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### Ohio EPA Total (Extracellular and Intracellular) Microcystin s

- ADDA by ELISA

Analytical Methodology

Version 2.0

January 2 0 1 5

1. SCOPE AND APPLICATION

This method is used for the determination of total (extracellular and intracellular) m icrocystin s

–

ADDA in surface water, ground water and finished drinking water.

Reporting Limit (RL) : 0.3 0 μg/L

2. SUMMARY OF METHOD

The Ohio EPA Total (Extracellular and Intracellular) M icrocystin s

– ADDA by ELISA Analytical

M ethod ology 1 is an immunoassay for the detection of microcystins in water samples. This test is

an indirect competitive ELISA allow ing the congener

- independent detection of microcystins and

nodularins. It is based on the recognition of microcystins, nodularins and their congeners by

specific antibodies. Microcystins, nodularins and their congeners when present in a sample and

a microcystin

- protein analogue immobilized on the plate compete for the binding sites of

antibodies in solution. After a washing step and addition of the substrate solution, a color signal is

generated. The intensity of the blue color is inversely proportional to the concentration of the

microcystins present in the sample. The color reaction is stopped after a specified time and the

color is evaluated using a microplate reader at 450 nm

.

3. SAMPLE COLLECTION AND PRESERVATION

3.1 A minimum of 100 mL should be collected in a glass or polyethylene terephthalate glycol

(PETG) container.

NOTE (1) : S amples treated with chlorine or any other oxidizer (e.g. KMnO 4 , NaMnO 4 )

must be quenched with 10 mg sodium thiosulfate or ascorbic acid added per 100 m L of

sample during collection.

NOTE (2) : Cleaning of approved sample collection containers is acceptable as long as

the lab oratory can demonstrate e ffectiveness of the cleaning procedure by collecting and

analyzing reagent water in 5% of the cleaned containers. The reagent water result s must

be less than the reporting limit. The laboratory must maintain these records.

3. 2 All samples must be protect ed from sunlight and cooled on ice at 0

- 4°C immediately after

collection and maintained at 0

- 4 o C until analysis. Sample s must be analyzed within 5

days from the time of collection. H olding time can be increased by freezing the sample

within 5 days of collection . When freezing, allow adequate volume for expansion and

place the sample container on i t s side to prevent breakage.

3.3 Field Duplicate (FD) Sample Collection : Collect enough volume in a container to split into

two separate sample containers of at least 100 mL. Vigorously shake the initial volume

of sample prior to pouring it into the two separate collection containers

. All quenching

requirements must be followed ( NOTE 1 )

.

4. INTERFERENCES

D ue to the high variability of compounds found in water samples, test interferences caused by

### Page 2 of 6 matrix effects cannot be completely ruled out

. Ohio EPA continues to work with U

. S

. EPA and

other experts to identify and provide more guidance on potential interferences

.

5. SAFETY

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

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

exposure should be as low as reasonably achievable. Cautions are included for

known extremely hazardous materials or procedures.

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

regulations regarding the safe handling of the ch emicals specified in this method. A

reference file of Material Safety Data Sheets ( M SDS) should be made available to all

personnel involved in the chemical analysis. The preparation of a formal safety

plan is also advisable.

6

. APPARATUS

6 .1 Class 'A' Volumetric Flask: 500 mL

6 .2 Log books

6 .3 Micropipette with disposable plastic tips : 10 to 100 μL

6 .4 Multi

- channel pipette: 50

- 300 μL

6 .5 Stepping pipette: 100

- 500 μL

6 .6 Pipette tips: 1

- 200 μL

6

. 7 Multi

- channel pipette r eagent r eservoir minimum 5 0 mL capacity

6 .8 Microplate reader capable of analyzing at 450 nm

6 .9 G lass vials: 4 mL

6.1 0 G lass vials: 40 mL

6 .1 1 Syringe filters: 0.45 μm pore size ( Glass Fiber )

6 .1 2 Glass G astight L u e r

- lock syringes: 5 mL

6 .1 3 ELISA sealing Teflon tape

6 .1 4 Freezer

6 .1 5 Refrigerator capable of 4oC

6.1 6 Chlorine meter

6.1 7 pH Test Strips (pH 0

– 14)

6.1 8 Hydrochloric Acid Solution ( 0. 1N)

6. 19 Sodium Hydroxide Solution ( 0. 1N)

### Page 3 of 6 6.20 Water Bath (35 o C)

7

. REAGENTS

7 .1 Liquid disinfectant

7 .2 Analysis Kit (capable of analyzing all m icrocystin congeners with the ADDA structure )

. Store kit according to manufacturer’s instructions. Standards and r eagents may be used

until the expiration date on the box.

