Leck Mitchell, Ph.D., PE 

 Instrumentation for Control and Monitoring

656 Independence Valley Drive; Grand Junction, CO 81507 

Phone: 970 244-8661; Fax: 970-244-8856; 

Leckmitchell@Bresnan.net 

 

MITCHELL ATP SUBMISSION FOR CHANGES IN 

METHOD 180.1 USING LED 

DETERMINATION OF TURBIDITY BY NEPHELOMETRY 

Mitchell Method M5331 

Alternate Test Procedures 

Leck Mitchell, Ph.D., P.E. 

Patricia Lacy Mitchell, Ph.D. 

Revision 1.0 

July 31, 2008 

 

 
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MITCHELL ATP SUBMISSION FOR CHANGES IN 

METHOD 180.1 USING LED 

DETERMINATION OF TURBIDITY BY NEPHELOMETRY 

Mitchell Method M5331 

Alternate Test Procedures

 

1.0 SCOPE AND APPLICATION

 

1.1 This method covers the determination of turbidity in drinking, ground, surface, and saline 

waters, domestic and industrial wastes and any colorless water with turbidity, less than 40 

NTU. 

1.2 This method introduces a solid state monochromatic LED light source which falls within the 

accepted range for EPA 180.1 and meets all quality control and performance characteristics. 

1.3 The applicable range is 0 to 40 nephelometric turbidity units (NTU). Higher values may be 

obtained with dilution of the sample. 

1.4 This method is to be used for compliance monitoring and reporting under the Safe Water 

Drinking Act (SDWA.) 

2.0 SUMMARY OF MATERIAL

 2.1 The method is based upon a comparison of the intensity of light scattered by the sample 

under defined conditions with the intensity of light scattered by a standard reference 

suspension. The higher the intensity of scattered light, the higher the turbidity. Readings in 

the NTUs are made in a nephelometer designed according to specifications given in sections 

6.1 and 6.2. A primary standard suspension is used to calibrate the instrument. A secondary 

standard is used as a calibration check and is monitored periodically for deterioration using 

one of the primary standards. 

2.1.1 Formazin polymer is used as a primary turbidity suspension for Water. 

2.1.2 A commercially available polymer primary standard is also approved for use in the 

National Interim Primary Drinking Water Regulations. This standard is identified as 

AMCO Clear. It is stable over longer periods of time than Formazin and is available 

from GFS Chemical Company. 

2.1.3 Stabilized Formazin, identified as Stab Cal, is an approved derivative of the 

Formazin standard and is stable in storage for two years. 

 
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3.0 DEFFINITIONS

3.1 CALIBRATION BLANK (CB) – A volume of reagent water fortified with the same matrix 

as the calibration standards, but without the analytes, internal standards, or surrogates’ 

analytes. 

3.2 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) – A solution of one or more 

method analytes, surrogates, internal standards, or other test substances used to evaluate the 

performance of the instrument system with respect to a defined set of criteria. 

3.3 LABORATORY REAGENT BLANK (LRB) – An aliquot of reagent water or other blank 

matrices that are treated exactly as a sample including exposure to all glassware, equipment, 

solvents, reagents, internal standards, and surrogates that are used with other samples. The 

LRB is used to determine if method analytes or other interferences are present in the 

laboratory environment, the reagents, or the apparatus. 

3.4 LINEAR CALIBRATION RANGE (LCR) – The concentration range over which the 

instrument response is linear. 

3.5 MATERIAL SAFETY DATA SHEET (MSDS) – Written information provided by vendors 

concerning a chemical/s toxicity, health hazards, physical properties, fire, and reactivity data 

including storage, spill, and handling precautions. 

3.6 PRIMARY CALIBRATION STANDARD (PCAL) – A suspension prepared from the 

primary dilution stock standard. The PCAL suspensions are used to calibrate the instrument 

response with respect to analyte concentration. 

3.7 QUALITY CONTROL SAMPLE (QCS) – A solution of the method analyte of known 

concentrations used to fortify an aliquot of LBR matrix. The QCS is obtained from a source 

external to the laboratory, and is used to check laboratory performance. 

3.8 SECONDARY CALIBRATION STANDARDS (SCAL) – commercially prepared, stabilized 

sealed liquid or gel turbidity standards, or other apparatus or mechanism calibrated against 

properly prepared and diluted Formazin or styrene divinylbenzene polymers (AMCO Clear). 

3.9 STOCK STANDARD SUSPENSION (SSS) – A concentrated suspension containing the 

analytic prepared in the laboratory using assayed reference materials or purchased from a 

reputable commercial source. Stock standard suspension is used to prepare calibration 

suspensions and other needed suspensions. 

