# APPENDIX H

Derivation of the 1985 Aquatic Life Criteria



# WATER QUALITY STANDARDS HANDBOOK

SECONDEDITION

# Derivation of the 1985

Aquatic Life Criteria

The following isasummary of the Guidelines for Derivation of Criteria for Aquatic Life. The complete text isfound in "Guidelines for Deriving Numerical National Water Quality Criteria for the Protection of Aquatic Organisms andTheir Uses” available from National Technical Information Service - PB85-227049. Derivation of numerical national water quality criteria for the protection of aquatic organisms and their usesisacomplex process that usesinformation from many areasof aquatic toxicology. When a national criterion is needed for aparticular material, all available information concerning toxicity to and bioaccumulation by aquatic organisms is collected, reviewed for acceptability, and sorted. If enough acceptable data on acute toxicity to aquatic animals areavailable, they are used to estimate the highest one-hour average concentration that should not result in unacceptable effectson aquatic organisms and their uses. If justified, this concentration is made a function of water quality characteristics such as pH, salinity, or hardness. Similarly, data on the chronic toxicity of the material to aquatic animals are used to estimate the highest four-day average concentration that should not cause unacceptable toxicity during a long-term exposure. If appropriate, this concentration is also related to awater quality characteristic. Data on toxicity to aquatic plants are examined to determine whether plants are likely to be unacceptably affected by concentrations that should not cause unacceptable effects on animals. Data on bioaccumulation by aquatic organisms are used to determine if residues might subject edible species to restrictions by the U.S. Food and Drug Administration (FDA), or if such residues might harm wildlife that consumes aquatic life. All other available data are examined for adverse effectsthat might bebiologically important. If athorough review of the pertinent information indicates that enough acceptable data exists, numerical national water quality criteria arederived for fresh water or salt water or both to protect aquatic organisms and their usesfrom unacceptable effectsdue to exposures to high concentrations for short periods of time, lower concentrations for longer periods of time, and combinations of the two.

I. Definition of Material of Concern
A. Each separate chemical that does not ionize substantially in most natural bodies of water should usually be considered aseparate material, except possibly for structurally similar organic compounds that exist only in large quantities as commercial mixtures of the various compounds and apparently have similar biological, chemical, physical, and toxicological properties. B. For chemicals that do ionize substantially in most natural waterbodies (e.g.,some phenols and organic acids, some salts of phenols and organic acids, and most inorganic salts and coordination complexes of metals), all forms in chemical equilibrium should usually be considered one material. Each different oxidation state of a metal and each different non-ionizable covalently bonded organometallic compound should usually be considered aseparate material. C. The definition of the material should include an operational analytical component. Identification of a material simply, for example, as "sodium" obviously implies "total sodium” but leaves room for doubt. If “total” is meant, it should be explicitly stated. Even"total" has different operational definitions, some of which do not necessarily measure

“all that is there” in all sample. Thus, it is also necessary to reference or describe one analytical method that is intended. The operational analytical component should take into account the analytical and environmental chemistry of the material, the desirability of using the same analytical method on samples from laboratory tests, ambient water and aqueous effluents, and various practical considerations such as labor and equipment requirements and whether the method would require measurement in the field or would allow measurement after samples aretransported to alaboratory. The primary requirements of the operational analytical component are that it be appropriate for use on samples of receiving water, compatible with the available toxicity and bioaccumulation data without making overly hypothetical extrapolations, and rarely result in underprotection or overprotection of aquatic organisms and their uses. Because an ideal analytical measurement will rarely beavailable, acompromise measurement will usually be used. This compromise measurement must fit with the general approach: if an ambient concentration is lower than the national criterion, unacceptable effects will probably not occur (i.e., the compromise measurement must not err on the side of underprotection when measurements are made on asurface water). Becausethe chemical and physical properties of an effluent are usually quite different from those of the receiving water, an analytical method acceptable for analyzing an effluent might not be appropriate for analyzing a receiving water, and vice versa. If the ambient concentration calculated from a measured concentration in an effluent is higher than the national criterion, an additional option isto measurethe concentration after dilution of the effluent with receiving water to determine if the measured concentration is lowered by such phenomena as complexation or sorption. A further option, of course, is to derive a site-specific criterion (1,2,3). Thus, the criterion should be based on an appropriate analytical measurement, but the criterion is not rendered useless if an ideal measurement either is not available or is not feasible. The analytical chemistry of the material might need to be considered when defining the material or when judging the acceptability of sometoxicity tests,but acriterion should not bebased on the sensitivity of an analytical method. When aquatic organisms are more sensitive than routine analytical methods, the proper solution is to develop better analytical methods, not to underprotect aquatic life.

