MOCON GreenLight® 910-930

Measurement of Toxicity or Inhibition from

Fractionation (Frac) Water

September 16, 2013

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Table of Contents

Introduction ................................................................................................................ 5

Equipment .................................................................................................................. 5

Chemicals ................................................................................................................... 5

Safety .......................................................................................................................... 6

Aerated Dilution Water .............................................................................................. 6

Hydration of PolySeed® Seed ................................................................................... 7

Wastewater Secondary Treatment Biomass Seed ...................................................... 7

Experimental Procedure ............................................................................................. 8

Frac Wastewater Sample ............................................................................................................ 8

Sample Collection, Storage, and Holding Time ...................................................................... 8

Analysis Pretreatment .............................................................................................................. 8

Prior to analyses .................................................................................................................. 8

GreenLight® Bacterial Activity Test Toxicity or Inhibition by Frac Wastewater .................... 9

Control Test Sample ................................................................................................................ 9

Frac Wastewater Test Sample ................................................................................................. 9

GreenLight® Media Addition ............................................................................................... 10

GreenLight® 910 or 930 Instrument Procedure ......................................................12

Table 1: Examples for Continuous Test............................................................................ 13

GreenLight® 910 ..................................................................................................................... 14

Continuous Test ..................................................................................................................... 14

Multi Point Test ....................................................................................................................... 14

GreenLight® 930 ..................................................................................................................... 18

Toxicity or Inhibition Evaluation.............................................................................20

Table 2: Frac Water Toxicity ............................................................................................ 20

Example 1: ............................................................................................................................... 21

Table 3: Frac Water Toxicity Measure with Polyseed®................................................... 21

Example 2: ............................................................................................................................... 22

Table 4: Frac Water Toxicity Measure with Wastewater Secondary Treatment Biomass 22

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Quality Control ........................................................................................................23

Duplicate Relative Percent Difference ..................................................................................... 23

Control Blank ........................................................................................................................... 24

References ................................................................................................................25

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Table of Figures

Figure 1: PolySeed® Aeration ........................................................................................................ 7

Figure 2: Sample Being Aerated ..................................................................................................... 9

Figure 3: Mixed Sample with Media A and Media B ................................................................... 10

Figure 4: Sample in a 15 mL Vial Being Transferred into a GreenLight® 910 ........................... 10

Figure 5: 15 mL GreenLight® Vials in the Hot Block Incubator ................................................. 11

Figure 6: GreenLight® 910 New Test Window ........................................................................... 12

Figure 7: GreenLight® 930 New Test Window ........................................................................... 12

Figure 8: GreenLight® 910 Analysis Window ............................................................................. 14

Figure 9: Vial Read screen for GreenLight® 910 Miultipoint test ............................................... 15

Figure 10: Vial Description screen for GreenLight® 910 Miultipoint test .................................. 16

Figure 11: GreenLight® 910 Multipoint Incubating Main Window ............................................ 16

Figure 12: GreenLight® 910 Multipoint Incubating Analyses Window ...................................... 17

Figure 13: GreenLight® 930 Vial Information Screen ................................................................. 18

Figure 14: GreenLight® 930 Analyses Screen ............................................................................. 18

Figure 15: Base Treatment Time Equation ................................................................................... 20

Figure 16: Fraction Frac Water Toxicity Equation ....................................................................... 20

Figure 17: Frac Water Toxicity Measured with Polyseed® ......................................................... 21

Figure 18: Frac Water Toxicity Measured with Wastewater Secondary Treatment Biomass ...... 22

Figure 19: Example of RPD Chart for Frac Water Toxicity ......................................................... 23

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Introduction

1. This procedure is for the use of the GreenLight® 910 or 930 to determine the toxicity of

Fractionation (Frac) Wastewater. This test will determine the Base Treatment Time for a

wastewater activated bacteria and the impact (Toxicity or Inhibition) of Frac water on this

wastewater activated bacteria. This test will allow the operations staff or industrial

pretreatment staff to determine in a rapid test whether the Frac wastewater is toxic to or

inhibits a wastewater treatment process. This information will provide an assessment of the

treatability of the pollutant and whether it may pass-through the wastewater treatment plant.

