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
9
11
13
15
17
19
21
23
25
27
29
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.
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