ASTM D5660-96(2009)
(Test Method)Standard Test Method for Assessing the Microbial Detoxification of Chemically Contaminated Water and Soil Using a Toxicity Test with a Luminescent Marine Bacterium (Withdrawn 2014)
Standard Test Method for Assessing the Microbial Detoxification of Chemically Contaminated Water and Soil Using a Toxicity Test with a Luminescent Marine Bacterium (Withdrawn 2014)
SIGNIFICANCE AND USE
This test method provides a rapid means of determining the acute toxicity of an aqueous waste, or waste extract, prior to and following biological treatment, and contributes to assessing the potential biodegradability of the waste (see 1.1, 1.2, and Note 1). The change in toxicity to the marine bacterium P. phosphoreum with respect to time may serve as an indication of the biodegradation potential. Sample analyses are usually obtained in 45 to 60 min, with as little as 5 mL of sample required (5).
Samples with high suspended solids concentrations may test nontoxic to the bacteria, while still exhibiting significant toxicity to freshwater organisms, due to those suspended solids.
The absorbance correction procedure included in this test method allows for the analysis of highly colored lightabsorbing samples, by providing a means for mathematically adjusting the light output readings to account for light lost due to absorption.
SCOPE
1.1 This test method (1) covers a procedure for the rapid evaluation of the toxicity of wastewaters and aqueous extracts from contaminated soils and sediments, to the luminescent marine bacterium Photobacterium phosphoreum, prior to and following biological treatment. This test method is meant for use as a means to assess samples resulting from biotreatability studies. Sensitivity data for P. phosphoreum to over 1300 chemicals have been reported in the literature (2). Some of the publications are very relevant to this test method (3). The data obtained from this test method, when combined with respirometry, total organic carbon (TOC), biochemical oxygen demand (BOD), chemical oxygen demand (COD), or spectrophotometric data, can assist in the determination of the degree of biodegradability of a contaminant in water, soil, or sediment (3). The percentage difference between the IC20 of treated and untreated sample is used to assess the progress of detoxification.
1.2 This test method is applicable to the evaluation of the toxicity (to a specific microbe) and its implication on the biodegradation of aqueous samples from laboratory research bio-reactors (liquid or soil), pilot-plant biological treatment systems, full-scale biological treatment systems, and land application processes (see Notes 1 and 2).
Note 1—If the biologically treated material is to be discharged in such a manner as to potentially impact surface waters and ground water, or both, then the user must consult appropriate regulatory guidance documents to determine the proper test species for evaluating potential environmental impact (4). Correlations between data concerning reduction in toxicity produced by this test method and by procedures for acute or short-term chronic toxicity tests, or both, utilizing invertebrates and fish (see Guides E729 and E1192E729E1192), should be established, wherever possible.
Note 2—Color (especially red and brown), turbidity, and suspended solids interfere with this test method by absorbing or reflecting light. In these situations data are corrected for these effects by use of an absorbance correction procedure included in this test method (see 5.3, 6.1, and 6.2).
1.3 The results of this test method are reported in terms of an inhibitory concentration (IC), which is the calculated concentration of sample required to produce a specific quantitative and qualitative inhibition. The inhibition measured is the quantitative reduction in light output of luminescent marine bacteria (that is, IC20 represents the calculated concentration of sample that would produce a 20 % reduction in the light output of exposed bacteria over a specified time).
1.4 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.
1.5 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...
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation:D5660 −96(Reapproved2009)
Standard Test Method for
Assessing the Microbial Detoxification of Chemically
Contaminated Water and Soil Using a Toxicity Test with a
Luminescent Marine Bacterium
This standard is issued under the fixed designation D5660; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
NOTE 1—If the biologically treated material is to be discharged in such
1. Scope
a manner as to potentially impact surface waters and ground water, or
1.1 This test method (1) covers a procedure for the rapid
both, then the user must consult appropriate regulatory guidance docu-
evaluationofthetoxicity ofwastewatersandaqueousextracts ments to determine the proper test species for evaluating potential
environmentalimpact (4).Correlationsbetweendataconcerningreduction
from contaminated soils and sediments, to the luminescent
4 in toxicity produced by this test method and by procedures for acute or
marine bacterium Photobacterium phosphoreum, prior to and
short-term chronic toxicity tests, or both, utilizing invertebrates and fish
following biological treatment. This test method is meant for
(see Guides E729 and E1192), should be established, wherever possible.
