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HJ 1016-2019 English PDF

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HJ 1016-2019: Water quality - Identification of mutagenicity - Vicia faba root-tip micronucleus test
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Basic data

Standard ID HJ 1016-2019 (HJ1016-2019)
Description (Translated English) Water quality - Identification of mutagenicity - Vicia faba root-tip micronucleus test
Sector / Industry Environmental Protection Industry Standard
Classification of Chinese Standard Z16
Classification of International Standard 13.060
Word Count Estimation 21,294
Date of Issue 2019
Date of Implementation 2019-09-01
Issuing agency(ies) Ministry of Ecology and Environment

HJ 1016-2019: Water quality - Identification of mutagenicity - Vicia faba root-tip micronucleus test


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Water quality - Identification of mutagenicity - Vicia faba root-tip micronucleus test National Environmental Protection Standard of the People's Republic Identification of water quality mutagenicity Vicia faba root tip micronucleus test Water quality-Identification of mutagenicity -Vicia faba root-tip micronucleus test 2019-04-13 released.2019-09-01 implementation Ministry of Ecology and Environment released

Content

Foreword...ii 1 Scope...1 2 Normative references...1 3 Terms and Definitions...1 4 Principles of the method...1 5 Reagents and materials...2 6 Instruments and Equipment...3 7 test organisms...3 8 Test organism preparation...3 9 samples...4 10 Analysis steps...4 11 Calculation and representation of results...6 12 precision...7 13 Quality Assurance and Quality Control...7 14 Waste treatment...8 Appendix A (informative appendix) mitotic cells, apical cell micronucleus counting area and micronucleus determination reference picture...9 Appendix B (Informative Appendix) Characteristics and Conservation of Songzi Green Beans...11 Appendix C (informative) apical treatment table reference picture...12 Appendix D (Normative) Pre-test for acute toxicity of samples...13 Appendix E (informative) Kruskal-Wallis test method...14 Appendix F (Informative Appendix) Original Record Table and Test Report Form...16

Foreword

Protect the ecology for the implementation of the Environmental Protection Law of the People's Republic of China and the Law of the People's Republic of China on Water Pollution Prevention and Control This standard is formulated for the environment, the protection of human health, and the identification of the mutagenicity of water samples. This standard specifies the micronucleus test of the broad bean root tip for the mutagenicity in surface water, groundwater, domestic sewage and industrial wastewater. Test. Appendix D of this standard is a normative appendix, Appendix A ~ Appendix C, Appendix E, Appendix F is an informative appendix. This standard is the first release. This standard is formulated by the Department of Eco-Environmental Monitoring, the Department of Regulations and Standards of the Ministry of Ecology and Environment. This standard was drafted. Jiangsu Changzhou Environmental Monitoring Center (Jiangsu Province Environmental Protection Water Environment Biological Monitoring Key Experiment Room), State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Academy of Environmental Sciences. The standard verification unit. Shanghai Environmental Monitoring Center, Zhejiang Environmental Monitoring Center, Jiangsu Environmental Monitoring Center, Nanjing Environmental Monitoring Center of Jiangsu Province, Suzhou Environmental Monitoring Center of Jiangsu Province and Taizhou Environmental Monitoring Center of Jiangsu Province. This standard is approved by the Ministry of Ecology and Environment on April 13,.2019. This standard has been implemented since September 1,.2019. This standard is explained by the Ministry of Ecology and Environment. Identification of water quality mutagenicity

1 Scope of application

This standard specifies the Vicia faba root tip micronucleus test method for identifying mutagenicity of water samples. This standard applies to the mutagenic identification of surface water, groundwater, domestic sewage and industrial wastewater.