7 .3 DPD free chlorine reagent

7.4 Dechlorination agent : S odium t hiosulfate or Ascorbic a cid

7.5 Reagent Water: Water free of contaminants

7.6 Saturated sodium chloride solution (optional) : Dissolve 300 g of sodium chloride in 900

m L reagent water.

8. SAMPLE PREP A RATION

8.1 Disinfect the work area

.

8.2 S ample pH must be adjusted w ithin the range of 5

- 11. Samples with pH level s outside of

this range may produce inaccurate (falsely low) results and must be adjusted as

necessary using hydrochloric acid (HCl) or sodium hydroxide (NaOH) solutions , prior to

analysis.

N OTE (3) : Pour off a portion of sample and use it to test pH

.

8. 3 S amples treated with chlorine: Check the sample for residual chlorine. If residual

chlorine is greater than 0.1 mg/L, add 10 mg of quenching agent (sodium thiosulfate or

ascorbic acid) per 100 mL of sample

. Samples treated with any other oxidizer must also

be checked for sufficient quenching. Any sample not sufficiently quenched must be

appropriately qualified (CL ) and noted in the final report. For all qualifier definitions , see

Section 13.

9. SAMPLE LYSING PROCEDURE BY FREEZE/THAW

9.1 S hake the sample and pour 20 mL of the sample in to two separate 40 mL vials to begin

the three freeze/thaw lysing cycles. The second 40 mL vial may be analyzed as a

laboratory duplicate (LD)

. Otherwise store second vi al as needed for future analysis.

9. 2 Place vials in the freezer until completely frozen ( T o speed up the process

, vial(s) may be

imme rse d in a saturated sodium chloride solution or dry ice ).

9. 3 Once sample is completely frozen, remove from freezer and thaw (To speed up the

process

, vial(s) may be immerse d in a 35oC water bath until it is completely thawed )

.

9. 4 Repeat steps 9. 2 and 9. 3 two more times.

9. 5 Filter 2 mL of sample in to two 5 mL vials. Samples are ready for immediate analysis.

Freeze remainder of samples on their side, to be analyzed at a later date.

### Page 4 of 6 NOTE (4): If sample is frozen to extend hold time, once the sample is thawed it has

undergone the first freeze/thaw cycle.

10. INITIAL DEMONSTRATION OF CAPABILITY

A Method Detection Limit ( MDL ) study for the Microcystins

– ADDA test kit must be calculated

annually by each analyst , when a new analyst begins work or whenever a change in analytical

performance caused by either a change in instrument hardware or operati ng conditions would

dictate the MDL must be re calculated

.

In order for a laboratory to maintain a r eporting limit of at least 0.30 μg/L , e ach analyst must

demonstrate the capability to achieve an MDL of < 0.30 μg/L.

MDLs must be established for microcystins using a standard with a concentration one to ten

times the reporting limit (0.30 μg/L) . To calculate the MDL value, take seven replicate aliquots of

the standard and process them through the entire analytical meth od. Once the results for the

seven replicates have been obtained, calculate the MDL as follows:

MDL = (t)*(S D R )

Where: t = Student’ s t value for a 99% confidence interval and a

standard deviation estimate with n

- 1 degrees of freedom

( t = 3.143 for the seven replicates )

S D R = S tandard deviation of the replicate aliquot analyses

The MDL study will be valid i f the resulting value of the MDL is no more than ten times lower than

the replicate standard concentration level and does not exceed the replicate standard

concentration level

. Save and p rint a copy of each MDL study as part of the laboratory’s record

maintenance protocol.

NOTE ( 5 ) : Ohio EPA can provide additional guidance on completing an MDL study and all

calculations upon request

.

11. ANALYSIS

The accuracy of ELISA analysis is highly dependent up on analyst technique, adequate storage

conditions of the test kit, pipetting sequence, accuracy of reagent volumes and maintenance of

constant/optimum laboratory temperature during the analysis. The ELISA analysis is a time

sensitive procedure

. Care must be taken to ensure the reagent addition steps are complete d in

a n efficient manner and incubation times are followed according to ma nufacturer’s instructions.

NOTE ( 6 ) : T he assay procedure must be performed away from direct sun light.

11.1 Verify kit standards and reagents are used prior to the expiration date.

11.2 Bring samples and standards to room temperature prior to analysis.

1 1. 3 Follow manufacturer’s instructions provided with the individual M icrocystin s

– ADDA kit

for calibration, quality control (QC) and sample analysis procedure s

.

11. 4 Sample analyses resulting in a higher concentration than the highest standard in the

calibration curve must be diluted within the calibration range and reanalyzed to obtain

accurate results. If a sample is diluted, the final values must be calculated by multiplying

the result by the proper dilution factor.

### Page 5 of 6 1 1. 5 Save and p rint a copy of the calibration curve and sample results as part of the

laboratory’s record maintenance protocol.