II. Collection of Data
A. Collect all available data on the material concerning toxicity to, and bioaccumulation by aquatic animals and plants; FDA action levels (compliance Policy Guide, U.S. Food & Drug Admin. 1981)and chronic feeding studies and long-term field studies with wildlife species that regularly consume aquatic organisms.
B. All data that are used should be available in typed, dated, and signed hard copy (publication, manuscript, letter, memorandum) with enough supporting information to indicate that acceptable test procedures were used and that the results are probably reliable. In some cases, additional written information from the investigator may be needed. Information that is confidential, privileged, or otherwise not available for distribution should not be used. C. Questionable data, whether published or unpublished, should not be used. Examples would bedata from teststhat did not contain acontrol treatment, tests in which too many organisms in the control treatment died or showed signs of stress or disease, and tests in which distilled or deionized water was used as the dilution water without addition of appropriate salts.
D. Data on technical grade materials may be used, if appropriate; but data on formulated mixtures and emulsifiable concentrates ofthe material may not be used.E. For some highly volatile, hydrolyzable, or degradable materials, only use data from flow-through tests in which the concentrations of test material were measured often enough with acceptable analytical methods.
F. Data should be rejected if obtained by using

• Brineshrimp- because they usually occur naturally only in water with salinity greater than 35g/kg;

• Speciesthat do not have reproducing wild populations in North America; or
• Organisms that were previously exposed to substantial concentrations of the test material or other contaminants. G. Questionable data, data on formulated mixtures and emulsifiable concentrates, and data obtained with nonresident species or previously exposed organisms may be used to provide auxiliary information but should not be used in the derivation of criteria.

III. Required Data A. Certain data should be available to help ensure that each of the four major kinds of possible adverse effects receives adequate consideration: results of acute and chronic toxicity tests with representative species of aquatic animals are necessary to indicate the sensitivities of appropriate untested species. However, since procedures for conducting testswith aquatic plants and interpreting the results are not aswell developed, fewer data concerning toxicity are required. Finally, data concerning bioaccumulation by aquatic organisms are required only with relevant information on the significance of residues in aquatic organisms. B. Toderive acriterion for freshwater aquatic organisms and their uses,the following should be available: 1. Results of acceptable acute tests (seesection IV) with at least one species of freshwater animal in at least eight different families including all of the following

• The family Salmonidae in the classOsteichthyes.

• Asecond family in the classOsteichthyes, preferably acommercially or recreationally important warmwater species,such asbluegill or channel catfish.

• Athird family in the phylum Chordata (may be in the classOsteichthyes or may bean amphibian etc.).

• A planktonic crustacean such asacladoceran or copepod.

• Abenthic crustacean (ostracod, isopod, amphipod, crayfish, etc.).

• An insect (mayfly, dragonfly, damselfly, stonefly, caddisfly, mosquito, midge, etc.).

• Afamily in aphylum other than Arthropoda or Chordata, such asRotifera, Annelida, Mollusca.

