1、Designation: D 5660 96 (Reapproved 2004)Standard Test Method forAssessing the Microbial Detoxification of ChemicallyContaminated Water and Soil Using a Toxicity Test with aLuminescent Marine Bacterium1This standard is issued under the fixed designation D 5660; the number immediately following the de
2、signation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method (1)2covers
3、 a procedure for the rapidevaluation of the toxicity3of wastewaters and aqueous extractsfrom contaminated soils and sediments, to the luminescentmarine bacterium Photobacterium phosphoreum,4prior to andfollowing biological treatment. This test method is meant foruse as a means to assess samples resu
4、lting from biotreatabilitystudies. Sensitivity data for P. phosphoreum to over 1300chemicals have been reported in the literature (2). Some of thepublications are very relevant to this test method (3). The dataobtained from this test method, when combined with respirom-etry, total organic carbon (TO
5、C), biochemical oxygen demand(BOD), chemical oxygen demand (COD), or spectrophotomet-ric data, can assist in the determination of the degree ofbiodegradability of a contaminant in water, soil, or sediment(3). The percentage difference between the IC20 of treated anduntreated sample is used to assess
6、 the progress of detoxifica-tion.1.2 This test method is applicable to the evaluation of thetoxicity (to a specific microbe) and its implication on thebiodegradation of aqueous samples from laboratory researchbio-reactors (liquid or soil), pilot-plant biological treatmentsystems, full-scale biologic
7、al treatment systems, and landapplication processes (see Notes 1 and 2).NOTE 1If the biologically treated material is to be discharged in sucha manner as to potentially impact surface waters and ground water, orboth, then the user must consult appropriate regulatory guidance docu-ments to determine
8、the proper test species for evaluating potentialenvironmental impact (4). Correlations between data concerning reductionin toxicity produced by this test method and by procedures for acute orshort-term chronic toxicity tests, or both, utilizing invertebrates and fish(see Guides E 729 and E 1192), sh
9、ould be established, wherever possible.NOTE 2Color (especially red and brown), turbidity, and suspendedsolids interfere with this test method by absorbing or reflecting light. Inthese situations data are corrected for these effects by use of an absorbancecorrection procedure included in this test me
10、thod (see 5.3, 6.1, and 6.2).51.3 The results of this test method are reported in terms ofan inhibitory concentration (IC), which is the calculatedconcentration of sample required to produce a specific quanti-tative and qualitative inhibition. The inhibition measured is thequantitative reduction in
11、light output of luminescent marinebacteria (that is, IC20 represents the calculated concentrationof sample that would produce a 20 % reduction in the lightoutput of exposed bacteria over a specified time).1.4 The values stated in SI units are to be regarded as thestandard. The values given in parent
12、heses are for informationonly.1.5 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitation
13、s prior to use. Specific hazardstatements are given in Section 9.2. Referenced Documents2.1 ASTM Standards:6D 888 Test Methods for Dissolved Oxygen in Water1This test method is under the jurisdiction of ASTM Committee D34 on WasteManagement and is the direct responsibility of Subcommittee D34.03.01
14、onThermal and Biological Treatment.Current edition approved March 10, 1996. Published May 1996. Originallypublished as D 5660 95. Last previous edition D 5660 95.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.3Toxicity measured as toxic inhibition of
15、bacterial light output.4Microbics Corp. is currently the only known supplier of the reagents (testorganism Photobacterium phosphoreum strain NRRL B-11177) specific to this testmethod. There are two known manufacturers of analyzers that can be used tomeasure bioluminescence under temperature control:
16、 Microbics Corp., 2232 Ruth-erford Road, Carlsbad, CA 92008 (Microtox Model 500 and Model 2055 Analyz-ers), and Pharmacia LKB, 9319 Gaither Road, Gaithersburg, MD 20877 (LKBWallac Model 1250 and Model 1251 Luminometers). Other instruments would beconsidered when they become available. Please notifyA
17、STM Subcommittee D34.09if you are aware of any additional systems or instruments capable of performing thistesting.5At present (1993) use of the color correction scheme described in thisprocedure is known to be effective only with the Microbics Corporations toxicityanalyzers, due to the fact that th