2 Normative references

This standard refers to the following documents or their terms. For undated references, the valid version applies to this standard. GB/T 6920 Determination of pH of water - Glass electrode method HJ/T 91 Surface Water and Wastewater Monitoring Technical Specifications HJ/T 164 Technical Specifications for Groundwater Environmental Monitoring

3 Terms and definitions

The following terms and definitions apply to this standard. 3.1 Micronucleus micronuclei In eukaryotic cells, small nuclei that are free from the main nucleus are micronuclei, whose material composition and characteristics are consistent with the main nucleus. Below 1/3 of the main nucleus, it is a manifestation of chromosomal aberrations. 3.2 Micronucleus rate A certain sample is used to detect several root tips, and each root tip observes 1 000 mitotic interphase cells. The ratio of the number of cells, in twips. 3.3 Mitotic index Observation of 1 000 cells at the same root tip, in the mitosis phase (including pre-, mid-, post-, and end-stage, see Appendix) A Figure A.1) The ratio of the number of cells to the total number of cells, in twips. 3.4 Reference substance A known positive control substance that verifies the sensitivity and effectiveness of the test method for determining the biological characteristics and test conditions of the test No significant changes have occurred, ensuring reliable and effective testing.

4 Principle of the method

The primary roots of broad bean grown after soaking and rooting are exposed to the sample for a certain period of time, and are restored, fixed, and dyed. After the microscopic examination, the micronucleus rate of the primary meristem of the Vicia faba root tip (see Appendix A, Figure A.2) was counted. Mutagens It can act on nuclear material, causing chromosome breaks during mitosis to form fragments, the entire chromosome is detached from the spindle, and the spindle The function of silk traction chromosome movement is impaired. These movements are affected by chromosome fragments or the entire chromosome cannot be stained with normal The color body moves toward the two poles of the cell to form a daughter cell nucleus, while the retained cytoplasm forms a daughter cell micronucleus and causes an increase in its number. Comparison Whether the micronucleus rate of the root tip cells of Vicia faba L. and the blank sample is significantly increased can determine whether the sample is mutagenic.

5 reagents and materials

Unless otherwise stated, analytically pure reagents that meet national standards are used for the analysis, and experimental water is newly prepared deionized Water or distilled water, conductivity (25 ° C) ≤ 0.50 mS/m, pH (25 ° C) between 5.5 ~ 7.5. 5.1 Temperature-controlled water. The experimental water used for soaking seeds (8.2), rooting (8.3), and root tip treatment (10.1) must be placed in advance. In a constant temperature incubator (6.3), equilibrate to a constant temperature reserve at 25 °C ± 1 °C. 5.2 Hydrochloric acid. ρ(HCl) = 1.18 g/ml. 5.3 sodium hydroxide (NaOH). 5.4 Acetic acid. ρ(C2H4O2) = 1.05 g/ml. 5.5 Anhydrous ethanol. ρ(C2H6O) = 0.79 g/ml. 5.6 Basic fuchsin (C20H20ClN3). 5.7 Sodium sulfite (Na2S2O5) or potassium sulfite (K2S2O5). 5.8 Activated carbon powder. 5.9 cyclophosphamide (C7H15Cl2N2O2P·H2O), biological reagent (BR), purity ≥99.0%, reference material of this standard. 5.10 Hydrochloric acid solution. c (HCl) = 1.0 mol/L. Measure 8.3 ml of hydrochloric acid (5.2), slowly add a small amount of water, stir and mix, and dilute to 100 ml with water. 5.11 Sodium hydroxide solution. c (NaOH) = 1.0 mol/L. Weigh 4.0 g of sodium hydroxide (5.3), add a small amount of water, stir to dissolve, and dilute to 100 ml with water. 5.12 Carnos's solution (acetic acid-anhydrous ethanol mixture). 13. Acetic acid (5.4) and absolute ethanol (5.5) were mixed in a volume ratio of 1.3, and the mixture was ready for use. 5.13 Ethanol solution. φ(C2H6O)=70%. Measure 70 ml of absolute ethanol (5.5) and dilute to 100 ml with water. 5.14 Schiff reagent. Commercially available finished reagents can be used. It can also be configured as follows. 5.0 ml of absolute ethanol (5.5) and 0.5 g of basic fuchsin (5.6) were added to a 100 ml flask and dissolved by shaking for 10 min. Dissolve 2.5 g of sodium metabisulfite or potassium metabisulfite (5.7) in 93 ml of water, add to the above flask, and mix. Continue to add 1.5 ml of hydrochloric acid (5.2), shake it to complete dissolution, and the solution is light yellow. Add 0.2 g more activity Carbon powder (5.8), shake for 3 min, filter the solution to make it colorless, and keep it for later use. This solution is refrigerated and protected from light below 4 °C. After 6 months, if the solution is pink or precipitated, it can not be reused. 5.15 SO2 rinse. w (Na2S2O5 or K2S2O5) = 0.5%. Weigh 10.0 g of sodium metabisulfite or potassium sulfite (5.7) dissolved in 90 ml of water to obtain w (Na2S2O5 or K2S2O5) = 10% solution. Pipette each 5.0 ml of this solution and hydrochloric acid solution (5.10) into a 100 ml volumetric flask. Make up to the mark with water and use it now. 5.16 Acetic acid dispersion. φ (C2H4O2) = 45%. Measure 45 ml of acetic acid (5.4), dilute to 100 ml with water, and use it immediately. 5.17 cyclophosphamide reference solution. ρ (C7H15Cl2N2O2P) = 20.0 mg/L. Weigh 0.107 g of cyclophosphamide (5.9) into a small amount of water, stir to dissolve, and dilute to 100 ml with water. Draw the solution 20.0 ml of liquid, make up to 1 000 ml with water, ready for use.