• A family in any order of insect or any phylum not already represented. 2. Acute-chronic ratios (seesection VI) with species of aquatic animals in at least three different families, provided that:

• At least one isafish;

• At least one is an invertebrate; and

• At least one is an acutely sensitive freshwater species (the other two may be saltwater species). 3. Results of at least one acceptable test with a freshwater alga or vascular plant (see section VIII). If the plants are among the aquatic organisms that are most sensitive to the material, testdata on aplant in another phylum (division) should alsobeavailable.4. At least one acceptable bioconcentration factor determined with an appropriate hshwater species, if a maximum permissible tissue concentration is available (see section IX). C. To derive acriterion for saltwater aquatic organisms and their uses,the following should beavailable: 1. Results of acceptable acute tests (seesection IV) with at least one species of saltwater animal in at least eight different families, including all of the following

l Two families in the phylum Chordata;

l A family in aphylum other than Arthropoda or Chordata;

l Either the Mysidae or Penaeidae family;

l Three other families not in the phylum Chordata (may include Mysidae or Penaeidae, whichever was not used previously); and

l Any other family. 2. Acute&r& ratios (sees&ion VI) with species of aquatic animals in at least three different families, provided that of the three species:

l Atleastoneisatih;

l At least one is an invertebrate; and

l At least one is an acutely sensitive saltwater species (the other may bean acutely sensitive freshwater species). 3. Results of at least one acceptable test with a saltwater alga or vascular plant (see section VIII). If plants are among the aquatic organisms most sensitive to the material+ results of atest with aplant in another phylum (division) should also beavailable. 4. At least one acceptable bioconcentration factor determined with an appropriate saltwater species, if a maximum permissible tissue concentration is available (see section DQ.
D. If all required data are available, a numerical criterion can usually be derived, except in special cases.For example, derivation of acriterion might not be possible if the available acute-chronic ratios vary by more than afactor of 10with no apparent pattern. Also, if a criterion is to be related to awater quality characteristic T (seesections V and VII), more data will be necessary. Similarly, if all required data are not available, a numerical criterion should not be derived except in special cases.For example, even if not enough acute and chronic data are available, it might bepossrble to derive acriterion if the available data clearly indicate that the Final Residue Value should be much lower than either the Final Chronic Value or the Final Plant Value.
E. Confidence in a criterion usually increases as the amount of available pertinent data increases. Thus, additional data areusually desirable.

IV. Final Acute Value A. Appropriate measures of the acute (short-term) toxicity of the material to a variety of species of aquatic animals are used to calculate the Final Acute Value. The Final Acute Value is an estimate of the concentration of the material, corresponding to a cumulative probability of 0.05 in the acute toxicity values for genera used in acceptable acute tests conducted on the material. Howevel; in some cases,if the Species Mean Acute Value of a commercially or recreationally important species is lower than the calculated Final Acute Value, then that Species Mean Acute Value replaces the calculated Final Acute Value to protect that important species.B. Acute toxicity tests should have been conducted using acceptable procedures (ASTM Standards E729and 724). C. Except for tests with saltwater annelids and mysids, do not use results of acute tests during which testorganisms were fed, unless data indicate that the food did not affect the toxicity of the test material. D. Results of acute tests conducted in unusual dilution water (dilution water in which total organic carbon or particulate matter exceeded 5 mg/L) should not be used unless a relationship is developed between acute toxicity and organic carbon or particulate matter or unless data show that the organic carbon or particulate matter does not affect toxicity. E. Acute values should be based on endpoints that reflect the total severe acute adverse impact of the test material on the organisms used in the test.Therefore, only the following kinds of data on acute toxicity to aquatic animals should be used: 1. Testswith daphnids and other cladocerans should bestarted with organisms lessthan 24-hours old, and tests with midges should be stressed with second- or third-in&u larvae. The result should be the 48-hour EC& based on percentage of organisms immobilized plus percentage oforganisms lolled. If such an ECs,-, is not available from atest,the 48-hour LCw should be used in place of the desired 48-hour ECm.An ECwor LCw of longer than 48 hours can be used aslong asthe animals were not fed and the control animals were acceptable atthe end of the test. 2. The result of a test with embryos and larvae of barnacles, bivalve molluscs (clams, mussels, oysters, and scallops), sea urchins, lobsters, crabs, shrimp, and abalones should be the %hour EC50based on the percentage of organisms with incompletely developed shells plus the percentage of organisms killed. If such an ECU, is not available from a test, the lower of the 96hour EC- based on the percentage of organisms with incompletely developed shells and the %-hour LCw should be used in place of the desired %hour EC50.If the duration of the test was between 48and % hours, the ECw or LCm atthe end of the test should beused. 3. The acute values from testswith all other freshwater and saltwater animal species and older life stages of barnacles, bivalve molluscs, seaurchins, lobsters, crabs, shrimps, and abalones should be the %-hour ECw based on the percentage of organisms exhibiting lossof equilibrium, plus the percentage of organisms immobilized, plus the percentage of organisms killed. If such an EC%is not available from atest,the %-hour LCm should be used in place of the desired %-hour ECm. 4. Testswith singlecelled organisms are not considered acute tests, even if the duration was 96hours or less. 5. If the tests were conducted properly, acute values reported as ‘greater than’ values and those above the solubility of the test material should be used because rejection of such acute values would unnecessarily lower the Final Acute Value by eliminating acute values for resistant species. F. If the acute toxicity of the material to aquatic animals apparently has been shown to be related to a water quality characteristic such as hardness or particulate matter for freshwater animals or salinity or particulate matter for salhvater animals, a Final Acute Equation should bederived based on that water quality characteristic. (Co to section V.) G. If the available data indicate that oneor more life stagesareatleastafactor of2more resistant than oneor more other life stagesofthe samespecies,the data for the more resistant life stages should not beused in the calculation ofthe SpeciesMean Acute Value becauseaspeciescanbe considered protected from acutetoxicity only if all life stagesareprotected. H. The agreement of the data within and between speciesshould beconsidered. Acute values that appear to be questionable in comparison with other acute and chronic data for the same species and for other species in the same genus probably should not be used inI. calculation of aSpeciesMean Acute Value. For example, if the acute values available for a

