HJ 775-2015 English PDF

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HJ 775-2015: Water quality. Ascarid ova determination. Nature sedimentation method
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Basic data

Standard ID: HJ 775-2015 (HJ775-2015)
Description (Translated English): Water quality. Ascarid ova determination. Nature sedimentation method
Sector / Industry: Environmental Protection Industry Standard
Word Count Estimation: 11,191
Date of Issue: 2015-12-04
Date of Implementation: 2016-01-01
Quoted Standard: HJ/T 91
Regulation (derived from): Ministry of Environment Announcement 2015 No.81
Issuing agency(ies): Ministry of Ecology and Environment
Summary: This Standard specifies the determination of Ascaris eggs in water precipitation egg collection method. This Standard is applicable to the determination of surface water and wastewater in Ascaris eggs. When the sample volume of 10 L, the detection limit of this standard 5/10 L.

HJ 775-2015: Water quality. Ascarid ova determination. Nature sedimentation method

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Water quality.Ascarid ova determination.Nity sedimentation method National Environmental Protection Standard of the People 's Republic of China Determination of water quality Ascaris eggs 2015-12-04 released 2016-01-01 Implementation Issued by the Ministry of Environmental Protection Directory Preface ⅱ

1 Scope of application

2 normative reference documents

3 Principle of the method

4 reagents and materials

5 instruments and equipment

6 samples .2

7 Analysis steps

8 Calculation and presentation of results

9 precision 4 10 quality assurance and quality control 4 11 Waste treatment 12 Precautions Appendix A (informative) Sodium nitrate water solubility, centrifuge centrifugal force and speed calculation formula. Appendix B (informative) Identification of Ascaris eggs Appendix C (informative) Preparation of Ascaris Egg Preparation Samples

Foreword

In order to implement the Environmental Protection Law of the People's Republic of China and the Law of the People's Republic of China on the Prevention and Control of Water Pollution, Protection of human health, regulate the determination of water Ascaris eggs, the development of this standard. This standard specifies the precipitation and egg collection methods for determination of Ascaris eggs in surface water and wastewater. This standard is the first release. Appendix A to Appendix C of this standard are informative. This standard is organized by the Ministry of Environmental Protection Science and Technology Standards Division. The main drafting unit of this standard. Changzhou City Environmental Monitoring Center. The standard verification unit. Shanghai Environmental Monitoring Center, Jiangsu Province Environmental Monitoring Center, Zhejiang Province Environmental Monitoring Center, Suzhou Environmental Monitoring Center, Xuzhou Environmental Monitoring Center Station and Taizhou Environmental Monitoring Center Station. This standard is approved by the Ministry of Environmental Protection on December 4,.2015. This standard has been implemented since January 1,.2016. This standard is explained by the Ministry of Environmental Protection. Determination of water quality Ascaris eggs

1 Scope of application

This standard specifies the determination of Ascaris eggs in water sedimentation egg collection method. This standard is applicable to the determination of Ascaris eggs in surface water and waste water. When the sampling volume is 10 L, the detection limit of this method is 5/10 L.

2 normative reference documents

The contents of this standard refer to the following documents or their terms. Those who do not specify the date of the reference file, the effective version of the appropriate For this standard. Technical specification for surface water and wastewater monitoring

3 Principle of the method

The sedimentation method is mainly determined by sample concentration, impurity separation, microscopic examination and so on. use 60 mesh screen filter to remove the larger impurities in the sample, the use of Ascaris eggs than the water and easy to precipitate the characteristics of overnight precipitation concentrated Shrinkage, siphon method to abandon the supernatant, with the surfactant Tween 80 as a residue and precipitation transfer cleaning agent, The concentrated residue was further concentrated by centrifugation, and the collected concentrate was mixed with acetic acid-sodium acetate buffer to control the pH of the solution, Get the best hydrophilic - lipophilic balance, add ethyl acetate to absorb the fat impurities in the water, so that Ascaris eggs more easily sink. Again away from Concentrating the sample and discarding the upper fat impurity layer and the buffer layer, the obtained egg-containing sediment layer is mixed with saturated sodium nitrate solution, In the count box after standing floating after the microscopic examination, you can measure the number of water samples in Ascaris eggs.