Equipment

1. GreenLight® 910 or 930 instrument

2. GreenLight® 2 mL or 15 mL vials

3. GreenLight® 2 mL or 15 mL Hot Block Incubator (if required for 910 Multipoint Analysis)

4. Computer with GreenLight® operational software loaded and all required cables and power

converters.

5. Magnetic Stir-bars

6. Magnetic Stir-plates

7. 2000, 1000, 2500 mL Glass Beakers

8. 10 mL and 100 mL Glass Graduated Cylinders

9. 1-10 mL adjustable pipette with disposable tips.

10. Aquarium Air Pump (if needed to aerate sample)

11. Aquarium Stainless Steel Diffuser Stone (if needed to aerate sample)

12. Plastic Tubing for Aquarium Air Pump and Diffuser Stone (if needed to aerate sample).

Chemicals

1. GreenLight® Media A (BD BBL™ Trypticase™ Soy Broth powder or suitable substitute)

2. GreenLight® Media B (Ethanol, absolute-200 Proof, Sigma-Aldrich #459844 or suitable

substitute)

3. Sterile Water (if needed to dilute sample or run control blank)

4. De-chlorination reagent (Ascorbic acid or Sodium thiosulfate) if needed.

5. PolySeed® bacteria seed or Wastewater Secondary Treatment Biomass (settled).

5.1. PolySeed® Buffering and Mineral Dilution Water Media

5.1.1. Buffer Solution pH 7.2 ± 0.2 at 20 ˚C, APHA for BOD (Hach® #431-49 or

suitable substitute)

5.1.2. Ferric Chloride Solution, APHA for BOD (Hach® #429-43 or suitable substitute)

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5.1.3. Magnesium Sulfate Solution, APHA for BOD (Hach® #430-49 or suitable

substitute)

5.1.4. Calcium Chloride Solution, APHA for BOD (Hach® #428-49 or suitable

substitute)

5.1.5. Distilled or Reverse Osmosis (RO) water.

Safety

1. PolySeed® Microbial Formulation: The product formulation consists of a range of naturally-

occurring microorganisms which are known to be non-pathogenic to humans, livestock, and

agricultural crops. Keep dry and at room temperature. Ensure containers remain sealed.

Avoid inhalation, ingestion and exposure to skin. Wash hand thoroughly after use.

2. Wastewater Secondary Treatment Biomass: This material consists of a range of naturally-

occurring microorganisms which are known to be pathogenic to humans, livestock, and

agricultural crops. Avoid inhalation, ingestion and exposure to skin. Wash hand thoroughly

after use.

3. Media A: Prevent dispersion of material. Wipe up with damp sponge or mop.

4. Media B: Avoid contact with skin and eyes. Avoid inhalation of vapor or mist. Keep away

from sources of ignition.

5. Buffer Solution pH 7.2 ± 0.2: Refer to Hach® MSDS

6. Ferric Chloride Solution: Refer to Hach® MSDS

7. Magnesium Sulfate Solution: Refer to Hach® MSDS

8. Calcium Chloride Solution: Refer to Hach® MSDS

Aerated Dilution Water

1. Prepare dilution water as needed.

2. Transfer the required amount of distilled or RO water into a suitable size inert container

containing a magnetic stir bar.

3. Aerate the water with an aquarium pump and stir. (~ 1 hour).

4. Transfer 1 mL of the following buffering and mineral chemical per liter of dilution water

while it is stirring:

1.1.1. Buffer Solution pH 7.2 ± 0.2 at 20 ˚C

1.1.2. Ferric Chloride Solution

1.1.3. Magnesium Sulfate Solution

1.1.4. Calcium Chloride Solution

For best results, the aerated dilution water should be used within three (3) hours of aeration.

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Hydration of PolySeed® Seed

1. Place the entire contents of one PolySeed® capsule (discard the gelatin capsule) into 500 mL

of dilution water in a 2000 mL beaker with a stir bar.