6 Instruments and equipment

Preparation of cyclophosphamide reference solution (5.17) and dilution of the sample using Class A glass gauges in accordance with national standards, other A Class B glass gauge that meets national standards can be used. 6.1 Refrigerated sampling box. 0 ° C ~ 8 ° C. 6.2 Biological microscope. Objective lens 10×, 20×, 40×, eyepiece 10× or 15×. 6.3 Constant temperature incubator. 25 °C ± 1 °C. 6.4 Refrigerator. Refrigeration temperature 0 °C ~ 8 °C, freezing temperature ≤ -18 °C. 6.5 Electronic balance. The division value is 0.000 1 g. 6.6 Constant temperature water bath. 20 ° C ~ 100 ° C, temperature control accuracy ± 1 ° C. 6.7 Tip treatment vessels. diameter ≥ 12 cm, height ≥ 3 cm, can be homemade (see Appendix C). 6.8 Equipment and equipment commonly used in biological laboratories.

7 test organisms

The test organism is Songzi green pea, which is a medium-sized broad bean (Vicia faba) native to Songzi County, Hubei Province. And long-term purification and conservation, its genetic characteristics are relatively stable, sensitive to mutagens. Can be purchased directly or self-protected, Characteristics and conservation should meet the requirements of Appendix B. This test uses its primary root as the test organ.

8 test organism preparation

8.1 Selection According to the number of 50 pieces of pine peas, each sample should be selected to be full of granules, green or blue-brown, large and small. Bean seeds with consistent, no pests and diseases and no obvious defects on the surface were washed with water. 8.2 Soaking seeds Soak seeds in the number of up to 300 beans per 1,000 ml beaker, add thermostat (5.1) over 1 000 ml in the beaker Scale, soak in the incubator at a temperature of 25 ° C ± 1 ° C in a constant temperature incubator (6.3) until the beans are fully swelled. Soaking seeds takes about 26 h to 30 h. Water can be changed at 18 h and 24 h, and if turbidity occurs, the frequency of water change is increased. 8.3 rooting Dip the medical gauze 3 times, wring it out, stack 4 layers into a clean enamel pan, add a small amount of warm water (5.1) to the yarn. The cloth is fully wetted. Adjust the amount of warm water (5.1) to tilt the enamel plate, and see a small amount of overflow at the lowest point. Bean species (8.2) After tempering water (5.1) is rinsed, it is evenly arranged in the enamel pan, covering 2 layers of fully wet gauze. The moisture content of the gauze is suspended. When hanging, it is just right to drip without water. Cover with enamel pan cover, avoiding light and rooting at 25 °C ± 1 °C in a constant temperature incubator (6.3) ~ 66 h. During the rooting period, check twice a day, pay attention to timely hydration to keep the gauze in a state of just being fully wetted, and remove it at any time. No bean roots, mildew, dead roots, apical morphology or abnormal color of the beans, the following gauze cloth appears sticky secretions, in a timely manner Cleaning and replacement.