4 reagents and materials

Unless otherwise stated, analytical pure chemical reagents conforming to national standards were used in the analysis, and the test water was freshly prepared. Water or deionized water. 4.1 Ethyl acetate (C4H8O2). 4.2 acetic acid - sodium acetate buffer. pH = 4.5. Weigh 15.0 g of sodium acetate trihydrate (CH3COONa · 3H2O), dissolved in about 800 ml of experimental water, add 3.6 ml Acetic acid (CH3COOH) to adjust the pH to 4.5, with the experimental water volume to 1000 ml. The shelf life of this solution is 30 d. 4.3 Tween 80 solution. φ (C24H44O6) = 1 ‰. Remove 1 ml Tween 80 (Tween 80), diluted with experimental water to set the volume of 1000 ml, with the current allocation. 4.4 Saturated sodium nitrate (NaNO3) solution. (NaNO3), which is slightly more than the experimental ambient temperature, is dissolved in 100 g of experimental water, Filter residue with filter paper, temporary with the distribution. The solubility of sodium nitrate at different temperatures is given in Appendix A.

5 instruments and equipment

Unless otherwise stated, the use of the national standard A-class glass gauge is used for the analysis. 5.1 microscope. objective lens 4 ×, 10 × times, eyepiece 10 × times. 5.2 refrigerator. 0 ℃ ~ 4 ℃. 5.3 Horizontal Rotor Centrifuge. with 50 ml screw tip tip centrifuge tube, centrifugal force above 1000 g. 5.4 Whirlpool Mixer. 5.5 Screen. 60 mesh, ׎250 μm. 5.6 stainless steel open straight wall container. 10 L. 5.7 open straight wall cylinder. 1000 ml. 5.8 siphon. 5.9 Wash bottles. 5.10 screw tip tip centrifuge tube. 50 ml. 5.11 disposable Pasteuria. 3 ml, 10 ml. 5.12 Micropipettor. 1000 μl. 5.13 Quantitative Counting Box. 1 ml (mesh), 5 ml (S type) Quantitative Counting Box.

6 samples

According to HJ/T 91 on the general requirements of the sampling of pollutants, with more than 10 L volume of plastic drum sampling. Sample before sampling (7.1) and precipitate (7.2) at room temperature.

