ASTM D5120-90(2009)
(Test Method)Standard Test Method for Inhibition of Respiration in Microbial Cultures in the Activated Sludge Process (Withdrawn 2014)
Standard Test Method for Inhibition of Respiration in Microbial Cultures in the Activated Sludge Process (Withdrawn 2014)
SIGNIFICANCE AND USE
The objectives of the respiration inhibition tests may be defined by the interests of the user, but the test method is designed primarily for examination of the inhibition response with operating microbial systems such as an activated sludge process treating domestic or industrial wastes.
Different apparatus exist that facilitate continuous or continual measurement of respiration in microbial systems and each may be used as the tool to observe respiration in this test method.
Respirometry may utilize any apparatus and technique that will achieve the determination of respiration rate. A number of devices are presented in Appendix X1. Equivalency in the experimental capability of each device is not implied. The analyst should select the respirometric approach that best suits his needs.
The inhibitory effect of a test candidate is identified more completely by examining inhibition over a range of concentrations, such as determining the EC50. The use of aerated containers permits concurrent management of a series of cell suspensions. A respirometer for each cell suspension might also be used.
SCOPE
1.1 This test method covers a batch procedure that evaluates the impact of selected wastewaters, materials, or specific compounds on the respiration rate of an aqueous microbial culture, such as activated sludge.
1.2 Alternative procedures for measurement of microbial activity, such as adenosine 5′ triphosphate (ATP), specific substrate utilization, etc. are not within the scope of this test method.
1.3 The results obtained are based on comparisons in a specific test series that examines a range of concentrations of the potentially inhibitory test candidate using batch methods in a laboratory. Results are completed in a short time frame (a few hours).
1.4 The test results are specific to the microbial culture used. Microbial culture from different wastewater treatment plants will differ in kinds and numbers of organisms, and performance capability. Thus, there is no basis for comparing results for microbial cultures from different treatment facilities.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
WITHDRAWN RATIONALE
This test method covered a batch procedure that evaluated the impact of selected wastewaters, materials, or specific compounds on the respiration rate of an aqueous microbial culture, such as activated sludge.
Formerly under the jurisdiction of Committee D34 on Waste Management, this test method was withdrawn in May 2014. This standard is being withdrawn without replacement due to its limited use by industry.
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: D5120 − 90(Reapproved 2009)
Standard Test Method for
Inhibition of Respiration in Microbial Cultures in the
Activated Sludge Process
This standard is issued under the fixed designation D5120; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
1.1 Thistestmethodcoversabatchprocedurethatevaluates 2.1 ASTM Standards:
the impact of selected wastewaters, materials, or specific D4478 TestMethodsforOxygenUptake(Withdrawn1994)
compounds on the respiration rate of an aqueous microbial
culture, such as activated sludge. 3. Terminology
1.2 Alternative procedures for measurement of microbial 3.1 Definitions:
3.1.1 respiration rate—the quantitative consumption of
activity, such as adenosine 5' triphosphate (ATP), specific
substrate utilization, etc. are not within the scope of this test oxygen by an aqueous microbial system. The consumption is
generally expressed as mg O /L/h.
method.
3.1.2 EC —the concentration of the test candidate in this
1.3 The results obtained are based on comparisons in a
procedure (volume percent or mg/L) that results in a reduction
specific test series that examines a range of concentrations of
of respiration rate to 50 % of that observed for the control.
the potentially inhibitory test candidate using batch methods in
alaboratory.Resultsarecompletedinashorttimeframe(afew
4. Summary of Test Method
hours).
4.1 This test method utilizes respiration rate as the indicator
1.4 Thetestresultsarespecifictothemicrobialcultureused.
of microbial activity.
Microbial culture from different wastewater treatment plants
willdifferinkindsandnumbersoforganisms,andperformance
4.2 A batch system that contains a microbial culture (re-
capability. Thus, there is no basis for comparing results for
turned activated sludge from the process or a culture main-
microbial cultures from different treatment facilities.
tained in the laboratory), selected nutrient dose, and a dilution
of a compound, substance, wastewater, etc. (test candidate) is
1.5 The values stated in SI units are to be regarded as
prepared in a container in the laboratory. The batch system is
standard. No other units of measurement are included in this
called a “cell suspension.”
standard.