4.0 INTERFERENCES

4.1 The presence of floating debris and coarse sediments which settle out rapidly will give low 

readings. Finely divided air bubbles can cause high readings. 

4.2 The presence of true color, that is, the color of water which is due to dissolved substances that 

absorb light, will cause turbidities to be low, although this effect is generally not significant 

with drinking waters. 
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4.3 Light absorbing materials such as activated carbon in significant concentrations can cause 

low readings. 

5.0 SAFETY

 

5.1 The toxicity or carcinogenicity of each reagent used in this method has not been fully 

established. Each chemical should be regarded as a potential health hazard and exposure 

should be as low as reasonably achievable. 

5.2 Each laboratory is responsible for maintaining a current awareness file of OSHA regulations 

regarding the safe handling of the chemicals specified in this method. A reference file of 

Material Safety Data Sheets (MSDS) should be made available to all personnel involved in 

the chemical analysis. The preparation of a formal safety plan is also advisable. 

5.3 Hydrazine Sulfate (7.2.1) is highly toxic and may be fatal if inhaled, swallowed, or absorbed 

through the skin. Formazin can contain residual hydrazine sulfate. Proper protection should 

be employed. 

6.0 EQUIPMENT AND SUPPLIES

6.1 The turbidimeter shall consist of a nephelometer, with light source for illuminating the 

sample and one or more photo-electric detectors with a readout device to indicate the 

intensity of light scattered at right angles to the path of the incident light. The turbidimeter 

should be designed so that little stray light reaches the detector in the absence of turbidity and 

should be free from significant drift after a short warm-up period. 

6.2 Differences in physical design of turbidimeters will cause differences in measured values for 

turbidity, even though the same suspension is used for calibration. To minimize such 

differences, the following design criteria should be observed: 

6.2.1 LED light source: Monochromatic source operated at nominal wavelength of 525 ± 

15nm. The source shall be used as a directly received reference for the scattered 

nephelometric signal. The beam shall not diverge and shall not converge over ± 1 

1/2 degrees. Alternate light sources may be acceptable if they fall within the 

proscribed wavelength specifications and meet all quality control and performance 

characteristics. In addition, the Mitchell designs allow a variation of 50% in light 

intensity of the source without variation in turbidity response. 

6.2.2 Distance traversed by incident light and scattered light not to exceed 10 cm. 

6.2.3 Detector/ Light Receiver: Centered at 90o ± 1 1⁄2 degrees to the incident light path; 

the light cone not to exceed ± 30 degrees from 90 degrees. The receiver, if used, may 

be coupled to a photo detector using an optical path. The photo detector must have a 

spectral output that encompasses a complete spectral response of the light source. A 

Mitchell/Mersch light trap shall be incorporated for on-line use. 

6.2.4 Instrument shall have incorporated a Mitchell/Mersch bubble trap for on-line use and 

Mitchell/Mersch anti-fog windows. Sensor shall be horizontal with vertical 
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windows. Both windows shall be immersed in the measurement fluid for on-line use. 

Instrument shall be capable of being pressurized to 30 psig. 

6.2.5 An optical device may be used to carry the light from the light source to the sample 

or to carry scattered light from the sample to the photo detector. 

6.2.6 Turbidimeter example meeting these specifications is the Mitchell (M5331). 

6.3 The sensitivity of the instrument should permit detection of a turbidity difference of .01 NTU 

or less, in waters having turbidities less than 1 unit. The instrument should measure from 0 to 

40 NTU turbidity. Several ranges may be necessary to obtain both adequate coverage and 

sufficient sensitivity for low turbidities. 

6.4 The sample tubes for laboratory, bench top or portable units to be used with the available 

instrument must be of clear, colorless glass or plastic. They should be kept scrupulously 

clean, both inside and out, and discarded when they become scratched or etched. A light 

coating of silicon oil may be used to mask minor imperfections in glass tubes. They must not 

be handled at all where the light strikes them, but should be provided with sufficient extra 

length, or with a protective case, so that they may be easily handled. Tubes should be 

checked, indexed and read at the orientation that produces the lowest background blank 

value. 

6.5 Balance – Analytical, capable of accurately weighing to the nearest 0.0001g. 

6.6 Glassware – Class A volumetric flasks and pipettes as required. 

7.0 REAGENTS AND STANDARDS

7.1 Reagent water, turbidity-free: Pass deionized distilled water through a 0.45 micron-pore size 

membrane filter, if such filtered water shows a lower turbidity than unfiltered distilled water. 