7 Analysis steps

7.1 Filtering Slightly shake the sample and transfer it to a stainless steel open-wall container (5.6) to 10 L. If the sample contains grass stalks, paper slag And other impurities, need to sample the screen (5.5) filter, and Tween 80 solution (4.3) cleaning screen (5.5) and impurities, The lotion was incorporated into the filtered 10 L sample. 7.2 precipitation The above sample (7.1) was allowed to stand overnight at room temperature for 15 h to 24 h at 15 ° C to 25 ° C and carefully with a siphon (5.8) Remove and discard the supernatant, avoid disturbing, keep 1 L of the sample containing the precipitate, turn into the 1000 ml open straight wall cylinder (5.7) Respectively, with 50 ml Tween 80 solution (4.3) thoroughly cleaned stainless steel open straight wall container 3 times, lotion into the open straight wall cylinder (5.7). In the 15 ℃ ~ 25 ℃ room temperature again after standing overnight for 12 h ~ 24 h, with a siphon (5.8) and discarded Supernatant, to avoid disturbance, to retain 90 ml ~ 100 ml samples containing sediment. Note 1. If the concentration of Ascaris eggs in the sample is more than 10 times the detection limit ( > 50/10 L), the sample can only take the total amount of not less than 1 L of the sample, Take 1 L of water directly to the second step of the cylinder precipitation concentrated. When the sample contains grass stalks, paper slag and other large impurities, the same should be the first sample with a screen (5.5), and the screen and impurities were washed with Tween 80 solution (4.3), and the lotion was precipitated in 1 L sample. 7.3 Centrifugal The precipitated sample (7.2) was carefully transferred to three screw tip tips (5.10), respectively, with 15 ml of Tween 80 solution (4.3) thoroughly clean the opening straight wall cylinder 3 times, lotion evenly into the 3 screw tip bottom centrifuge tube. To 1000 g Centrifugal force (centrifuge speed calculation see Appendix A) centrifugal 15 min, with a pasteurized tube (5.11) carefully suck and discard the supernatant, A small amount of stay, to avoid precipitation. The precipitate in the three centrifuge tubes was suspended with a small amount of Tween 80 solution (4.3), and the two centrifugal tubes with few precipitates Of the suspension into the largest amount of precipitation in a centrifuge tube; respectively, with 3 ml ~ 5 ml Tween 80 solution thoroughly washed by the suspension Floating the two centrifuge tubes, each cleaning 3 times, to ensure that no precipitation was discarded, lotion incorporated into the largest amount of precipitation centrifuge tube. Centrifuge for 15 min with centrifugal force of 1000 g and carefully aspirate and discard the supernatant with a pasteurized tube (5.11). Free of precipitation. 7.4 Separation and removal of lipid impurities The precipitate was suspended with an equal volume of acetic acid-sodium acetate buffer (4.2) after centrifugation (7.3) (i.e., after centrifugation Sedimentation volume of 2 ml, adding 2 ml buffer). If the volume of centrifugation after centrifugation is less than 2 ml, add buffer to 4 ml. To ensure that after extraction with ethyl acetate (4.1), there is a sufficient volume of buffer above the precipitate to facilitate complete pouring of the lipid- And to avoid the loss of Ascaris eggs. Centrifuge the precipitated (7.3) volumes of ethyl acetate (4.1) after centrifugation twice, and mix thoroughly with a vortex mixer (5.4). To 1000 g centrifugal centrifugation 15 min, the sample is divided into clear three layers. All non-fatty substances, heavy debris, including roundworms Eggs, larvae and protozoa at the bottom; the middle layer is the buffer layer; fat and other fat-soluble substances form black above Thick layer. The upper and middle layer of liquid was dumped and dumped, record the bottom sediment volume. If necessary, use fine needles in the centrifuge tube Wall around the upper layer of fat layer loose. 7.5 microscopic examination Add 5 times the bottom of the precipitate (7.4) volume of saturated sodium nitrate solution (4.4), the separation of lipid impurities after removal of eggs The bottom layer is suspended. The suspension can be placed in 0 ℃ ~ 4 ℃ refrigerator (5.2) frozen storage, preparation, within 1 week Finished, the test should be allowed to stand to room temperature. Shake the suspension sufficiently, transfer it to the quantitative counter box (5.13), allow to stand for 5 min, Under the microscope to count until all the suspension microscopy is completed. Separate the mirror with 1 ml of saturated sodium nitrate solution (4.4) Check the centrifuge tube 3 times, the lotion into the quantitative count box (5.13), according to the above steps microscopic examination. All detected roundworm eggs All counts. Mirror, the need to maintain the stability of environmental conditions, no vibration and wind disturbance, slow moving count box, top-down "U" number. The method of identification of roundworm eggs is given in Appendix B. 7.6 blank control test Take 10 L with the batch of experimental water, according to the above sampling and analysis procedures for the whole process of blank sample determination. Calculation and representation of results 8.1 Results calculation Count the number of roundworm eggs counted by the number, according to formula (1) to calculate and report 10 L water samples of Ascaris eggs. NC = (1) Where. C - water samples of Ascaris eggs concentration (a/10 L) N - number of all roundworm eggs detected Q - Actual water sample volume (10 L) 8.2 The result is shown Water quality in the determination of Ascaris eggs, the final results of the experiment, said the "a/10 L" as a unit.

9 precision

(20/10 L), medium concentration (40/10 L), high concentration (80 & lt; RTI ID = 0.0 & gt; / 10 L) from the samples of Ascaris eggs were measured, the relative standard deviation of the laboratory were. 6.5% to 18.9%, respectively, 10.1% ~ 22.0%, 12.8% ~ 23.8% and 8.9% ~ 17.4% respectively. The relative standard deviations were 5.8%, 5.5% 4.9%, 4.8%; repeatability limit (per/10 L) was 244,3,7,11; reproducibility limit (per/10 L) was 260,3,7,11. 10 quality assurance and quality control Each batch of samples at least one full program blank test, and take 10% of the samples for parallel sample determination. Full program blank Tests may not be detected roundworm eggs, parallel to the relative deviation between the sample shall not exceed 30%. 11 Waste treatment Experiments in the siphon boiling 10 min after the general waste treatment, with ethyl acetate waste liquid should be centralized custody, the Commission Qualified units for processing. 12 Precautions Experiments used equipment are required to be high temperature inactivation, placed in the water boiled 10 min before re-use. Experimental operators should pay attention to their own protection, the test to wear uniforms, latex gloves and masks, but also to avoid Sample contamination of roundworm eggs free of laboratory environment.

