based on critical, independent and collective evaluation of information summarized herein, the tested compound fulfil the B criterion but does not fulfil the P and T criterion and has therefore not been classified as a PBT compound within Annex XIII.