use as a means to assess samples resulting from biotreatability
NOTE 2—Color (especially red and brown), turbidity, and suspended
studies. Sensitivity data for P. phosphoreum to over 1300 solids interfere with this test method by absorbing or reflecting light. In
thesesituationsdataarecorrectedfortheseeffectsbyuseofanabsorbance
chemicals have been reported in the literature (2). Some of the
correction procedure included in this test method (see 5.3, 6.1, and 6.2).
publications are very relevant to this test method (3). The data
1.3 The results of this test method are reported in terms of
obtained from this test method, when combined with
an inhibitory concentration (IC), which is the calculated
respirometry, total organic carbon (TOC), biochemical oxygen
concentration of sample required to produce a specific quanti-
demand (BOD), chemical oxygen demand (COD), or spectro-
tativeandqualitativeinhibition.Theinhibitionmeasuredisthe
photometric data, can assist in the determination of the degree
quantitative reduction in light output of luminescent marine
ofbiodegradabilityofacontaminantinwater,soil,orsediment
bacteria (that is, IC20 represents the calculated concentration
(3).The percentage difference between the IC20 of treated and
of sample that would produce a 20% reduction in the light
untreated sample is used to assess the progress of detoxifica-
output of exposed bacteria over a specified time).
tion.
1.4 The values stated in SI units are to be regarded as the
1.2 This test method is applicable to the evaluation of the
standard. The values given in parentheses are for information
toxicity (to a specific microbe) and its implication on the
only.
biodegradation of aqueous samples from laboratory research
bio-reactors (liquid or soil), pilot-plant biological treatment
1.5 This standard does not purport to address all of the
systems, full-scale biological treatment systems, and land
safety concerns, if any, associated with its use. It is the
application processes (see Notes 1 and 2).
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. Specific hazard
This test method is under the jurisdiction ofASTM Committee D34 on Waste
statements are given in Section 9.
ManagementandisthedirectresponsibilityofSubcommitteeD34.03onTreatment,
Recovery and Reuse.
2. Referenced Documents
Current edition approved Sept. 1, 2009. Published November 2009. Originally
approved in 1995. Last previous edition approved in 2004 as D5660–96(2004).
2.1 ASTM Standards:
DOI: 10.1520/D5660-96R09.
D888Test Methods for Dissolved Oxygen in Water
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard.
3 5
Toxicity measured as toxic inhibition of bacterial light output. At present (1993) use of the color correction scheme described in this
Microbics Corp. is currently the only known supplier of the reagents (test procedure is known to be effective only with the Microbics Corporation’s toxicity
organism Photobacterium phosphoreum strain NRRL B-11177) specific to this test analyzers, due to the fact that the correction mathematics involve the detailed
method. There are two known manufacturers of analyzers that can be used to geometry of both theACC and the light meter. Please notifyASTM Subcommittee
measure bioluminescence under temperature control: Microbics Corp., 2232 Ru- D34.09ifyouareawareofanyothersourceofequipmentcapableofprovidingcolor
therford Road, Carlsbad, CA 92008 (Microtox Model 500 and Model 2055 or turbidity correction, or both, for the P. phosphoreum test. Data validating the
Analyzers), and Pharmacia LKB, 9319 Gaither Road, Gaithersburg, MD 20877 absorbance correction procedure are available from Microbics Corp.
(LKB Wallac Model 1250 and Model 1251 Luminometers). Other instruments For referenced ASTM standards, visit the ASTM website, www.astm.org, or
would be considered when they become available. Please notify ASTM Subcom- contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
mittee D34.09 if you are aware of any additional systems or instruments capable of Standards volume information, refer to the standard’s Document Summary page on
performing this testing. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D5660−96 (Reapproved2009)
D1125Test Methods for Electrical Conductivity and Resis- exposed bacteria, allows for an assessment of the reduction in
tivity of Water toxicity to the marine bacterium P. phosphoreum (see 1.1, 1.2,
D1129Terminology Relating to Water and Note 1).