9 samples

9.1 Sample Collection Sample collection according to the requirements of HJ/T 91 or HJ/T 164, and the capacity of 1 000 ml and above is selected according to the nature of the sample. Sampling bottle. The sampling bottle can be a brown glass bottle or a container made of polypropylene, Teflon or polyethylene. When sampling Note that the sample is slowly poured into the sampling bottle along the wall of the bottle, leaving no gap between the sample and the stopper. Note. In order to ensure that the test is carried out as soon as possible, the sampling time should be arranged as far as possible at the end of the rooting (8.3) step. 9.2 Sample storage After the sample was collected, it was stored in the dark at 8 ° C or less and stored in the test within 48 h. If the sample needs to be stored for a long time, it must be Mix well and leave a proper space. Store at -18 °C for less than 2 months. 9.3 Sample preparation The sample is equilibrated to a constant temperature at 25 ° C ± 1 ° C in a constant temperature incubator (6.3), fully mixed, and then filled up to the apical processing dish (6.7), leave a gap between the cover and the cover to be tested. The pH was measured and recorded according to the method of GB/T 6920 before the test. At -18 ° C The samples preserved below should be firstly thawed in a water bath not exceeding 25 ° C or thawed overnight at 8 ° C. Note. The appropriate pH range for the sample is 5.5 to 8.5. A pH value that is too high or too low may have an effect on the test. When the test results need to be packaged Containing this effect, or adjusting the pH will cause physical denaturation or chemical reaction of the sample, then the pH value of the sample will not be adjusted; when the pH of the sample For < 5.5 or >8.5, adjust the pH to the appropriate range with hydrochloric acid solution (5.10) or sodium hydroxide solution (5.11). According to the supervisor For the purpose of the test, the test samples before and after the test can be tested separately or simultaneously. 9.4 Control sample 9.4.1 Replace the sample with the test water and prepare the empty test for the negative control test in the same manner as the sample preparation (9.3). White sample. 9.4.2 Replace the sample with a cyclophosphamide reference solution (5.17) and prepare for the same procedure as sample preparation (9.3). Reference sample for positive control test. 10 Analysis steps 10.1 apical processing Select the bean species (8.3) with a root length of 1.5 cm to 2.0 cm and a good growth, at each apex of the 9.3 step Insert 10 to 12 beans in the dish to ensure that the root tip is at least 1.0 cm, and in a constant temperature incubator (6.3) at 25 °C ± 1 °C Leave it in the dark for 6 h. Remove the bean seeds, dip them with warm water (5.1) for 3 times (5 min each time), and restore the culture according to the conditions specified in 8.3. 24 h. During the treatment, if the root tip appears as acute toxicity symptoms described in Appendix D, increase the sample according to the requirements of Appendix D. Acute toxicity pretest; otherwise, follow-up trials are performed directly. 10.2 apical fixation Cut the tip of the top l.0 cm (10.1) into the cap tube (the tip of the same sample is placed in a finger tube). Gacano solution (5.12) was fixed for 2 h. The fixed time is no longer than 24 hours. If it is not possible to dye the tablet in time, discard the Carnox solution, wash it with experimental water, and immerse it in ethanol solution (5.13). Refrigerated in the refrigerator and stained for microscopic examination within 72 hours. 10.3 Feulgen staining The root tip (10.2) was digested twice with water for 5 min each time. Take a hydrochloric acid solution (5.10) equilibrated in a water bath at 60 ° C, Quickly add into the tube until the root tip is immersed. After the tube is capped, it is placed in a 60 °C water bath for about 10 minutes. The specific time is The root tip is softened and the white is slightly transparent. The tweezers are lightly pinched and elastic. Quickly discard the hydrochloric acid solution. Immediately dip the above-mentioned apex twice with experimental water for 5 min each time. Add Schiff reagent (5.14) to the vial until Immerse the root tip and stain it for 1 h to 2 h in a dark room or protected from light. Discard the dye solution and dip the root tip with SO2 rinse (5.15) 2 times, each time 5 min; then rinse the root tip twice with experimental water for 5 min each time. If it is not possible to make a film immediately, soak the root tip in the newly exchanged experimental water, refrigerate in the refrigerator, and make a microscopic examination within 48 hours. 