Bran, which acts as the carrier for the microorganisms, will neither dissolve nor inhibit

microbial activity, but must be settled out of the PolySeed® solution prior to use.

2. Next, aerate the PolySeed® solution with an aquarium pump with a diffuser stone and stir the

PolySeed® solution for at least sixty (60) minutes.

Figure 1: PolySeed® Aeration

3. Allow the PolySeed® bran suspension to settle and decant the supernatant carefully into a

1000 mL beaker with as stir bar so as not to allow any bran to carry over.

4. Stir and aerate the PolySeed® suspension. Remove the aliquots of bacteria suspension as

needed from the aerated mixed suspension.

For best results, the PolySeed® solution should be used within six (6) hours of rehydration of

the capsule.

Wastewater Secondary Treatment Biomass Seed

1. Collect sufficient Final Effluent from the Secondary Treatment system of the wastewater

plant after the final clarifier and before the disinfection process in a HDPE or brown glass

bottle.

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2. Keep out of direct sunlight.

3. Cool to 1 ˚C to 6 ˚C until analyses.

4. Maximum storage time: 6 hours

5. Next, aerate the Final Effluent solution with an aquarium pump with a diffuser stone and stir

for at least thirty (30) minutes.

6. Allow any suspended solids to settle and decant the supernatant carefully into a 1000 mL

beaker with as stir bar so as not to allow any suspended solids to carry over.

7. Stir and aerate the Final Effluent suspension. Remove the aliquots of bacteria suspension as

needed from the aerated mixed suspension.

For best results, the Final Effluent solution should be used within six (6) hours of collection.

Experimental Procedure

Frac Wastewater Sample

Sample Collection, Storage, and Holding Time

1. Collect a minimum 100 mL of Frac wastewater water from a sample point in a HDPE or

brown glass sample bottle.

2. Add a de-chlorination reagent to remove any chlorine based disinfection chemicals if needed.

3. Keep out of direct sunlight.

4. Cool to 1 ˚C to 6 ˚C until analyses.

5. Maximum storage time: 14 days

Analysis Pretreatment

Prior to analyses:

1. Bring Frac Wastewater up to room temperature.

2. Transfer up to 1000 mL of Frac Wastewater sample to a 2000 mL glass beaker with a stir

bar.

3. Aerate the Frac Wastewater samples with stirring for 15 minutes at room temperature.

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Figure 2: Sample Being Aerated

GreenLight® Bacterial Activity Test Toxicity or Inhibition by Frac Wastewater

Control Test Sample

1. Transfer 90 mL of aerated dilution water with a 100 mL graduated cylinder into a 250 mL

glass beaker that has a magnetic stir bar.

2. Add 10 mL of Hydrated Polyseed® or Wastewater Secondary Treatment Biomass Seed with

a 10 mL graduated cylinder

3. Stir to mix.

Frac Wastewater Test Sample

1. Transfer 80 mL of aerated dilution water with a 100 mL graduated cylinder into a 250 mL

glass beaker that has a magnetic stir bar.

2. Add 10 mL of Hydrated Polyseed® or Wastewater Secondary Treatment Biomass Seed with

a 10 mL graduated cylinder.

3. Add 10 mL of the Frac Wastewater with a 10 mL graduated cylinder.

Note: Frac Wastewater sample can be diluted into serial dilutions with aerated dilution water

in needed.

4. Stir to mix

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GreenLight® Media Addition

1. Add ½ teaspoon of Nutrient A to the stirring Control Test or Frac Wastewater Test sample.

2. Once Nutrient A has dissolved, add 1 mL of Nutrient B with an adjustable pipet while

stirring.

Figure 3: Mixed Sample with Media A and Media B

3. Transfer 2 ml or 15 mL of sample to a GreenLight® 2 mL or 15 mL vial and run the Frac

Water Toxicity GreenLight® test on either the 910 or 930.