1.6 This standard does not purport to address all of the 4.3 The nutrient dose introduces a large excess of biode-
gradable substrate thereby putting the culture at a high meta-
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro- bolic rate. Inhibition of respiration by the test candidate is
observed under these conditions.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
1 2
This Test Method is under the jurisdiction ofASTM Committee D34 on Waste For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Management and is the direct responsibility of Subcommittee D34.03 on Treatment, contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Recovery and Reuse. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved Sept. 1, 2009. Published November 2009. Originally the ASTM website.
approved in 1990. Last previous edition approved in 2004 as D5120–90(2004). The last approved version of this historical standard is referenced on
DOI: 10.1520/D5120-90R09. www.astm.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D5120 − 90 (2009)
4.4 The prepared cell suspension is aerated for a 2-h period. microbial system damage. The degree of damage is not
At the end of the period, the respiration rate is determined quantified by comparison of respiration rates for the test
using a respirometric or an oxygen uptake technique. candidate and the control. Whether the cause is due to
uncoupled electron transfer or lysis of cells can be determined
4.5 A lower respiration rate for a cell suspension that has
by comparing the filtered Dissolved Organic Carbon (DOC) of
received the test candidate compared to the respiration rate of
the experimental cell suspension with the sum of the DOC of
a control cell suspension indicates inhibition of respiration.
the control plus that added by the test candidate.Ahigher DOC
5. Significance and Use represents cell lysis.
6.3 Where industrial wastewaters in the sewer system are
5.1 The objectives of the respiration inhibition tests may be
defined by the interests of the user, but the test method is continually introducing inhibitory components to the collective
wastewaters, it may not be feasible to utilize the returned
designed primarily for examination of the inhibition response
with operating microbial systems such as an activated sludge sludge from the process directly as the microbial culture. The
maintenance of a protected culture of organisms in the labo-
process treating domestic or industrial wastes.
ratory may be necessary.
5.2 Different apparatus exist that facilitate continuous or
continual measurement of respiration in microbial systems and
7. Apparatus
each may be used as the tool to observe respiration in this test
7.1 Respirometer or an Oxygen probe— An apparatus ca-
method.
pableofmeasuringtherespirationrateoroxygenuptakerateof
5.3 Respirometry may utilize any apparatus and technique
the cell suspension.
that will achieve the determination of respiration rate. A
7.1.1 Respirometer—A device that receives the cell
number of devices are presented in Appendix X1. Equivalency
suspension, or an aliquot and provides a technique for mea-
in the experimental capability of each device is not implied.
surement of oxygen utilization to be interpreted as respiration
The analyst should select the respirometric approach that best
rate (see Appendix X1).
suits his needs.
7.1.2 Dissolved Oxygen Probe and Instrumentation—An
alternate device for the measurement of respiration rate as
5.4 The inhibitory effect of a test candidate is identified
more completely by examining inhibition over a range of oxygen uptake rate.
concentrations, such as determining the EC . The use of
7.2 Culture Tank—If it is deemed necessary to maintain a
aerated containers permits concurrent management of a series
microbial culture in the laboratory, the apparatus required is a
of cell suspensions. A respirometer for each cell suspension
container with adequate mixing and oxygen transfer. The
might also be used.
container should hold at least four times the volume of culture
that might be used in one day.
6. Interferences
7.2.1 The culture tank should be adequately mixed to insure
6.1 This test method is most readily applied to substances
that the culture remains in suspension and that sufficient
which, due to water solubility and low volatility, are likely to
mechanical or bubble aeration occurs to maintain the desired
remain in the aqueous system.
dissolved oxygen (DO) concentration.
6.2 Results have been observed where cell suspensions
NOTE 1—Energy input should not be such that the biological floc is
containing the test candidate had a respiration rate greater than
sheared to sizes smaller than that which exists in the large-scale process.
the blank, particularly at shorter aeration periods of the cell Mixing and aeration provided through diffused aeration in a laboratory-
sized container may result in an excessive power input. Consider
suspensions (less than 1 h). Thus, a minimum aeration period
controlling the power input per unit volume to approximately that which
for the cell suspensions before determinations of respiration
exists in the large-scale process. For example, pure oxygen for aeration in
rate is 2 h.
combination with mechanical mixing may be utilized to achieve a balance
6.2.1 One reason for increased oxygen uptake rate in an
between oxygen transfer and mixing. Determine the mixer power input by
measuring the electrical power consumed at different operating speed, and
experimental cell suspension may be that severe physical or
adjust the mixer speed to achieve a power input that is equivalent to that
chemical reactions with the test candidate cause a fraction of
which exists in the large-scale system.
the microbial culture to be lysed. The release of very readily
NOTE2—Culturesgrownatlow(0.5to2mg/L)andhigh(>5mg/L)DO
biodegradable soluble organic material from the lysed cells
concentrations possess different kinetic capabilities. Thus, to maintain a
may support a higher oxygen uptake rate by the cell suspen-
laboratory culture with performance capabilities similar to those of the
full-scale culture, the DO concentration should be maintained at the level
sion.
appropriate for the full-scale process. The probable explanation for the
6.2.2 Analternatereasonforincreasedoxygenuptakerateis
difference in culture performance is that higher concentrations of oxygen
that certain test candidates (2,4-dichlorophenol for example)
penetrate more completely through the floc particles.
may uncouple the transfer of electrons involved in the process
7.3 A pH Probe and Instrumentation.
calledoxidativephosphorylationinwhichadenosine5'triphos-
phate (ATP) is formed by the phosphorylation of adenosine 5' 7.4 Dissolved Oxygen Probe—If utilized, the following
diphosphate (ADP).The result of the uncoupling is an increase apparatus is needed:
in the rate of oxygen consumption that is not related to 7.4.1 Biochemical Oxygen Demand (BOD) bottles.
substrate stabilization. 7.4.2 Agitation Device, may be used with the dissolved
6.2.3 A respiration rate by an experimental cell suspension oxygen probe in the BOD bottle. The device must provide
that is greater than the respiration rate of the control represents complete mixing of the microbial culture in the BOD bottle.