species or genus differ by more than a factor of 10,some or all of the values probably should not be used in calculations. For each species for which at least one acute value is available, the Species Mean Acute Value should becalculated asthe geometric mean of the results of all flow-through tests in which the concentrations of test material were measured. For aspecies for which no such result is available, the Species Mean Acute Value should be calculated asthe geometric mean of all available acute values - i.e., results of flow-through tests in which the concentrations were not measured and results of static and renewal tests based on initial concentrations of test material. (Nominal concentrations are acceptable for most test materials if measured concentrations are not available.) NOTE Data reported by original investigators should not be rounded off. Resultsof all intermediate calculations should berounded tofour significant digits. NOTE The geometric meanof N numbers isthe Nth root of the product of the N numbers. Alternatively, the geometric mean can be calculated by adding the logarithms of the N numbers,dividing thesumby N, andtaking theantilog ofthequotient. Thegeometric mean oftwo numkn isthesquarerootoftheproduct ofthetwo numbers,andthegeometricmean ofonenumber isthat number. Either natural (base0)orcommon (base10)logarithms canbe usedtocalculategeometricmeansaslong astheyareusedconsistently within eachsetofdata (i.e.,theantilog usedmustmatchthelogarithm used). NOTE:Geometricmeansratherthanarithmetic meansareusedherebecause thedistributions ofindividual organisms’sensitivities in toxicity testson mostmaterials,andthedistributions ofspecies’sensitivities within agenus,aremorelikely tobelognormal than normal. Similarly, geometric means ate used for acutethronic ratios and bioconcentration factors because quotients arelikely tobecloserto lognormal than normal distributions. In addition, division of the geometric mean of a set of numerators by the geometric mean of the set of corresponding denominaton will result in the geometric meanof thesetof corresponding quotients. J. The Genus Mean Acute Value should be calculated asthe geometric mean of the Species Mean Acute Values available for eachgenus. K. Order the Genus Mean Acute Value from high to low. L. Assign ranks, R, to the Genus Mean Acute Value from “1” for the lowest to “N” for the highest. If two or more Genus Mean Acute Values are identical, arbitrarily assign them successive ranks.
M. Calculate the cumulative probability, P,for eachGenus Mean Acute Value asR/ (N+l).
N. Select the four Genus Mean Acute Values that have cumulative probabilities closest to 0.05. (If there are less than 59 Genus Mean Acute Values, these will always be the four lowest Genus Mean Acute Values).
0. Using the selected Genus Mean Acute Values and Ps,calculate: s2=Z((ln GMAV)2) - ((Z(ln CMAv&q

