ASTM D5660-96
(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
Standard Test Method for Assessing the Microbial Detoxification of Chemically Contaminated Water and Soil Using a Toxicity Test with a Luminescent Marine Bacterium
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 E1192), 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 practices and determine the applicability of regulatory limitations prior to use. Specific hazard statements are given in Section 9.
General Information
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Standards Content (Sample)
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
Designation: D 5660 – 96
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 D 5660; 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 (e) 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
evaluation of the toxicity of wastewaters and aqueous extracts
environmental impact (4). Correlations between data concerning reduction
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
(see Guides E 729 and E 1192), should be established, wherever possible.
following biological treatment. This test method is meant for
NOTE 2—Color (especially red and brown), turbidity, and suspended
use as a means to assess samples resulting from biotreatability
solids interfere with this test method by absorbing or reflecting light. In
studies. Sensitivity data for P. phosphoreum to over 1300
these situations data are corrected for these effects by use of an absorbance
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 respirom-
an inhibitory concentration (IC), which is the calculated
etry, total organic carbon (TOC), biochemical oxygen demand
concentration of sample required to produce a specific quanti-
(BOD), chemical oxygen demand (COD), or spectrophotomet-
tative and qualitative inhibition. The inhibition measured is the
ric data, can assist in the determination of the degree of
quantitative reduction in light output of luminescent marine
biodegradability of a contaminant in water, soil, or sediment
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
1.5 This standard does not purport to address all of the
bio-reactors (liquid or soil), pilot-plant biological treatment
safety concerns, if any, associated with its use. It is the
systems, full-scale biological treatment systems, and land
responsibility of the user of this standard to establish appro-
application processes (see Notes 1 and 2).
priate safety and health practices and determine the applica-
NOTE 1—If the biologically treated material is to be discharged in such
bility of regulatory limitations prior to use. Specific hazard
statements are given in Section 9.
2. Referenced Documents
This test method is under the jurisdiction of ASTM Committee D34 on Waste
Management and is the direct responsibility of Subcommittee D34.07 on Municipal
2.1 ASTM Standards:
Solid Waste. 6
D 888 Test Methods for Dissolved Oxygen in Water
Current edition approved March 10, 1996. Published May 1996. Originally
D 1125 Test Methods for Electrical Conductivity and Re-
published as D 5660 – 95. Last previous edition D 5660 – 95.
The boldface numbers in parentheses refer to the list of references at the end of sistivity of Water
this standard.
D 1129 Terminology Relating to Water
Toxicity measured as toxic inhibition of bacterial light output.
Microbics Corp. is currently the only known supplier of the reagents (test
organism Photobacterium phosphoreum strain NRRL B-11177) specific to this test
method. There are two known manufacturers of analyzers that can be used to At present (1993) use of the color correction scheme described in this
measure bioluminescence under temperature control: Microbics Corp., 2232 Ruth- procedure is known to be effective only with the Microbics Corporation’s toxicity
erford Road, Carlsbad, CA 92008 (Microtox Model 500 and Model 2055 Analyz- analyzers, due to the fact that the correction mathematics involve the detailed
ers), and Pharmacia LKB, 9319 Gaither Road, Gaithersburg, MD 20877 (LKB geometry of both the ACC and the light meter. Please notify ASTM Subcommittee
Wallac Model 1250 and Model 1251 Luminometers). Other instruments would be D34.09 if you are aware of any other source of equipment capable of providing color
considered when they become available. Please notify ASTM Subcommittee D34.09 or turbidity correction, or both, for the P. phosphoreum test. Data validating the
if you are aware of any additional systems or instruments capable of performing this absorbance correction procedure are available from Microbics Corp.
testing. Annual Book of ASTM Standards, Vol 11.01.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
D 5660
D 1193 Specification for Reagent Water 4.5 Samples that are highly colored, or contain solids that
D 1293 Test Method for pH of Water cannot be removed without seriously compromising sample
D 3370 Practices for Sampling Water integrity, can be analyzed using an absorbance correction
E 729 Guide for Conducting Acute Toxicity Tests with procedure. This procedure determines the amount of light
Fishes, Macroinvertebrates, and Amphibians absorbed by the wastewater at a concentration near the nominal
E 943 Terminology Relating to Biological Effects and En- IC20 versus the baseline light output established by measuring
vironmental Fate the light absorbed by the clear diluent.