10.4 Production Use a pair of tweezers to pick up the root tip (10.3) with good dyeing effect, absorb the surface moisture on the filter paper, place it on the glass slide, and use The scalpel cuts the top apex from about 1.0 mm to 2.0 mm (see Appendix A, Figure A.2), and drops 1 drop of acetic acid dispersion. (5.16) Immerse the root tip, fully smash the scalpel, cover the slide (to avoid air bubbles), stack the filter paper on the cover slip, light Tap to spread the apical tissue, and press the thumb to make a thin layer, so that the apical cells are scattered in a single layer. 10.5 Microscopic examination The prepared pellets (10.4) were placed under a biological microscope (6.2), and the root tip cells were found to be close to each other under low magnification. Shaped or elliptical, evenly distributed, non-overlapping areas, transferred to high magnification microscopic examination. Observe at least 6 roots per sample Point to the following statistics for each root tip according to the 10.6 decision rule. a) the number of cells in the mitosis phase of 1 000 cells; b) The number of micronuclei in 1 000 mitotic cells. 10.6 Determination of mitotic cells and micronuclei 10.6.1 The process of cell mitosis consists of two phases, the interphase and the mitosis, in which mitotic cells are present. See Appendix A, Figure A.1 for identification. 10.6.2 Determine the microkernel according to the following rules. a) the size is less than 1/3 of the main nucleus, and is separated or tangent to the main nucleus; b) The coloration reaction and refractive index are consistent with the main nucleus, and there are obvious chromatin particles inside, and the color is slightly lighter or phased than the main nucleus. when; c) The shape is round, elliptical or irregular. Morphologically similar to micronuclei, but does not meet the above characteristics, especially the refractive index is inconsistent with the main nucleus, and there is no obvious chromatin inside. Particles, darker or lighter particles are pseudomicronuclei. For micronucleus and pseudo micronucleus decisions, see Appendix A, Figure A.3. 10.7 Controlled trial 10.7.1 Negative control For each batch of samples tested, the blank sample (9.4.1) was subjected to a negative control test in accordance with the steps 10.1 to 10.6. 10.7.2 Positive control For each batch of samples tested, the reference sample (9.4.2) was subjected to a positive control test in accordance with the steps 10.1 to 10.6. 11 Calculation and representation of results 11.1 Calculation of results a) Each tip mitotic index is calculated according to formula (1). I 1000 =  ‰ (1) Where. I--single apical mitotic index (preserving one decimal), ‰; M-- Observe the number of cells in the mitosis phase of 1 000 cells. b) The micronucleus rate of the sample to be tested is calculated according to formula (2). 1MCN 1000 n 1000 ==  ‰ (2) Where. MCN--the micronucleus rate of the sample to be tested (preserving one decimal), ‰; Ri--the number of micronuclei in the 1st apical cell of the i-th apical test sample; n--The total number of root tips (≥6) observed in the same sample. Note. The micronucleus rate of the sample, the micronucleus rate of the blank sample, and the micronucleus rate of the reference sample are represented by the MCN sample, the MCN blank, and the MCN reference, respectively. 