4-n - w~h 

## L=@On GMAV) =S@(fl)))l4 A=S(m+L FAV =eA (Seeoriginal document, referenced at beginning of this appendix, for development of the calculation procedure and Appendix 2for example calculation and computer program.) NOTE: Natural logarithms (logarithms to basee,denoted as In) are used herein merely because theyareeasiertouseonsomehandcalculatorsandcomputen thancommon(base10) logarithms. Consistentuseofeitherwill producethesameresult.F’. If for acommercially or recreationally important species the geometric mean of the acute values from flow-through tests in which the concentrations of test material were measured islower than the calculated Final Acute Value, then that geometric mean should be used asthe Final Acute Value instead of the calculated Final Acute Value. Q. Go to section VI.

V. Final Acute Equation
A. When enough dataareavailable toshow that acutetoxicity totwo or more speciesissimilarly related to a water quality characteristic, the relationship should be taken into account as described in @on IV,stepsBthrough G,or using analysis of covaxiance. The two methods areequivalent and produce identical results. The manual method described below provides an understanding of this application of covariance analysis, but computerized versions of covariance analysis aremuch more convenient for analyzing large data tests.If two or more factors affecttoxicity, multiple regression analysis should beused. B. For each species for which comparable acute toxicity values are available at two or more different values of the water quality characteristic, perform a least squares regression of the acute toxicity values on the corresponding values of the water quality characteristic to obtain the slope and its 95percent confidence limits for eachspecies. NOTE: Because

thebestdocumented relationship fitting thesedataisthat betweenhardness and acutetoxicity of metalsin freshwater and a log-log relationship, geometric meansand natural logarithms ofboth toxicity and water quality areusedin the restof this section.For relationships basedonotherwaterquality characteristicssuchaspH, temperature,orsalinity, no transformation or a different transformation might fit the data better, and appropriate changeswill benecessary. C. Decide whether the data for each species are useful, taking into account the range and number of the tested values of the water quality characteristic and the degree of agreement within and between species. For example, a slope based on six data points might be of limited value if based only on data for avery narrow range of water quality characteristic values. A slope based on only two data points, however, might be useful if consistent with other information and if the two points cover abroad enough range of the water quality characteristic. In addition, acute values that appear to be questionable in comparison with other acute and chronic data available for the same species and for other species in the same genus probably should not be used. For example, if after adjustment for the water quality characteristic the acute values available for aspecies or genus differ by more than afactor of 10, probably some or ail of the values should be rejected. If useful slopes are not available for at least one fish and one invertebrate, or if the available slopes axe too dissimilar, or if too few data are available to adequately define the relationship between acute toxicity and the water quality characteristic, return to section lV.G, using the results of tests conducted under conditions and in waters similar to those commonly used for toxicity testswith the species. D. Individually for each species, calculate the geometric mean of the available acute values and then divide eachof these acute values by the mean for the species.This normalizes the values sothat the geometric mean of the normalized values for eachspecies, individually, and for any combination of species is 1.O.
E. Similarly normalize the values of the water quality characteristic for each species, individually. F. Individually for each species, perform a least squares regression of the normalized acute toxicity values on the corresponding normalized values ofthe water quality characteristic. The resulting slopes and 95percent confidence limits will be identical to those obtained instep B.However, now, if the data areactually plotted, the line of bestfit for eachindividual

specie will go thmugh the point 1,l in the center of the graph. G. Treat normalized data asif they were all for the same species and perform aleast squares regression of all the normalized acute values on the corresponding normalized values of the water quality characteristic to obtain the pooled acute slope, V and its 56 percent confidence limits. If ail the normalized data are actuaIly plotted, the line of best fit will go thmugh the point 1,l in the center of the graph H. For each species, calculate the geometric mean, W, of the acute toxicity values and the geometric mean, X,ofthe values ofthe water quahty characteristic. (Thesewere calculated in steps D and E.) I. For eachspecies, calculate the logarithm, Y,of the Species Mean Acute Value at aselected value, Z of the water quality characteristic using the equation: Y=lnW-V(lnX-1nZ).
J. For eachspecies, calculate the SMAV at2 using the equation: SMAV =e”. NOTE: Alternatively, theSpecies MeanAcuteValuesatZcanbeobtained byskipping stepH using the equations in steps I and Jto adjust eachacutevalue individually to Z, and then calculating thegeometricmeanoftheadjustedvaluesforeachspecksindividually. This akrnative procedure allows an examination of the range of the adjusted acute values for eachspecies. K. Obtain the Final Acute Value at Z by using the procedure described in section IV steps J through 0. L. If the SpeciesMean Acute Value atZof acomrnercialIy or recreationally important species is lower than the calculated Final Acute Value at Z, then that Species Mean Acute Value should be used asthe Final Acute Value atZ instead ofthe calculated Final Acute Value.
M. The Final Acute Equation iswritten as:

