ASTM D5660-96(2004)

Standard Test Method for Assessing the Microbial Detoxification of Chemically Contaminated Water and Soil Using a Toxicity Test with a Luminescent Marine Bacterium

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.5
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 Note 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 E 729 and E 1192), 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 safety and health...

General Information

• Status: Historical
• Publication Date: 09-Mar-1996
• Technical Committee: D34 - Waste Management
• Drafting Committee: D34.03 - Treatment, Recovery and Reuse

Relations

• Replaces: ASTM D5660-96 - Standard Test Method for Assessing the Microbial Detoxification of Chemically Contaminated Water and Soil Using a Toxicity Test with a Luminescent Marine Bacterium
• Effective Date: 10-Mar-1996
• Replaced By: ASTM D5660-96(2009) - 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)
• Effective Date: 01-Sep-2009
• Referred By: ASTM E2552-23 - Standard Guide for Assessing the Environmental and Human Health Impacts of New Compounds for Military Use
• Effective Date: 10-Mar-1996
• Referred By: ASTM D5681-22e1 - Standard Terminology for Waste and Waste Management
• Effective Date: 10-Mar-1996

Standards Content (Sample)

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 (Reapproved 2004)
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.
a manner as to potentially impact surface waters and ground water, or
1. Scope
both, then the user must consult appropriate regulatory guidance docu-
1.1 This test method (1) covers a procedure for the rapid
ments to determine the proper test species for evaluating potential
evaluationofthetoxicity ofwastewatersandaqueousextracts
environmentalimpact(4).Correlationsbetweendataconcerningreduction
from contaminated soils and sediments, to the luminescent
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
(seeGuidesE729andE1192E729E1192),shouldbeestablished,wherever
following biological treatment. This test method is meant for
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
chemicals have been reported in the literature (2). Some of the
thesesituationsdataarecorrectedfortheseeffectsbyuseofanabsorbance
publications are very relevant to this test method (3). The data
correction procedure included in this test method (see 5.3, 6.1, and 6.2).
obtainedfromthistestmethod,whencombinedwithrespirom-
1.3 The results of this test method are reported in terms of
etry, total organic carbon (TOC), biochemical oxygen demand
an inhibitory concentration (IC), which is the calculated
(BOD), chemical oxygen demand (COD), or spectrophotomet-
concentration of sample required to produce a specific quanti-
ric data, can assist in the determination of the degree of
tativeandqualitativeinhibition.Theinhibitionmeasuredisthe
biodegradability of a contaminant in water, soil, or sediment
quantitative reduction in light output of luminescent marine
(3).The percentage difference between the IC20 of treated and
bacteria (that is, IC20 represents the calculated concentration
untreated sample is used to assess the progress of detoxifica-
of sample that would produce a 20% reduction in the light
tion.
output of exposed bacteria over a specified time).
1.2 This test method is applicable to the evaluation of the
1.4 The values stated in SI units are to be regarded as the
toxicity (to a specific microbe) and its implication on the
standard. The values given in parentheses are for information
biodegradation of aqueous samples from laboratory research
only.
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-
NOTE 1—If the biologically treated material is to be discharged in such
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. Specific hazard
statements are given in Section 9.
This test method is under the jurisdiction ofASTM Committee D34 on Waste
ManagementandisthedirectresponsibilityofSubcommitteeD34.03onTreatment,
2. Referenced Documents
Recovery and Reuse.
Current edition approved Sept. 28, 2004. Published May 1996. Originally
2.1 ASTM Standards:
published as D5660–95. Last previous edition D5660–95. DOI: 10.1520/D5660-
D888 Test Methods for Dissolved Oxygen in Water
96R04.
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard.
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 Ruth-
D34.09ifyouareawareofanyothersourceofequipmentcapableofprovidingcolor
erford Road, Carlsbad, CA 92008 (Microtox Model 500 and Model 2055 Analyz-
or turbidity correction, or both, for the P. phosphoreum test. Data validating the
ers), and Pharmacia LKB, 9319 Gaither Road, Gaithersburg, MD 20877 (LKB
