Reactive Blue 4 was effectively decolorized and biotransformed by aerobic bacterial granules (ABGs) under in vitro conditions.
The ABGs decolorized Reactive Blue 4 at a wide range of pH (4.0 to 11.0) and temperature (20 to 55 °C).
The granules tolerated and degraded high dye concentrations up to 1000 mg L⁻¹, with a maximum decolorization rate (Vmax) of 6.16 ± 0.82 mg L⁻¹ h⁻¹ and a Michaelis constant (Km) of 227 ± 41 mg L⁻¹.
The decolorization rate was higher under static conditions (5.01 mg L⁻¹ h⁻¹) compared to shaking conditions (3.71 mg L⁻¹ h⁻¹).
During continuous exposure cycles, the decolorization rate increased from 1.27 mg L⁻¹ h⁻¹ (61% decolorization) in the 1st cycle to 1.79 mg L⁻¹ h⁻¹ (86% decolorization) by the 9th cycle, then decreased to 1.15 mg L⁻¹ h⁻¹ (55% decolorization) by the 16th cycle, indicating retained tolerance.
Biotransformation was confirmed by HPLC, FT-IR, and GC-HRMS analysis, identifying metabolites such as 4-amino-9,10-dihydro-9,10-dioxoanthracene-2-sulfonic acid, 2-(4,6-dichloro-1,3,5-triazin-2-ylamino)-4-aminophenol, and 1-aminoanthracene-9,10-dione.
Significant reduction in organic load was observed: Chemical Oxygen Demand (COD), Biological Oxygen Demand (BOD), and Total Dissolved Solids (TDS) were reduced by 90%, 91%, and 77%, respectively, after 72 h under static conditions.[2]

Cytotoxicity Assay: The cytotoxicity of Reactive Blue 4 and its biotransformation metabolites was evaluated on HaCat (human keratinocyte) and FHM (fish epithelial) cell lines using the MTT assay.
Cells were seeded in 96-well plates (HaCat: 10⁵ cells mL⁻¹; FHM: 2×10⁵ cells mL⁻¹) in their respective media (DMEM/F12 for HaCat, MEM for FHM).
The dye or its metabolites (final concentration 0.01 mg mL⁻¹), obtained at different biotransformation time points, were added to the wells.
Plates were incubated for 48 h in a CO₂ incubator (5% CO₂, 37°C).
After incubation, media was removed, wells were washed with PBS, and MTT solution (40 µg per well) was added followed by a 4 h incubation.
The resulting formazan crystals were dissolved in dimethyl sulphoxide, and absorbance was measured at 570 nm (with background subtraction at 690 nm).
Cell viability was calculated as a percentage relative to untreated control cells.[2]
Genotoxicity Assay (Comet Assay): The genotoxicity of Reactive Blue 4 and its metabolites was assessed on human blood lymphocytes, HaCat, and FHM cell lines using the alkaline comet assay.
For lymphocytes, cells (10⁴ cells mL⁻¹) were incubated with the dye (0.25 mg mL⁻¹) or its metabolites in RPMI medium for 1 h at 37°C. H₂O₂ (100 µM, 5 min, 4°C) served as the positive control.
For HaCat and FHM cells, cells (4×10⁵ cells mL⁻¹) were treated with the dye or metabolites (0.01 mg L⁻¹) in 96-well plates for 48 h, then detached using trypsin-EDTA.
Treated cells were washed, suspended in a small volume of medium, mixed with low-melting-point agarose, and layered onto microscope slides pre-coated with normal-melting-point agarose.
Cells were lysed (in a solution containing NaCl, EDTA, Tris, DMSO, and Triton X-100, pH 10), followed by alkaline unwinding (in electrophoresis buffer: NaOH, EDTA, pH >13) and electrophoresis.
Slides were neutralized, stained with ethidium bromide (20 µg mL⁻¹), and visualized under a fluorescence microscope.
DNA damage was quantified for ~300 cells per sample using image analysis software, with percent tail DNA as the parameter.[2]