18、e correction mathematics involve the detailedgeometry of both the ACC and the light meter. Please notify ASTM SubcommitteeD34.09 if you are aware of any other source of equipment capable of providing coloror turbidity correction, or both, for the P. phosphoreum test. Data validating theabsorbance co
19、rrection procedure are available from Microbics Corp.6For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyrig
20、ht ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.D 1125 Test Methods for Electrical Conductivity and Re-sistivity of WaterD 1129 Terminology Relating to WaterD 1193 Specification for Reagent WaterD 1293 Test Method for pH of WaterD 3370 Pract
21、ices for Sampling WaterE 729 Guide for Conducting Acute Toxicity Tests withFishes, Macroinvertebrates, and AmphibiansE 943 Terminology Relating to Biological Effects and En-vironmental FateE 1192 Guide for Conducting Acute Toxicity Tests onAqueous Effluents with Fishes, Macroinvertebrates, andAmphib
22、ians3. Terminology3.1 DefinitionsThe IC20 is defined in terms of a modifi-cation of the definition of IC50 as it appears in TerminologyE 943. The terms turbidity and volatile matter are defined inaccordance with Terminology D 1129. These terms are asfollows:3.1.1 colorthat is, the presence of dissol
23、ved matter thatabsorbs the light emitted by P. phosphoreum (that is, wave-length of 490 6 100 nm).3.1.2 IC20a statistically or graphically estimated concen-tration of test material that, under specified conditions, isexpected to cause a 20 % inhibition of a biological process(such as growth, reprodu
24、ction, or bioluminescence) for whichthe data are not dichotomous.3.1.3 turbidityreduction of transparency of a sample dueto the presence of particulate matter.3.1.4 volatile matterthat matter that is changed underconditions of the test to the gaseous state.4. Summary of Test Method4.1 This test meth
25、od covers the determination of acutetoxicity of aqueous samples to luminescent marine bacteria, P.phosphoreum.4.2 Wastewater samples are osmotically adjusted to theappropriate salinity for the test species P. phosphoreum.Asodium chloride (NaCl) concentration of 2 % has been foundoptimal for this tes
26、t organism for freshwater tests, or about3.4 % NaCl for seawater samples. This provides the necessaryosmotic protection for the bacteria, which are of marine origin.4.3 Samples should not be pH adjusted unless the user is notconcerned about toxic effects related directly to pH. Alteringthe sample pH
27、 will usually alter the solubility of both organicand inorganic constituents of the sample. Altering the pH canalso cause chemical reactions that will change the integrity ofthe sample, and greatly alter the exhibited toxicity of thesample. If sample pH is considered secondary to organismresponse, t
28、hen the optimal pH for the bacterium Photobacte-rium phosphoreum is 6.7.4.4 Comparison of inhibitory concentrations (IC20s) foruntreated wastewater (or extracts of untreated soils) versusthose for biologically treated wastewater (or extracts of treatedsoils), calculated from measured decreases in li
29、ght output ofexposed bacteria, allows for an assessment of the reduction intoxicity to the marine bacterium P. phosphoreum (see 1.1, 1.2,and Note 1).4.5 Samples that are highly colored, or contain solids thatcannot be removed without seriously compromising sampleintegrity, can be analyzed using an a
30、bsorbance correctionprocedure. This procedure determines the amount of lightabsorbed by the wastewater at a concentration near the nominalIC20 versus the baseline light output established by measuringthe light absorbed by the clear diluent.5. Significance and Use5.1 This test method provides a rapid
31、 means of determiningthe acute toxicity of an aqueous waste, or waste extract, priorto and following biological treatment, and contributes toassessing the potential biodegradability of the waste (see 1.1,1.2, and Note 1). The change in toxicity to the marinebacterium P. phosphoreum with respect to t
32、ime may serve as anindication of the biodegradation potential. Sample analyses areusually obtained in 45 to 60 min, with as little as 5 mL ofsample required (5).5.2 Samples with high suspended solids concentrations maytest nontoxic to the bacteria, while still exhibiting significanttoxicity to fresh
33、water organisms, due to those suspendedsolids.5.3 The absorbance correction procedure included in thistest method allows for the analysis of highly colored lightab-sorbing samples, by providing a means for mathematicallyadjusting the light output readings to account for light lost dueto absorption.5
34、6. Interferences6.1 Some test samples that are highly colored (especiallyred and brown) interfere with this test method, but theabsorbance correction procedure can be used to correct for thisinterference.56.2 Turbidity due to suspended solids interferes with thistest method. The absorbance correctio