Appendix A

(Informative) Dissolution of Sodium Nitrate in Water, Centrifuge Force and Calculation Formula of Centrifuge A.1 Solubility of Sodium Nitrate in Water Table A.1 Solubility in Sodium Nitrate Water Temperature (° C) Solubility (g) A.2 Calculation formula of centrifugal force The centrifugal force is calculated according to the formula (A.1). RpmrRCF 2) (⋅ = (A.1) Where. RCF - relative centrifugal force (g) R - Centrifuge radius (distance from centrifuge tube center to centrifuge shaft, cm) Rpm - Centrifuge speed (r/min) K - 89456 A.3 Centrifugal force is converted to speed formula Centrifuge speed is scaled according to formula (A.2). RCFkrpm ⋅ = (A.2)

Appendix B

(Informative) Identification of Ascaris eggs B.1 Morphological characteristics of Ascaris eggs B.1.1 fertilized roundworm eggs The eggs were short oval and had a size of 45 μm to 75 μm × 35 μm to 50 μm. Egg shell is very thick, from outside to inside is divided into three layers. Fertilized membrane, crust layer and roundworm layer, thick shell layer, the other two very thin, difficult to distinguish under the ordinary microscope. There are eggs inside A large and round split of the fertilized egg cells, and eggshells common between the crescent-shaped gap. There is a layer of eggs outside the body of the uterus Secreted protein membrane, the surface of the rugged waves. Emulsions discharged with feces are often dyed yellow or brown by bile color. B.1.2 Unfertilized Ascaris Eggs The eggs were elliptic or irregular, with sizes ranging from 88 μm to 94 μm × 39 μm to 44 μm. Both eggshells and protein membranes More fertilized roundworm eggs thin, pale yellow, no round sugaride layer, egg shell contains many different sizes of refractive particles. B.1.3 Infection period Ascaris eggs The egg contains a curled larva. B.1.4 Protein membrane shedding of Ascaris eggs If the Ascaris egg protein membrane off, egg shell was colorless and transparent, should pay attention to the identification of other nematode eggs. B.2 Ascaris eggs reference picture B.2.1 Ascaris egg pattern Figure B.1 Ascaris egg pattern B.2.2 Development of various stages of Ascaris eggs Figure B.2 Development of various stages of Ascaris eggs Fertilized roundworm eggs off the protein membrane of fertilized roundworm eggs (left) Unwanted Ascaris Egg Infected Ascaris Eggs

Appendix C

(Informative) Preparation of Ascaris Egg Samples Ascaris suum live adults collected from the slaughterhouse, washed with distilled water, placed in 4% formaldehyde Solution, 0 ℃ ~ 4 ℃ preservation reserve, long-term preservation. Preparation of roundworm eggs, the choice of 2 to 3 female roundworm, with 75% ethanol disinfection of the scalpel, (2 mm ~ 3 mm) near each other in the uterus of each roundworm, after anatomy, the contents and tissues of Ascaris eggs Debris into the 50 ml centrifuge tube, add a few diameter 1 mm glass beads and 10 ml of saline, mix in the vortex after mixing Shaking 3 min ~ 5 min, with 260 mesh screening, take 40 ml of physiological saline several times the centrifuge tube and screen, at this time The ascorbate eggs in the filtrate are present in a single state. Filtrate by adding formaldehyde to the final concentration of 4%, 0 ℃ ~ 4 ℃ preservation reserve, can be Save for 1 month. Ready to use when you do not add formaldehyde. Preparation Preparation of samples, shake the roundworm eggs with liquid, with 1000 μl micro-pipette, immediately absorb the right amount of Ascaris egg preparation The cells were counted in a 1 ml dosing box (mesh) and counted with 250 μl of microtransfer to add or remove roundworms to the counting box Egg to the required quantity. All the liquid in the counting box was rinsed into the formulated sample with Tween 80 solution. And then under the microscope Count the box to ensure that no roundworm eggs remain. ......

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