12. QUALITY CONTROL (QC) AND DATA REPORTING

T he Ohio EPA requires at a minimum the following program specific analy tical QC requirements

be met.

12.1 Analyze all calibration standards, QC standar ds and samples in at least two well

r eplicate s . T he mean of the well replicates must be used in all analytical calculations and

reporting of sample results

.

12.2 The curve generation must include a calibration concentration point ≤ 0.30 μg/L ( RL )

.

12.3 Calibration curve Correlation Coefficient (R) must be > 0.99 0 or calibration curve

Coefficient of Determination (R 2 ) > 0.9 8 0 to be acceptable.

12. 4 Coefficient of Variation (%CV) for well replicate absorbance values for calibration

s tandards and QC standards should be < 10%. If %CV for more than one calibration

standard is > 10%, the analytical run is not acceptable. C orrective action and re analysis

of the sample batch is required.

NOTE ( 7 ) : A n analytical run may be accepted if %CV for only one calibration standard is

> 10% but < 15% as long as all other calibration standards in the analytical run are

< 10%.

Calculate %CV as follows:

%CV = (S D A

/Mean A )*100

Where: SD A = Standard deviation of well replicate absorbances

Mean A = Mean of well replicate absorbances

12

. 5 %CV for replicate absorbance values for samples must be < 15%, if the value is > 15%

then reanalyze or qualify the results with the appropriate qualifier (J) and noted in the

final report

.

12

. 6 Laboratory Reagent Blank (LRB) / Diluent Blank : A n LRB must be analyzed with each

batch of samples to verify laboratory and reagents are free of contaminants

. Values

exceed ing the reporting limit require corrective action and re analysis of the sample batch

. If reanalysis is not possible, the results must be appropriately qualified (B) and noted in

the final report

.

12. 7 Low Calibration Range Check (LCRC): An LCRC must be analyzed with each batch o f

samples to verify accuracy of the calibration curve near the reporting limit. The LCRC

may be one of the curve calibration points and the concentration must be ≥ 0.3 0 μg/L and

≤ 0.50 μg/L. A cceptance limits m ust be within ± 4 0% of the true value. LCRC v alues

exceed ing the acceptance limit s require corrective action and re analysis of the sample

batch

. If reanalysis is not possible, all sample concentration results less than an

acceptable QCS analyzed in the same batch must be appropriately qualified (J) and

noted in the final report

.

12.8 Quality Control Standard (QCS) : A secondary source QCS must be analyzed with each

batch of samples to verify the concentration of the calibration curve

. If a QCS is already

### Page 6 of 6 included in the kit, it may be used if it has a different lot number than the calibration

standards and was prepared from a separate primary stock. Acceptance limits must be

within ±25% of true value. QCS v alues exc eed ing the acceptance limit s require corrective

action and re analysis of the sample batch

. If reanalysis is not possible, the results must

be appropriately qualified (J) and noted in the final report

.

12. 9 Lab oratory Duplicate (LD): LDs must be analyzed on 5 % of samples in any analytical

batch and both results must be reported to the Ohio EPA

. The R elative P ercent

D ifference (RPD) must be calculated and records must be maintained.

Calculate RPD as follows:

RPD = [ (D 2 – D 1 )/D Avg ] *100

Where: D 1 = Laboratory D uplicate 1

D 2 = Laboratory Duplicate 2

D Avg = Average of Laboratory Duplicates 1and 2

12. 10 Field Duplicates (FD) : FDs should be analyzed on 5 % of samples in any analytical batch

and both results should be reported

. The RPD must be calculated and records must be

maintained.

Calculate RPD as follows:

RPD = [(D 2 – D 1 )/D Avg ]*100

Where: D 1 = Field Duplicate 1

D 2 = Field Duplicate 2

D Avg = Average of Field Duplicates 1 and 2

12. 1 1 Samples not analyzed within the required holding time should be recollected. If

recollection is not possible, the results must be appropriately qualified (PT) and noted in

the final report

.

12. 1 2 Samples received with residual chlorine concentrations greater than the acceptable limit

indicate ineffective quenching and should be recollected. If recollection is not possible,

the results must be appropriately qualified (CL) and noted in the final report

.

13. Q UALIFIERS

B Analytical result is estimated. Analyte was detected in associated reagent blank as well

as the samples.

CL Analytical result is estimated due to i neffective quenching

.

J Analyte was positively identified; the associated numerical value is estimated.

PT The reported result is estimated because the sample was not analyzed within required

holding time.

1 The Ohio EPA Total (Extracellular and Intracellular) Microcystins

- ADDA by ELISA Analytical

Methodology in raw and finished drinking water samples has been developed in conjunction with

U

. S

. EPA, public water system laboratories, Ohio State University

- Stone Laboratory and other

national experts. Ohio EPA will continue to work with these groups to evaluate and revise the

method as additional information becomes available.