Final Acute Value = e(V[lll(Watcfqldity chanctcrlstic)] +hl A-V[hl ZD where V =pooled acute slope A =Final Acute Value atZ. Because V, A, and Z are known, the Final Acute Value can be calculated for any selected value of the water quality characteristic.

VI. FinaI Chronic VaIue A. Depending on the data that are available concerning chronic toxicity to aquatic animals, the Final chronic Value might be calculated in the same manner asthe Final Acute Value or by dividing the Final Acute Value by the Final Acute-Chronic Ratio. In some cases,it may not be possible to calculate aFinal Chronic Value. NOTE: As the nameimplies, theAcute-Chronic Ratio isaway of relating acuteand chronic toxkitks. The Acute-Chronic Ratio isbasically the inverse of theapplication factor,but this new nameisbetterKrause it ismoredescriptive andshould help prevent confusion between ‘applkation factors’ and ‘safety factors.’ Acute-Chronic Ratiosand application factomare ways of relating the acuteand chronic toxicities of a material to aquatic organisms. Safety factor ate used to provide an extra margin of safety beyond the known or estimated sensitivities ofaquaticorganisms.Another advantageoftheAcuteChronic Ratioisthat it will usually bepater than 1;this should avoid the confusion astowhether a large

## lpplkation factor &s onethat iscloeetounity oronethat hasadenominator that ismuch greaterthan the numerator.B. Chronic values should be based on results of flow- through chronic tests in which the concentrations of test material in the testsolutions were properly measured atappropriate times during the test. (Exception: renewal, which is acceptable for daphnids.) C. Results of chronic testsin which survival, growth, or reproduction in the control treatment was unacceptably low should not be used. The limits of acceptability will depend on the SptXieS. D. Results of chronic testsconducted in unusual dilution water (dilution water in which total organic carbon or particulate matter exceeded 5 mg/L) should not be used, unless a relationship is developed between chronic toxicity and organic carbon or particulate matter, or unless data show that organic carbon, particulate matter (and so forth) do not affect toxicity.
E. Chronic values should bebased on endpoints and lengths of exposure appropriate to the species. Therefore, only results of the following kinds of chronic toxicity tests should be used: 1. Lifecycle toxicity tests consisting of exposures of each of two or more groups of individuals of aspecks to adifferent concentration of the test material throughout a life cycle. To ensure that all life stages and life processes are exposed, tests with Rsh should begin with embryos or newly hatched young lessthan 48-hours old, continue through maturation and reproduction, and end not less than 24 days (90 days for salmonids) after the hatching of the next generation. Tests with daphnids should begin with young lessthan 24-hours old and last for not lessthan 21days. Testswith mysids should begin with young lessthan 24-hours old and continue until seven days past the median time of first brood release in the controls. For fish, data should beobtained and analyzed on survival and growth of adults and young maturation of males and females, eggsspawned per female, embryo viability (salmonids only), and hatchability. For daphnids, data should beobtained and analyzed on survival and young per female. For mysids, data should be obtained and analyzed on survival, growth, and young per female. 2.