7.2 Stock standard suspension (Formazin) 

7.2.1 Dissolve 1.00 g hydrazine sulfate (CASRN 10034-93-2) in reagent water and dilute 

to 100 mL in a volumetric flask. (toxic) 

7.2.2 Dissolve 10.00 g hexamethylenetetramine (CASRN 100-97-0) in reagent water and 

dilute to 100 mL in a volumetric flask. In a 100 mL volumetric flask, mix 5.0 mL of 

each solution (7.2.1 + 7.2.2). Allow to stand 24 hours at 25 ± 3 degree C, then dilute 

to the mark with reagent water. 

7.3 Primary calibration standards: Mix and dilute 10.00 mL of stock standard suspension (7.2) to 

100 mL with reagent water. The turbidity of this suspension is defined as 40 NTU. For other 

values, mix and dilute portions of this suspension as required. 

7.3.1 A new stock standard suspension (7.2) should be prepared each month. Primary 

calibration standards (7.3) should be prepared daily by dilution of the stock standard 

suspension. 
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7.4 Formazin in commercially prepared, certified, primary concentrated stock standard 

suspension (SSS) may be diluted and used as required. Dilute turbidity standards should be 

prepared daily. 

7.5 AMCO Clear, styrene divinylbenzene polymer, primary standards are available for specific 

instruments and require no preparation or dilution prior to use. This is an approved EPA 

standard. 

7.6 Stabilized Formazin, (StablCal) turbidity standards, certified, primary, polymer suspensions 

have long-term storage stability. 

7.7 Secondary standards may be acceptable as a calibration check, but must be monitored on a 

routine basis for deterioration and replaced as required. 

8.0 SAMPLE COLLECTION, PRESERVATION AND STORAGE

8.1 Samples for bench top and portable laboratory units should be collected in plastic or glass 

bottles. All bottles must be thoroughly cleaned and rinsed with turbidity free water. Volume 

collected should be sufficient to insure a representative sample, allow for replicate analysis (if 

required), and minimize waste disposal. 

8.2 No chemical preservation is required for samples. Cool sample to 4 °C. 

8.3 Samples for bench top and portable laboratory units should be analyzed as soon as possible 

after collection. If storage is required, samples maintained at 4 °C may be held for up to 48 

hours. 

8.4 Online instrumentation does not require sample cooling, preservation or storage. 

9.0 QUALITY CONTROL

9.1 Each laboratory using this method is required to operate a formal quality control (QC) 

program. The minimum requirements of this program consist of an initial demonstration of 

laboratory capability and analysis of laboratory reagent blanks and other solutions as a 

continuing check on performance. The laboratory is required to maintain performance 

records that define the quality of data generated. 

9.2 INITIAL DEMONSTRATION OF PERFORMANCE 

9.2.1 The initial demonstration of performance is used to characterize instrument 

performance (determined of LCRs and analysis of QCS). 

9.2.2 Linear Calibration Range (LCR) – The LCR must be determined initially and verified 

whenever a significant change in instrument response is observed or expected. The 

initial demonstration of linearity must use sufficient standards to insure that the 

resulting curve is linear. The verification of linearity must use a minimum of a blank 

and three standards. If any verification data exceeds the initial values by ± 10%, 

linearity must be reestablished. If any portion of the range is shown to be nonlinear, 

sufficient standards must be used to clearly define the nonlinear portion. 
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9.2.3 Quality Control Sample (QCS) – When beginning the use of this method, on a 

quarterly basis or as required to meet data-quality needs, verify the calibration 

standards and acceptable instrument performance with the preparation and analysis of 

a QCS. If the determined concentrations are not within ± 10 % of the stated values, 

performance of the determinative step of the method is unacceptable. The source of 

the problem must be identified and corrected before continuing with on-going 

analyses. 

9.3 ASSESSING LABORATORY PERFORMANCE 

9.3.1 Laboratory Reagent Blank (LRB) – The laboratory must analyze at least one LRB 

with each batch of samples. Data produced are used to assess contamination from the 

laboratory environment. 

9.3.2 Instrument Performance Check Solution (IPC) – For all determinations, the 

laboratory must analyze the IPC (A midrange check standard) and a calibration blank 

immediately following daily calibration, after every tenth sample [or more frequently 

if required] and at the end of the sample run. Analysis of the IPC solution and 

calibration blank immediately following calibration must verify that the instrument is 

within ± 10 % of calibration. Subsequent analyses of the IPC solution must verify the 

calibration is within ± 10 %. If the calibration cannot be verified within the specified 

limits, reanalyze the IPC solution. If the second analysis of the IPC solution 

confirms calibration to be outside the limits, sample analysis must be discontinued, 

the cause determined and/or in the case of drift the instrument recalibrated. All 

samples, following the last acceptable IPC solution, must reanalyzed. The analysis 

data of the calibration blank and IPC solution must be kept on file with the sample 

analyses data. NOTE: Secondary calibration standards (SS) may also be used as the 

IPC. 