Figure 4: Sample in a 15 mL Vial Being Transferred into a GreenLight® 910

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4. If a Multipoint test on the 910 is run, then the 2 mL or 15 mL vials must be kept incubated in

the Hot Block Incubator. Set the incubator temperature to 40 ˚C and allow it to come to

equilibrium before starting the GreenLight® tests.

Figure 5: 15 mL GreenLight® Vials in the Hot Block Incubator

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GreenLight® 910 or 930 Instrument Procedure

1. Open the GreenLight® 910 or 930 software program on the computer.

2. The program can be opened before you start sample analyses so as to allow the instrument to

warm up to its operational temperature (40 ºC)

3. Select the New Test tab in either the GreenLight® 910 or 930.

Figure 6: GreenLight® 910 New Test Window

Figure 7: GreenLight® 930 New Test Window

4. Enter the following data in the GreenLight® 910 or 930 fields

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Table 1: Examples for Continuous Test

Field Name 910 930

Instrument Not Required.

Select the Instrument. If only one

instrument is used, default

instrument number will be

displayed.

Test ID Enter your test ID number. Enter your test ID number.

Test Type Enter “Continuous” or

“Multipoint” Enter “Continuous”

Method Select: Frac Water Toxicity. Select: Frac Water Toxicity

Rack ID Only Required for Multipoint.

Example::010113A Not Required

Sample Template Select Sample template “Frac

Water or Default” for Multipoint Select “Default 24”

Vial Description

Enter in information prior to

analyses for Continuous or after

initial reading for Multi Point

Enter in information prior to

analyses for Continuous or Multi

Point

5. Select the Next Button

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GreenLight® 910

Continuous Test

1. Place the 2 mL or 15 mL tube in the instrument and the internal bar code reader will start the

analysis.

2. Once the GreenLight® signal for a sample vial has crossed the threshold in the method, the

times for either the Base Treatment Time or the Frac Water Time will be reported.

Note: Use the threshold crossover time for the calculations

Figure 8: GreenLight® 910 Analysis Window

Multi Point Test

1. Select “Assign Vial Description” check box in the new test window.

2. Place each 2 mL or 15 mL tube in the multipoint sample set into the GreenLight® 910

instrument and the internal bar code reader will beep, read the bar code and initial signal for

each vial.

NOTE: If the vial is not read by the bar code reader

2.1. Select “Read” block in the test window.

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Figure 9: Vial Read screen for GreenLight® 910 Miultipoint test

2.2. Enter the vial number from the screen in the AP Check vial window. Include the “0s” in

front of each vial number.

2.3. Select “Read” in this window to enter the vial # and the initial signal value.

3. A “Vial Description” window will appear after the vials have been read. Additional

descriptive information for each vial can be entered.

4. Select “Save” in this window to save your vial description information.

5. Select the “Begin Incubation” block to start the tests

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Figure 10: Vial Description screen for GreenLight® 910 Miultipoint test

6. Remove each of the 2 mL or 15 mL tubes after they have been read and place it in the Hot

Block Incubator.

7. Observe the incubating time countdown window on the GreenLight® 910 screen. Once the

indicator bar has reached read, reinsert each 2 mL or 15 mL tube in the multipoint sample set

into the GreenLight® 910 instrument and the internal bar code reader will read the barcode

and read the oxygen concentration in the sample tube.

Figure 11: GreenLight® 910 Multipoint Incubating Main Window

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Figure 12: GreenLight® 910 Multipoint Incubating Analyses Window

8. Repeat steps 2 and 3 until the oxygen concentration has passed the threshold for all of the

samples in the multipoint sample set.

9. Once the GreenLight® signal for a sample vial has crossed the threshold in the method, the

times for either the Base Treatment Time or the Frac Water Time will be reported.