D1193Specification for Reagent Water
4.5 Samples that are highly colored, or contain solids that
D1293Test Methods for pH of Water
cannot be removed without seriously compromising sample
D3370Practices for Sampling Water from Closed Conduits
integrity, can be analyzed using an absorbance correction
E729Guide for Conducting Acute Toxicity Tests on Test
procedure. This procedure determines the amount of light
Materials with Fishes, Macroinvertebrates, and Amphib-
absorbedbythewastewaterataconcentrationnearthenominal
ians
IC20 versus the baseline light output established by measuring
E943Terminology Relating to Biological Effects and Envi-
the light absorbed by the clear diluent.
ronmental Fate
E1192Guide for ConductingAcute Toxicity Tests onAque-
5. Significance and Use
ous Ambient Samples and Effluents with Fishes,
5.1 This test method provides a rapid means of determining
Macroinvertebrates, and Amphibians
the acute toxicity of an aqueous waste, or waste extract, prior
3. Terminology
to and following biological treatment, and contributes to
assessing the potential biodegradability of the waste (see 1.1,
3.1 Definitions—The IC20 is defined in terms of a modifi-
1.2, and Note 1). The change in toxicity to the marine
cation of the definition of IC50 as it appears in Terminology
bacteriumP. phosphoreumwithrespecttotimemayserveasan
E943. The terms turbidity and volatile matter are defined in
indicationofthebiodegradationpotential.Sampleanalysesare
accordance with Terminology D1129. These terms are as
usually obtained in 45 to 60 min, with as little as 5 mL of
follows:
sample required (5).
3.1.1 color—that is, the presence of dissolved matter that
absorbs the light emitted by P. phosphoreum (that is, wave-
5.2 Sampleswithhighsuspendedsolidsconcentrationsmay
length of 490 6 100 nm).
test nontoxic to the bacteria, while still exhibiting significant
3.1.2 IC20—a statistically or graphically estimated concen- toxicity to freshwater organisms, due to those suspended
tration of test material that, under specified conditions, is
solids.
expected to cause a 20% inhibition of a biological process
5.3 The absorbance correction procedure included in this
(such as growth, reproduction, or bioluminescence) for which
test method allows for the analysis of highly colored lightab-
the data are not dichotomous.
sorbing samples, by providing a means for mathematically
3.1.3 turbidity—reduction of transparency of a sample due
adjusting the light output readings to account for light lost due
to the presence of particulate matter.
to absorption.
3.1.4 volatile matter—that matter that is changed under
conditions of the test to the gaseous state. 6. Interferences
6.1 Some test samples that are highly colored (especially
4. Summary of Test Method
red and brown) interfere with this test method, but the
4.1 This test method covers the determination of acute
absorbancecorrectionprocedurecanbeusedtocorrectforthis
toxicity of aqueous samples to luminescent marine bacteria, P. 5
interference.
phosphoreum.
6.2 Turbidity due to suspended solids interferes with this
4.2 Wastewater samples are osmotically adjusted to the
test method. The absorbance correction procedure can be used
appropriate salinity for the test species P. phosphoreum.A
to correct for this interference and is preferable to other
sodium chloride (NaCl) concentration of 2% has been found
alternatives. Pressure filtration, or centrifuging and decanting,
optimal for this test organism for freshwater tests, or about
will also remove this interference. Some toxics may be lost
3.4% NaCl for seawater samples. This provides the necessary
through adsorption and volatilization during filtration or
osmoticprotectionforthebacteria,whichareofmarineorigin.
centrifugation, thus impacting the exhibited toxicity.
4.3 SamplesshouldnotbepHadjustedunlesstheuserisnot
concerned about toxic effects related directly to pH. Altering
7. Apparatus
the sample pH will usually alter the solubility of both organic
7.1 Fixed or Adjustable Volume Pipetter, 10 µL, with
and inorganic constituents of the sample. Altering the pH can
disposable tips.
also cause chemical reactions that will change the integrity of
the sample, and greatly alter the exhibited toxicity of the 7.2 Variable Volume Pipetter, 10 to 1000 µL, with dispos-
sample. If sample pH is considered secondary to organism
able tips.
response, then the optimal pH for the bacterium Photobacte-
7.3 Variable Volume Pipetter, 1 to 5 mL, with disposable
rium phosphoreum is 6.7.
tips.