D5120 − 90 (2009)
7.4.3 Magnetic Stirrer and Magnetic Stirring Bar, ing inhibition studies. Store the prepared solution in a refrig-
alternatively, may be used to mix the BOD bottle. erator at 4°C. Warm the portion of the feed to be used in tests
to the operating temperature of the test before use. Replace the
7.5 Beakers, 2-L size, (or other containers of suitable size).
solution after 14 days of storage or earlier if it becomes
7.6 Clean, Oil-Free Air Supply, to provide cell suspension
odorous.
mixing and aeration.
Ingredient Quantity (g)
Bacto peptone 32
7.7 Fritted Glass Diffusers or Pasteur-Pipets, as air diffus-
Beef extract 22
ers.
Ammonium chloride 11
Sodium chloride 1.4
Calcium chloride (CaCl ·2H O) 0.8
2 2
8. Reagents
Potassium dihydrogen phosphate 3.5
8.1 Microbial Culture—The microbial culture to be used is Potassium monohydrogen phosphate 4.5
Distilled water make up to 1 L
the returned sludge from the full-scale facility. For those
8.3 Stock Inhibitor Solution—Dissolve 0.5 g of 3,5-
activated sludge system where industrial contributions regu-
dichlorophenol in 10 mL of 1N NaOH, dilute to 30 mL with
larly cause microbial inhibition, direct use of the returned
distilled water, add 1N H SO to the point of incipient
sludge may be impractical. For those systems where microbial
2 4
precipitation (approximately 8 mL of 1N H SO will be
inhibitionisnotacontinuousproblem,thereturnedsludgemay
2 4
required), bring the volume to 950 mL with distilled water,
be used directly if, by observed system performance, it appears
adjust the pH to the range of 7 to 8, and bring the volume to 1
to be healthy.
L. The EC of 3,5-dichlorophenol for relatively non-
8.1.1 A microbial culture may be maintained in the labora-
acclimatized microbial cultures from domestic wastewater
tory.Thecultureshouldbemaintainedatthetemperatureofthe
plantsisabout10to30mg/Lbutmaybeoutsidethisrange.For
full-scale mixed liquor and approximately at the concentration
acclimatized microbial cultures, the EC will be higher, and
of the full-scale process returned sludge.
values <200 mg/L has been reported. The stock solution may
8.1.2 Ifmaintenanceofamicrobialcultureistobepracticed
be used to check experimental technique and possibly the
in the laboratory, and if the inhibition tests are to be related to
susceptivity of a microbial culture.
a specific activated sludge wastewater treatment process, the
initial microbial culture should be taken from the process
9. Procedure
returned sludge.
8.1.2.1 Care should be taken to obtain the microbial culture
9.1 Prepare experimental and control cell suspensions so
when, by appearance and performance, the culture is consid-
that each is identical in its concentration of microbial culture
ered to be healthy.
and Marlene’s mix.
8.1.2.2 The microbial culture should be fed daily with the
9.1.1 When the test is related to an operating activated
actual process wastewater if the wastewater is of suitable
sludge process, the microbial culture concentration, and the
qualityandnotinhibitory.Determinethewastewaterqualityby
initial pH, make sure that the temperature of the cell suspen-
following the procedure in Section 9. Prepare a control cell
sion (throughout the experimental period) is the same as that in
suspension and a wastewater cell suspension. If the wastewater
the process mixed liquor. As an example of the control cell
cell suspension does not show inhibition, it is suitable for use
suspension, the following table applies if a microbial concen-
as feed material.
tration of 2000 mg/L is desired and the microbial culture
8.1.2.3 If wastewater of good quality is not available, a
concentration is 10 000 mg/L.
synthetic feed, such as Marlene’s Mix (see 8.2) or other feed
Percent of
similar in character to the wastewater should be used (sucrose
Component Preparation Volume
A
Microbial Culture (at 10 000 mg/L) 20
has been used successfully with domestic wastewater activated
Marlene’s Mix 3.8
sludge). The feed application should not be excessive. For
Tap Water 76.2 or as required to equal 100
example, preferably it should be equal to about one-half of the
A
food-to-microorganism ratio that exists in the full-scale pro-
If the culture is not at 10 000 mg/L, adjust the volume percent to obtain the
desired microbial concentration in the cell suspension.
cess.
8.1.2.4 When the full-scale system is considered to be in
9.1.2 Make sure that the concentration of readily biodegrad-
goodcondition,replenishonethirdtoonehalfofthevolumeof
able organic material in the nutrient dose (such as Marlene’s
the microbial culture daily with the returned sludge from the
Mix) is high enough that an additional inc
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