E 1192 Guide for Conducting Acute Toxicity Tests on
5. Significance and Use
Aqueous Effluents with Fishes, Macroinvertebrates, and
5.1 This test method provides a rapid means of determining
Amphibians
the acute toxicity of an aqueous waste, or waste extract, prior
3. Terminology
to and following biological treatment, and contributes to
3.1 Definitions—The IC20 is defined in terms of a modifi- assessing the potential biodegradability of the waste (see 1.1,
1.2, and Note 1). The change in toxicity to the marine
cation of the definition of IC50 as it appears in Terminology
bacterium P. phosphoreum with respect to time may serve as an
E 943. The terms turbidity and volatile matter are defined in
accordance with Terminology D 1129. These terms are as indication of the biodegradation potential. Sample analyses are
usually obtained in 45 to 60 min, with as little as 5 mL of
follows:
3.1.1 color—that is, the presence of dissolved matter that sample required (5).
5.2 Samples with high suspended solids concentrations may
absorbs the light emitted by P. phosphoreum (that is, wave-
length of 490 6 100 nm). test nontoxic to the bacteria, while still exhibiting significant
toxicity to freshwater organisms, due to those suspended
3.1.2 IC20—a statistically or graphically estimated concen-
tration of test material that, under specified conditions, is solids.
5.3 The absorbance correction procedure included in this
expected to cause a 20 % inhibition of a biological process
(such as growth, reproduction, or bioluminescence) for which test method allows for the analysis of highly colored lightab-
sorbing samples, by providing a means for mathematically
the data are not dichotomous.
3.1.3 turbidity—reduction of transparency of a sample due adjusting the light output readings to account for light lost due
to absorption.
to the presence of particulate matter.
3.1.4 volatile matter—that matter that is changed under
6. Interferences
conditions of the test to the gaseous state.
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
absorbance correction procedure can be used to correct for this
4.1 This test method covers the determination of acute
interference.
toxicity of aqueous samples to luminescent marine bacteria, P.
6.2 Turbidity due to suspended solids interferes with this
phosphoreum.
test method. The absorbance correction procedure can be used
4.2 Wastewater samples are osmotically adjusted to the
to correct for this interference and is preferable to other
appropriate salinity for the test species P. phosphoreum.A
alternatives. Pressure filtration, or centrifuging and decanting,
sodium chloride (NaCl) concentration of 2 % has been found
will also remove this interference. Some toxics may be lost
optimal for this test organism for freshwater tests, or about
through adsorption and volatilization during filtration or cen-
3.4 % NaCl for seawater samples. This provides the necessary
trifugation, thus impacting the exhibited toxicity.
osmotic protection for the bacteria, which are of marine origin.
4.3 Samples should not be pH adjusted unless the user is not
7. Apparatus
concerned about toxic effects related directly to pH. Altering
7.1 Fixed or Adjustable Volume Pipetter, 10 μL, with
the sample pH will usually alter the solubility of both organic
disposable tips.
and inorganic constituents of the sample. Altering the pH can
7.2 Variable Volume Pipetter, 10 to 1000 μL, with dispos-
also cause chemical reactions that will change the integrity of
able tips.
the sample, and greatly alter the exhibited toxicity of the
7.3 Variable Volume Pipetter, 1 to 5 mL, with disposable
sample. If sample pH is considered secondary to organism
tips.
response, then the optimal pH for the bacterium Photobacte-
7.4 Timer or Stopwatch.
rium phosphoreum is 6.7.