35、n procedure can be usedto correct for this interference and is preferable to otheralternatives. Pressure filtration, or centrifuging and decanting,will also remove this interference. Some toxics may be lostthrough adsorption and volatilization during filtration or cen-trifugation, thus impacting the
36、 exhibited toxicity.57. Apparatus7.1 Fixed or Adjustable Volume Pipetter, 10 L, withdisposable tips.7.2 Variable Volume Pipetter, 10 to 1000 L, with dispos-able tips.7.3 Variable Volume Pipetter, 1 to 5 mL, with disposabletips.7.4 Timer or Stopwatch.7.5 Glass Cuvettes, 11.75 mm OD, 10.5 mm ID by 50
37、mmheight, 4-mL volume.7.6 Absorbance Correction Cuvettes (ACC)Optionalitem, but required to analyze highly colored samples or thosecontaining suspended particulates.57.7 Variable Voltage Chart Recorder (optional)Usefulwhen using some types of light meters.7.8 Computer (optional)Useful with some ligh
38、t meters,for which software is also available, to facilitate data captureand reduction.D 5660 96 (2004)27.9 Light Meter, for cuvettes listed in 7.5.4,57.10 Temperature Control Devices (temperature-controlledroom, water bath, refrigerators, or other device)One capableof maintaining 5.5 6 1C and one c
39、apable of maintaining 15 60.5C.8. Reagents and Materials8.1 Test Reagents:8.1.1 For purposes of this test method, test reagents aredefined as the reagents actually used in performance of the testmethod. The necessary requirement with regard to qualificationof test reagents is that this test method p
40、rovide acceptableresults when reference toxicants are tested using the testreagents.They are then considered to be non-toxic for purposesof this test method.8.1.2 Microbial ReagentFreeze-dried Photobacteriumphosphoreum. This is the only test reagent that is currently(1993) available from only one so
41、urce.4While other acceptablemeans of preservation may become available in the future,freeze-dried P. phosphoreum is specified in this test methodbecause a large number of users concur in the opinion that thestrain is well standardized by this method of preservation, andthat the same strain does not
42、provide comparable response toreference toxicants when preserved by other methods, or whenfreshly cultured and harvested at the users laboratory, asdescribed by Anthony A. Bulich, et al (1). Another consider-ation is that a large body of published results, for whichfreeze-dried P. phosphoreum was us
43、ed, has accumulated sinceabout 1980 (1,2,3,5,6).8.1.3 Reconstitution SolutionNontoxic water.8.1.4 DiluentNontoxic 2 % sodium chloride (NaCl), or3.4 % NaCl, reconstituted seawater or sea water (dependingupon the type of sample and purpose of the test). The P.phosphoreum test has been performed at osm
44、otic pressuresequivalent to 1 to 6 % NaCl, but has long been standardized at2 % for freshwater samples. The major requirement is that theosmotic pressure be held constant within each test, to minimizetransient variations in luminescence due to variations inosmotic pressure. The higher salinity (and
45、osmotic pressure) ofmarine samples dictate the use of a diluent other than 2 %NaCl. Both reconstituted seawater and clean seawater havebeen used as diluent. A procedure for preparing reconstitutedsalt water, and formula, are given in Table 3 of Guide E 729.Actual seawater has also been collected at
46、remote sites andused as diluent for testing aqueous samples of marine origin.The most important requirement is that the diluent must bequalified for use with this test method (see 8.1.1).8.2 Reagent ChemicalsReagent grade chemicals are rec-ommended for use in preparation of test reagents and referen
47、cetoxicants. Unless otherwise indicated, it is intended that allreagents shall conform to the specifications of the Committeeon Analytical Reagents of the American Chemical Society.7Other grades may be used, but there will be more risk that theresulting test reagents will fail to qualify (see 8.1.1)
48、.8.2.1 Sodium Chloride (NaCl)Used in preparation ofdiluent, and for adjusting the osmotic pressure of samples tothat of the chosen diluent.8.2.2 Phenol, or Other Common Organic ToxicantUsedas a reference toxicant.8.2.3 Zinc Sulfate Heptahydrate, or Other Common Inor-ganic ToxicantUsed as reference t
49、oxicant.8.3 Purity of Water Unless otherwise indicated, refer-ences to water shall be understood to mean reagent waterconforming to Specification D 1193, Reagent Water, Type I orII, SubtypeA. Test reagents prepared from reagent water are tobe qualified for use with this test method (see 8.1.1).8.4 When this test method is used in conjunction with othertests employing higher organisms, appropriate dilution waterfor bulk samples should meet the acceptability criteria estab-lished in Section 8 of Guide E 729. In addition, all suchdilution water used for compar