11.2 Result determination Under the present measurement method, the mutagenicity of the sample is judged by the following conditions. a) MCN sample ≤ laboratory history cumulative MCN blank upper limit (reference value 6.6 ‰), then the sample is not mutagenic; b) MCN sample > laboratory history cumulative MCN blank upper limit (reference value 6.6 ‰), and this MCN sample is smaller than MCN blank Significantly increased, the sample is mutagenic; c) MCN sample > laboratory history cumulative MCN blank upper limit (reference value 6.6 ‰), but this time MCN sample is MCN blank Without a significant increase, the sample was suspected of being mutagenic. Comparing the micronucleus rate of the Vicia faba root tip of the blank and the blank sample, there is a significant difference, and appropriate statistics can be used. Methods such as nonparametric test (recommended Kruskal-Wallis test method), see Appendix E. Note 1. The cumulative upper limit of MCN in the laboratory history is the historical effective data of the micronucleus rate of the blank sample obtained by the laboratory according to this standard method. 99% confidence upper limit. Note 2. This method qualitatively determines the mutagenicity of the sample. For samples with mutagenicity, a stepwise dilution can also be used on this basis. Method, according to the presence or absence of mutagenicity of samples with different dilution concentrations, the maximum non-mutagenic effect of the sample is obtained. degree. 11.3 Test report See Appendix F for the format of the original record form and test report form. The test report requirements include but are not limited to the following information. a) the source and harvest year of the pine peas; b) the name, type, source, preservation method, sampling time, value before and after the pH adjustment of the sample, etc.; c) the presence of acutely toxic samples for the maximum concentration of acute toxicity-free effects for formal testing; d) the range of mitotic index of all microscopic apical cells corresponding to each sample; e) the number of micronuclei of all microscopic root tips corresponding to each sample; f) micronucleus rate of blank sample (negative control) and reference sample (positive control) in the control test results; g) The micronucleus rate of the sample, the statistical method, the judgment standard, and the calculation knot of the difference between the sample and the blank sample Fruit, and sample mutagenicity identification results. 12 precision 6 laboratories were blank samples (mean MCN=3.6‰), ρ=2.0 mg/L (mean MCN=11.1‰), Self-made cyclophosphamide reference with ρ=20.0 mg/L (mean MCN=23.3‰) and ρ=50.0 mg/L (mean MCN=30.1‰) Sample, negative actual sample (surface water, average MCN = 3.9 ‰), positive actual sample (chemical wastewater, average MCN = 8.6 ‰) The micronucleus rate of Vicia faba root tip was measured, and each sample was measured in parallel six times. Relative standard deviation range in the experimental room Do not. 16% ~ 38%, 10% ~ 16%, 8.7% ~ 16%, 7.3% ~ 13%, 25% ~ 45%, 15% ~ 34%; experiment The relative standard deviations between the chambers were 7.4%, 8.9%, 3.9%, 3.6%, 7.4%, and 4.2%, respectively. 13 Quality Assurance and Quality Control 13.1 mitotic index. all the root tips of the microscopic examination after treatment, such as samples, blank samples, reference samples, etc. The mitotic index of the apical cell micronucleus counting region should be >20‰, otherwise the apical micronucleus counting result is invalid. 13.2 Control test. The results of the control test meet the following requirements, and the test results are valid. Otherwise, after identifying the cause, New trials. a) Negative control After apical treatment, the apical apex must not have acute toxicity symptoms, and MCN blank ≤ laboratory history cumulative MCN blank upper limit (see The test value is 6.6‰). b) positive control After apical treatment, the apical apex must not have acute toxicity symptoms, and the MCN reference = 23.3 ‰ ± 7.5 ‰, that is, 15.8 ‰ ~ 30.8 ‰. 14 Waste treatment The wastes with acute toxicity or mutagenicity produced in the experiment are classified as hazardous wastes, and others are classified according to general wastes. Collect and mark accordingly, and entrust a qualified unit to handle it.

Appendix A

(informative appendix) Mitochondrial cells, apical cell micronucleus counting area and micronucleus determination reference picture The mitotic stages of Vicia faba mitotic cells, cell micronucleus counting area and micronucleus determination reference pictures, see Figure A.1 ~ Figure A.3. Figure A.1 ...

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