7.5 Glass Cuvettes, 11.75 mm OD, 10.5 mm ID by 50 mm
4.4 Comparison of inhibitory concentrations (IC20s) for
height, 4-mL volume.
untreated wastewater (or extracts of untreated soils) versus
7.6 Absorbance Correction Cuvettes (ACC)—Optional
those for biologically treated wastewater (or extracts of treated
item, but required to analyze highly colored samples or those
soils), calculated from measured decreases in light output of
containing suspended particulates.
exposed bacteria, allows for an assessment of the reduction in
7.7 Variable Voltage Chart Recorder (optional)—Useful
toxicity to the marine bacterium P. phosphoreum (see 1.1, 1.2,
when using some types of light meters.
and Note 1).
7.8 Computer (optional)—Useful with some light meters,
for which software is also available, to facilitate data capture
Annual Book of ASTM Standards, Vol 11.05. and reduction.
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
D 5660
,
4 5
7.9 Light Meter, for cuvettes listed in 7.5. Other grades may be used, but there will be more risk that the
7.10 Temperature Control Devices (temperature-controlled resulting test reagents will fail to qualify (see 8.1.1).
room, water bath, refrigerators, or other device)—One capable 8.2.1 Sodium Chloride (NaCl)—Used in preparation of
of maintaining 5.5 6 1°C and one capable of maintaining 15 6 diluent, and for adjusting the osmotic pressure of samples to
0.5°C. that of the chosen diluent.
8.2.2 Phenol, or Other Common Organic Toxicant—Used
8. Reagents and Materials
as a reference toxicant.
8.1 Test Reagents: 8.2.3 Zinc Sulfate Heptahydrate, or Other Common Inor-
8.1.1 For purposes of this test method, test reagents are ganic Toxicant—Used as reference toxicant.
defined as the reagents actually used in performance of the test 8.3 Purity of Water— Unless otherwise indicated, refer-
method. The necessary requirement with regard to qualification ences to water shall be understood to mean reagent water
of test reagents is that this test method provide acceptable conforming to Specification D 1193, Reagent Water, Type I or
results when reference toxicants are tested using the test II, Subtype A. Test reagents prepared from reagent water are to
reagents. They are then considered to be non-toxic for purposes be qualified for use with this test method (see 8.1.1).
of this test method. 8.4 When this test method is used in conjunction with other
tests employing higher organisms, appropriate dilution water
8.1.2 Microbial Reagent—Freeze-dried Photobacterium
phosphoreum. This is the only test reagent that is currently for bulk samples should meet the acceptability criteria estab-
lished in Section 8 of Guide E 729. In addition, all such
(1993) available from only one source. While other acceptable
means of preservation may become available in the future, dilution water used for comparative testing with this test
method and invertebrates and fish is to be assayed on P.
freeze-dried P. phosphoreum is specified in this test method
because a large number of users concur in the opinion that the phosphoreum (minimally once per month).
strain is well standardized by this method of preservation, and
9. Hazards
that the same strain does not provide comparable response to
9.1 The handling of wastewaters entails potential hazards
reference toxicants when preserved by other methods, or when
due to exposure to chemical and biological contaminants.
freshly cultured and harvested at the user’s laboratory, as
Appropriate safety measures, such as the wearing of protective
described by Anthony A. Bulich, et al (1). Another consider-
clothing (gloves, apron, face shield, respirator, etc.) and main-
ation is that a large body of published results, for which
taining proper hygiene, are utilized to minimize the chance of
freeze-dried P. phosphoreum was used, has accumulated since
exposure. This test method is to be performed in a well-
about 1980 (1,2,3,5,6).
ventilated area.
8.1.3 Reconstitution Solution—Nontoxic water.
9.2 Appropriate, environmentally safe procedures pre-
8.1.4 Diluent—Nontoxi
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