absorbance correction procedure are available from Microbics Corp.
Wallac Model 1250 and Model 1251 Luminometers). Other instruments would be
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
consideredwhentheybecomeavailable.PleasenotifyASTMSubcommitteeD34.09
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
ifyouareawareofanyadditionalsystemsorinstrumentscapableofperformingthis
Standards volume information, refer to the standard’s Document Summary page on
testing.
the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D5660 – 96 (2004)
D1125 Test 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,
and Note 1).
D1129 Terminology Relating to Water
4.5 Samples that are highly colored, or contain solids that
D1193 Specification for Reagent Water
cannot be removed without seriously compromising sample
D1293 Test Methods for pH of Water
integrity, can be analyzed using an absorbance correction
D3370 Practices for Sampling Water from Closed Conduits
procedure. This procedure determines the amount of light
E729 Guide for Conducting Acute Toxicity Tests on Test
absorbedbythewastewaterataconcentrationnearthenominal
Materials with Fishes, Macroinvertebrates, and Amphib-
IC20 versus the baseline light output established by measuring
ians
the light absorbed by the clear diluent.
E943 Terminology Relating to Biological Effects and Envi-
ronmental Fate
5. Significance and Use
E1192 GuideforConductingAcuteToxicityTestsonAque-
5.1 This test method provides a rapid means of determining
ousAmbient Samples and Effluents with Fishes, Macroin-
the acute toxicity of an aqueous waste, or waste extract, prior
vertebrates, and Amphibians
to and following biological treatment, and contributes to
assessing the potential biodegradability of the waste (see 1.1,
3. Terminology
1.2, and Note 1). The change in toxicity to the marine
3.1 Definitions—The IC20 is defined in terms of a modifi-
bacterium P. phosphoreumwithrespecttotimemayserveasan
cation of the definition of IC50 as it appears in Terminology indicationofthebiodegradationpotential.Sampleanalysesare
E943. The terms turbidity and volatile matter are defined in
usually obtained in 45 to 60 min, with as little as 5 mL of
accordance with Terminology D1129. These terms are as sample required (5).
follows:
5.2 Sampleswithhighsuspendedsolidsconcentrationsmay
test nontoxic to the bacteria, while still exhibiting significant
3.1.1 color—that is, the presence of dissolved matter that
absorbs the light emitted by P. phosphoreum (that is, wave- toxicity to freshwater organisms, due to those suspended
solids.
length of 490 6 100 nm).
5.3 The absorbance correction procedure included in this
3.1.2 IC20—a statistically or graphically estimated concen-
test method allows for the analysis of highly colored lightab-
tration of test material that, under specified conditions, is
sorbing samples, by providing a means for mathematically
expected to cause a 20% inhibition of a biological process
adjusting the light output readings to account for light lost due
(such as growth, reproduction, or bioluminescence) for which
to absorption.
the data are not dichotomous.
3.1.3 turbidity—reduction of transparency of a sample due
6. Interferences
to the presence of particulate matter.
6.1 Some test samples that are highly colored (especially
3.1.4 volatile matter—that matter that is changed under
red and brown) interfere with this test method, but the
conditions of the test to the gaseous state.
absorbancecorrectionprocedurecanbeusedtocorrectforthis
interference.
4. Summary of Test Method
6.2 Turbidity due to suspended solids interferes with this
4.1 This test method covers the determination of acute
test method. The absorbance correction procedure can be used
toxicity of aqueous samples to luminescent marine bacteria, P.
to correct for this interference and is preferable to other
phosphoreum.
alternatives. Pressure filtration, or centrifuging and decanting,
4.2 Wastewater samples are osmotically adjusted to the
will also remove this interference. Some toxics may be lost
appropriate salinity for the test species P. phosphoreum.A
through adsorption and volatilization during filtration or cen-
sodium chloride (NaCl) concentration of 2% has been found
trifugation, thus impacting the exhibited toxicity.
optimal for this test organism for freshwater tests, or about
7. Apparatus
3.4% NaCl for seawater samples. This provides the necessary
osmoticprotectionforthebacteria,whichareofmarineorigin.
7.1 Fixed or Adjustable Volume Pipetter, 10 µL, with
4.3 SamplesshouldnotbepHadjustedunlesstheuserisnot
disposable tips.
concerned about toxic effects related directly to pH. Altering