4.4 Comparison of inhibitory concentrations (IC20s) for
7.4 Timer or Stopwatch.
untreated wastewater (or extracts of untreated soils) versus
thoseforbiologicallytreatedwastewater(orextractsoftreated 7.5 Glass Cuvettes, 11.75 mm OD, 10.5 mm ID by 50 mm
soils), calculated from measured decreases in light output of height, 4-mL volume.
D5660−96 (Reapproved2009)
7.6 AbsorbanceCorrectionCuvettes(ACC)—Optionalitem, toxicants. Unless otherwise indicated, it is intended that all
but required to analyze highly colored samples or those reagents shall conform to the specifications of the Committee
5 7
containing suspended particulates. on Analytical Reagents of the American Chemical Society.
Other grades may be used, but there will be more risk that the
7.7 Variable Voltage Chart Recorder (optional)—Useful
resulting test reagents will fail to qualify (see 8.1.1).
when using some types of light meters.
8.2.1 Sodium Chloride (NaCl)—Used in preparation of
7.8 Computer (optional)—Useful with some light meters,
diluent, and for adjusting the osmotic pressure of samples to
for which software is also available, to facilitate data capture
that of the chosen diluent.
and reduction.
8.2.2 Phenol,orOtherCommonOrganicToxicant—Usedas
4,5
7.9 Light Meter, for cuvettes listed in 7.5. a reference toxicant.
8.2.3 Zinc Sulfate Heptahydrate, or Other Common Inor-
7.10 Temperature Control Devices (temperature-controlled
ganic Toxicant—Used as reference toxicant.
room, water bath, refrigerators, or other device)—One capable
8.3 PurityofWater—Unlessotherwiseindicated,references
ofmaintaining5.5 61°Candonecapableofmaintaining15 6
0.5°C. towatershallbeunderstoodtomeanreagentwaterconforming
to Specification D1193, Reagent Water, Type I or II, Subtype
8. Reagents and Materials
A.Testreagentspreparedfromreagentwateraretobequalified
for use with this test method (see 8.1.1).
8.1 Test Reagents:
8.1.1 For purposes of this test method, test reagents are
8.4 When this test method is used in conjunction with other
definedasthereagentsactuallyusedinperformanceofthetest
tests employing higher organisms, appropriate dilution water
method.Thenecessaryrequirementwithregardtoqualification
for bulk samples should meet the acceptability criteria estab-
of test reagents is that this test method provide acceptable
lishedinSection8ofGuideE729.Inaddition,allsuchdilution
results when reference toxicants are tested using the test
water used for comparative testing with this test method and
reagents.Theyarethenconsideredtobenon-toxicforpurposes
invertebrates and fish is to be assayed on P. phosphoreum
of this test method.
(minimally once per month).
8.1.2 Microbial Reagent—Freeze-dried Photobacterium
phosphoreum. This is the only test reagent that is currently 9. Hazards
(1993)availablefromonlyonesource. Whileotheracceptable
9.1 The handling of wastewaters entails potential hazards
means of preservation may become available in the future,
due to exposure to chemical and biological contaminants.
freeze-dried P. phosphoreum is specified in this test method
Appropriatesafetymeasures,suchasthewearingofprotective
because a large number of users concur in the opinion that the
clothing (gloves, apron, face shield, respirator, etc.) and main-
strain is well standardized by this method of preservation, and
taining proper hygiene, are utilized to minimize the chance of
that the same strain does not provide comparable response to
exposure. This test method is to be performed in a well-
reference toxicants when preserved by other methods, or when
ventilated area.
freshly cultured and harvested at the user’s laboratory, as
9.2 Appropriate, environmentally safe procedures pre-
described by Anthony A. Bulich, et al (1). Another consider-
scribed by regulatory agencies are utilized in the disposal of
ation is that a large body of published results, for which
used waste samples.
freeze-dried P. phosphoreum was used, has accumulated since
about 1980 (1,2,3,5,6).
9.3 Due to the presence of aqueous samples and electrical
8.1.3 Reconstitution Solution—Nontoxic water.
instrumentation in
...
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