Phytotoxicity: Reactive Blue 4 exhibited phytotoxicity to common wheat (Triticum aestivum). At a concentration of 100 mg L⁻¹, it reduced seed germination rate by 27%, root length by 48%, shoot length by 29%, chlorophyll a content by 76%, and chlorophyll b content by 53%.
Biotransformation metabolites (especially after 48 hours of static treatment) did not show significant phytotoxicity, and germination rate, growth, and chlorophyll content were close to normal levels. [2]
Cytotoxicity: Reactive Blue 4 (0.01 mg mL⁻¹) significantly reduced the cell viability of HaCat cells and FHM cells, with cell viability decreasing to 53.33% and 44.97%, respectively.
The cytotoxicity of its metabolites decreased over time. Metabolites after 48 hours of static treatment showed that the survival rate of HaCat cells was 95.83% and that of FHM cells was 100.80%, indicating a significant detoxification effect. [2]
Genetic toxicity: Reactive Blue 4 induced significant DNA damage. Compared with the control group, Reactive Blue 4 increased the percentage of tail DNA in human lymphocytes, HaCat cells, and FHM cells by 3.26-fold, 2.02-fold, and 11.5-fold, respectively.
Biotransformation metabolites (from 24-hour static treatment, 48-hour static treatment, and 24-hour static treatment + 24-hour shaking treatment) showed significantly reduced DNA damage, indicating the disappearance of genotoxicity. [2]

[1]. Reactive Blue 4 as a Single Colorimetric Chemosensor for Sequential Determination of Multiple Analytes with Different Optical Responses in Aqueous Media: Cu2+-Cysteine Using a Metal Ion Displacement and Cu2+-Arginine Through the Host-Guest Interaction.

[2]. Effective biotransformation and detoxification of anthraquinone dye reactive blue 4 by using aerobic bacterial granules. Water Res. 2017 Oct 1;122:603-613.

Reactive Blue 4 (RB4) is a water-soluble anthraquinone dye used as a colorimetric chemical sensor. The sensor operates sequentially: free RB4 detects Cu²⁺ ions via a binding site-signal subunit mechanism, forming a 1:1 RB4-Cu²⁺ complex with an association constant of (4.46 ± 0.12) × 10⁵ L mol⁻¹. This complex then distinguishes between the detection of L-arginine (Arg) and L-cysteine (Cys). The detection of Arg is achieved through host-guest interactions (binding constant 1.09 × 10⁴ L mol⁻¹) involving the binding of its amino and carboxyl groups to the complex. Cysteine (Cys) has a higher affinity for Cu²⁺ and can be detected via a metal substitution mechanism, where Cu²⁺ releases free RB4. In a HEPES aqueous buffer solution at pH 7.0, the limits of detection (LODs) for Cu²⁺, arginine (Arg), and Cys were 1.96 μmol L⁻¹, 1.06 μmol L⁻¹, and 1.33 μmol L⁻¹, respectively. The sensor operates effectively in the pH range of 6.0 to 9.0, with optimal response at pH 7.0. Practical applications include the detection of Cu²⁺ in water samples (tap water, river water) and serum samples, Arg in commercial dietary supplements, and Cys in human serum samples, with good recovery rates. The system has also been formulated into paper test strips for visual detection of Arg and Cys. Furthermore, the absorbance change at 607 nm was used to simulate molecular logic gates: a NAND gate for inputs of Cu²⁺ and Arg, and an IMPLICATION gate for inputs of Cu²⁺ and Cys. [1]
Reactive Blue 4 is an anthraquinone dye used in the textile industry and is also a recalcitrant water pollutant.
This study shows that aerobic bacterial particles (ABGs) are an effective system for its bioremediation and detoxification.
Microbial community analysis (16S rRNA metagenomics) revealed changes in particle composition during dye exposure, with increased abundance of phyla such as Firmicutes and Bacteroidetes, as well as families such as Porphyromonas and Cercibacteria, indicating their role in degradation.
Metagenomic analysis inferred from PICRUSt predicted upregulation of exogenous degradation pathways (e.g., naphthalene and benzoic acid degradation) and environmental information processing systems during dye decolorization.
The proposed biotransformation pathway involves reductive cleavage and subsequent decomposition into simpler, less toxic aromatic compounds.
This study highlights the potential of ABGs in treating textile wastewater containing high concentrations of anthraquinone dyes under various environmental conditions. [2]

C23H14CL2N6O8S2

637.4287

635.969

13324-20-4

4499-01-8 (di-hydrochloride salt)

25863

Blue to dark blue solid powder

1.86g/cm3

1.774

6.405

5

14

6

41

1220

0

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~83.33 mg/mL (~130.73 mM)

Solubility in Formulation 1: ≥ 4.17 mg/mL (6.54 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 41.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 4.17 mg/mL (6.54 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 41.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 4.17 mg/mL (6.54 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 41.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: ≥ 2.08 mg/mL (3.26 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 5: ≥ 2.08 mg/mL (3.26 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

 (Please use freshly prepared in vivo formulations for optimal results.)