Note: Use the threshold crossover time for the calculations

10. The test will continue until all vials have crossed the threshold.

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GreenLight® 930

1. Place the 2 mL or 15 mL tubes in the GreenLight® 930 carousel.

2. Close the instrument lid. The Instrument will read the tube barcodes.

3. A table will appear listing the tube location in the carousel and a fields to enter Vial

Description

Figure 13: GreenLight® 930 Vial Information Screen

4. Select the Next button after you have entered any addition vial description and the analyses

will start

Figure 14: GreenLight® 930 Analyses Screen

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5. Once the GreenLight® signal for a sample vial has crossed the threshold in the method, the

times for either the Base Treatment Time or the Frac Water Time will be reported.

Note: Use the threshold crossover time for the calculations

6. The test will continue until all vials have crossed the threshold.

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Toxicity or Inhibition Evaluation

1. Determine the time it takes for the Plant Control to reach the threshold. This is the normal

treatment time for your plant.

2. Determine the treatment time for each pollutant tested.

3. Determine Base Treatment Time

Base Treatment Time= Time for Control to Cross Threshold

Figure 15: Base Treatment Time Equation

4. Determine Fraction (decimal) of Frac Water Toxicity

Time for Control to Cross ThresholdFraction of FracWater Toxicity=

Time for Frac Water to Cross Threshold

Figure 16: Fraction Frac Water Toxicity Equation

Table 2: Frac Water Toxicity

Fraction of Frac Water Toxicity Value

≥ 1 Non Toxic

0.99 -0.85 Inhibitory

< 0.85 Toxic

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Example 1:

1. Toxicity or Inhibition of a Frac Water Matrix utilizing Polyseed

Figure 17: Frac Water Toxicity Measured with Polyseed®

Table 3: Frac Water Toxicity Measure with Polyseed®

% Frac Water Frac Water

Time (min.)

Control Time

(min.)

Frac Water

Fraction (%)

10% 139 145 104% Non Toxic

25 % 160 145 91% Inhibitory

50 % 191 145 76% Toxic

75 % 310 145 47% Toxic

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Example 2:

1. Toxicity or Inhibition of a Frac Water Matrix utilizing Wastewater Secondary Treatment

Biomass.

Figure 18: Frac Water Toxicity Measured with Wastewater Secondary Treatment Biomass

Table 4: Frac Water Toxicity Measure with Wastewater Secondary Treatment Biomass

% Frac Water Frac Water

Time (min.)

Control Time

(min.)

Frac Water

Fraction (%)

10% 361 333 92% Inhibitory

25 % 421 333 79% Toxic

50 % 594 333 56% Toxic

75 % 1049 333 32% Toxic

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Quality Control

Duplicate Relative Percent Difference

1. It is suggested that a duplicate of one sample per batch be run. Acceptable control limits

are developed per Frac Wastewater geological site.

2. Calculate the Relative Percent Difference (RPD).

Sample1 - Sample1Duplicate*100 = RPD

Sample1 + Sample1Duplicate

2

Sample1 = Frac Water Sample 1

Sample 1 Duplicate = Frac Water Sample 1 Duplicate

Figure 19: Example of RPD Chart for Frac Water Toxicity

3. Run control charts of the RPD as per Standard Methods 1020 (Eaton 2012)

4. Set control limits for the geological matrix.

0

5

10

15

20

25

1

3

5

7

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11

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15

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19

21

23

25

27

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31

Rel

ati

ve

Per

cen

t D

iffe

ren

ce

Test Number

Frac Water Toxicity RPD

UAL UWL RPD

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Control Blank

1. Run 100 mL of dilution water as a sample when bacteria cross contamination is suspected in

the dilution water or media.

2. If the blank sample crosses the threshold within 24 hours, then there is a bacteria cross

contamination.

3. Run a Root Cause Analyses to determine source of bacterial contamination.

4. Replace all suspected sources of bacteria cross contamination.

5. Correct bacteria contamination problem as per company QC policy. Further information can

be obtained in Standard Methods for the Examination of Water and Wastewater Part 9000

(Eaton 2012).

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References

Eaton, A., D., Baird R.B.., Rice, E., W. (2012). Standard Methods for the Examination of Water

and Wastewater 22nd Edition. Washington D.C., APHA, AWWA, WEF.