9.3.3 Where additional reference materials such as Performance Testing samples are 

available, they should be analyzed to provide additional performance data. The 

analysis of reference samples is a valuable tool for demonstrating the ability to 

perform the method acceptably. 

10.0 CALIBRATION AND STANDARDIZATION

10.1 Turbidity Calibration: The manufacturer’s operating instructions should be followed. 

Measure standards on the turbidimeter covering the range of interest. If the instrument is 

already calibrated in standard turbidity units, this procedure will check the accuracy of the 

calibration scales. At least one standard should be run in each instrument range to be used. 

Some instruments permit adjustments of sensitivity so that scale values will correspond to 

turbidities. Solid standards, if used must be protected from surface scratches which may 

potentially cause calibration changes. If a pre-calibrated scale is not supplied, calibration 

curves should be prepared for each range of the instrument. Calibration must be performed 

under identical optical conditions as the operational conditions. The unit may incorporate an 

optional Mitchell/Mersch self-check system for verification of operation. 

 
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11.0 PROCEDURE

11.1 For on-line use, all instrument parameters should be checked to insure constant sample flow 

in the instrument. This will insure bubble interferences are minimized. Refer to the 

manufacturer’s instrument manual for the set up of measurement parameters and flow rates. 

11.2 Turbidities less than 40 NTU: For instruments other than on-line, if possible, allow samples 

to come to room temperature before analysis. Mix the sample to thoroughly disperse the 

solids. Wait unit air bubbles disappear, and then pour the sample into the turbidimeter tube. 

Read the turbidity directly from the instrument scale or from the appropriate calibration 

curve. 

 

11.3 Turbidities exceeding 40 NTU: Dilute the sample with one or more turbidity-free water until 

the turbidity falls below 40 NTUs. The turbidity of the original sample is then computed 

from the turbidity of the diluted sample and the dilution factor. 

11.3.1 Some turbidimeters are equipped with several separate scales. The higher scales are 

to be used only as indicators of required dilution volumes to reduce readings to less 

than 40 NTU. 

 

12.0 DATA ANALYSIS AND CALCULATIONS

12.1 Multiply sample readings by appropriate dilution to obtain final reading. 

 

12.2 Report results as follows: 

NTU Record to Nearest

0.0 – 1.0 .01 

1 – 10 0.1 

10 – 40 1. 

40 – 100 5. 

100 – 400 10 

400 – 1,000 50 

≥1,000 100 

 

13.0 METHOD PERFORMANCE

In an intra laboratory test facility the following readings for precision and accuracy were 

established for M5331 
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 True Value Sample Instrumentation Average Standard Deviation 

 0 .015 .0012 

 .1 .100 .0006 

 .3 .294 .0007 

 .5 .506 .0007 

 5 5.014 .008 

 10 9.975 .005 

 20 19.993 .088 

 40 39.966 .149 

 

14.0 POLLUTION PREVENTION 

14.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity or 

toxicity of waste at the point of generation. Numerous opportunities for pollution prevention 

exist in laboratory operation. The EPA has established a preferred hierarchy of 

environmental management techniques that places pollution prevention as the management 

option of first choice. Whenever feasible, laboratory personnel should use pollution 

prevention techniques to address their waste generation. When wastes cannot be feasibly 

reduced at the source, the Agency recommends recycling as the next best option. 

14.2 The quantity of chemicals purchased should be based on expected usage during its shelf life 

and disposal cost of unused material. Actual reagent preparation volumes should reflect 

anticipated usage and reagent stability. 

 

15.0 WASTE MANAGEMENT

The U.S. Environmental Protection Agency requires that laboratory waste management practices be 

conducted consistent with all applicable rules and regulations. Excess reagents, samples and 

method process wastes should be characterized and disposed of in an acceptable manner. The 

Agency urges laboratories to protect the air, water and land by minimizing and controlling all 

releases from hoods, and bench operations, complying with the letter and spirit of any waste 

discharge permit and regulations, and by complying with all solid and hazardous waste regulations, 

particularly the hazardous waste identification rules and land disposal restrictions. For further 

information on waste management consult the “Waste Management Manual for Laboratory 

Personnel”, available from the American Chemical Society. 

16.0 REFERENCES 

1. American Society for Testing and Materials (ASTM). 1993. Annual Book of ASTM 

Standards, Volume 11.01. Water (1), Standard D1889-88A, p. 359. West Conshohocken, PA.

2. Standard Methods. 1992. Standard Methods for the Examination of Water and Wastewater. 

Eighteenth Edition, pp. 2-9, Method 2130B. APHA, AWWA, and WEF. Port City Press, 

Baltimore, MD. 

3. McKee, Gerald F., (1985) Turbidity Standard. EPA Record