7.2 Variable Volume Pipetter, 10 to 1000 µL, with dispos-
the sample pH will usually alter the solubility of both organic
able tips.
and inorganic constituents of the sample. Altering the pH can
7.3 Variable Volume Pipetter, 1 to 5 mL, with disposable
also cause chemical reactions that will change the integrity of
tips.
the sample, and greatly alter the exhibited toxicity of the
7.4 Timer or Stopwatch.
sample. If sample pH is considered secondary to organism
7.5 Glass Cuvettes, 11.75 mm OD, 10.5 mm ID by 50 mm
response, then the optimal pH for the bacterium Photobacte-
height, 4-mL volume.
rium phosphoreum is 6.7.
7.6 Absorbance Correction Cuvettes (ACC)—Optional
4.4 Comparison of inhibitory concentrations (IC20s) for item, but required to analyze highly colored samples or those
untreated wastewater (or extracts of untreated soils) versus containing suspended particulates.
those for biologically treated wastewater (or extracts of treated 7.7 Variable Voltage Chart Recorder (optional)—Useful
soils), calculated from measured decreases in light output of when using some types of light meters.
D5660 – 96 (2004)
7.8 Computer (optional)—Useful with some light meters, on Analytical Reagents of the American Chemical Society.
for which software is also available, to facilitate data capture Other grades may be used, but there will be more risk that the
and reduction. resulting test reagents will fail to qualify (see 8.1.1).
,
8.2.1 Sodium Chloride (NaCl)—Used in preparation of
7.9 Light Meter, for cuvettes listed in 7.5.
diluent, and for adjusting the osmotic pressure of samples to
7.10 Temperature Control Devices (temperature-controlled
that of the chosen diluent.
room, water bath, refrigerators, or other device)—One capable
8.2.2 Phenol, or Other Common Organic Toxicant—Used
ofmaintaining5.5 61°Candonecapableofmaintaining15 6
as a reference toxicant.
0.5°C.
8.2.3 Zinc Sulfate Heptahydrate, or Other Common Inor-
ganic Toxicant—Used as reference toxicant.
8. Reagents and Materials
8.3 Purity of Water— Unless otherwise indicated, refer-
8.1 Test Reagents:
ences to water shall be understood to mean reagent water
8.1.1 For purposes of this test method, test reagents are
conforming to Specification D1193, Reagent Water, Type I or
defined as the reagents actually used in performance of the test
II,SubtypeA.Testreagentspreparedfromreagentwaterareto
method.Thenecessaryrequirementwithregardtoqualification
be qualified for use with this test method (see 8.1.1).
of test reagents is that this test method provide acceptable
8.4 When this test method is used in conjunction with other
results when reference toxicants are tested using the test
tests employing higher organisms, appropriate dilution water
reagents.Theyarethenconsideredtobenon-toxicforpurposes
for bulk samples should meet the acceptability criteria estab-
of this test method.
lishedinSection8ofGuideE729.Inaddition,allsuchdilution
8.1.2 Microbial Reagent—Freeze-dried Photobacterium water used for comparative testing with this test method and
phosphoreum. This is the only test reagent that is currently
invertebrates and fish is to be assayed on P. phosphoreum
(1993)availablefromonlyonesource. Whileotheracceptable (minimally once per month).
means of preservation may become available in the future,
9. Hazards
freeze-dried P. phosphoreum is specified in this test method
because a large number of users concur in the opinion that the 9.1 The handling of wastewaters entails potential hazards
strain is well standardized by this method of preservation, and due to exposure to chemical and biological contaminants.
that the same strain does not provide comparable response to Appropriatesafetymeasures,suchasthewearingofprotective
reference toxicants when preserved by other methods, or when clothing (gloves, apron, face shield, respirator, etc.) and main-
freshly cultured and harvested at the user’s laboratory, as taining proper hygiene, are utilized to minimize the chance of
described by Anthony A. Bulich, et al (1). Another consider- exposure. This test method is to be performed in a well-
ation is that a large body of published results, for which ventilated area.
freeze-dried P. phosphoreum was used, has accumulated since
9.2 Appropriate, environmentally safe procedures pre-
about 1980 (1,2,3,5,6). scribed by regulatory agencies are utilized in the disposal of
used waste samples.
8.1.3 Reconstitution Solution—Nontoxic water.
9.3 Due to the presence of aqueous samples and electrical
8.1.4 Diluent—Nontoxic 2% sodium chloride (NaCl), or
instrumentation in close proximity, care must be taken to
3.4% NaCl, reconstituted seawate
...