3. Partial life-cycle toxicity testsconsisting ofexposures of eachof two or more groups of individuals in a fish species to a concentration of the test material through most portions of alife cycle. Partial lifecycle testsare allowed with fish species that require more than ayear to reach sexual maturity sothat all major life stagescanbeexposed to the test material in lessthan 15months. Exposure to the test material should begin with immature juveniles at least two months prior to active gonad development, continue through maturation and reproduction, and end not lessthan 24days (90days for salmonids) after the hatching of the next generation. Data should beobtained and analyzed on survival and growth of adults and young, maturation of males and females, eggs spawned per female, embryo viability (salmonids only), and hatchability. Early life stage toxicity tests consisting of 2& to 32-day (60 days post hatch for salmonids) exposures of the early life stages of a fish species from shortly after fertilization through embryonic, larval, and early juvenile development. Data should beobtained and analyzed on survival and growth. NOTE: Resultsofanearly life stagetestareusedaspredictions ofresultsoflifecycle and partial lifeqck testswith thesamespecies.Therefore,when results of atotal or partial lifecycle testareavailable, resultsofanearly life stagetestwith thesamespecksshould not beused.Also, resultsof early life stagetestsin which the incidence of mortalities or abnormalities increased substantially near the end should not be used becausethese results arepossibly not good predictions ofthe resultsof comparable total or partial life cyclecxpartial life cycletests.F. A chronic value canbeobtained by calculating the geometric mean of the lower and upper

chronic limits from achronic test or by analyzing chronic data using regression analysis. A lower chronic limit isthe highest tested concentration in an acceptable chronic test that did not cause an unacceptable amount of adverse effect on any of the specified biological measurements and below which no tested concentration caused an unacceptable effect. An upper chronic limit is the lowest tested concentration in an acceptable chronic test that did cause an unacceptable amount of adverse effect on one or more of the specified biological measunxnenb and above which alI tested concentrations alsocaused such aneffect. NOTE Because

various authm haveusedavariety oftermsanddefinitions to interpret and report resulb of chronic tests,reported resultsshould bereviewed carefully. Theamount of effectthat isconsidered unacceptableisoftenbasedonastatistical hypothesis testbut might alsobedefined in termsof aspecified percentreduction from the controls. Asmall percent rtduction (e.g.,3percent)might beconsideredacceptableevenif it isstatistically significantly diffeerentfrom the control whereas a large percent reduction (e.g., 30 percent) might be consideredunacceptableevenif it isnotstatistically significant. G. If the chronic toxicity of the material to aquatic animals apparently has been shown to be related to a water quality characteristic such as hardness or particulate matter for fnxhwater animals or salinity or particulate matter for saltwater animals, a Final chronic Equation should bederived based on that water quaI.@ characteristic. Go to section VII. H. If chronic values areavailable for species in eight families asdescribed in sections BIB.1 or 1II.C.1,aSpecies Mean Chronic Value should also becalculated for eachspecies for which at least one chronic value is available by calculating the geometric mean of all chronic values available for the species; appropriate Genus Mean Chronic Values should also be calculated. The Final Chronic Value should then be obtained using the procedure described in section III, stepsJthrough 0. Then go to section VIM. I. For each chronic value for which at least one corresponding appropriate acute value is available, calculate an acute-chronic ratio using for the numerator the geometric mean of the results of aII acceptable flow-through acute tests in the same dilution water and in which the concentrations were measured. (Exception: static isacceptable for daphnids.) For fish, the acute test(s) should have been conducted with juveniles and should have been part of the samestudy asthe chronic test. If acute tests were not conducted aspart of the same study, acute tests conducted in the same laboratory and dilution water but in a different study may be used. If no such acute tests are available, results of acute tests conducted in the same dilution water in a different laboratory may be used. If no such acute testsareavailable, an acute-chronic ratio should not becalculated. J. For each species, cakulate the species mean acute-chronic ratio asthe geometric mean of aII acutechronic ratios available for that species. K. For some materials, the acutechronic ratio seems to be the same for all species, but for other materials, the ratio seemsto increase or decrease asthe Species Mean Acute Value increases. Thus the Final Acute-Chronic Ratio can beobtained in four ways, depending on the data available: 1. If the Species Mean Acute-Chronic ratio seemsto increase or decrease asthe Species Mean Acute Value increases, the Final Acute-Chronic Ratio should becalculated asthe geometric mean of the acute-chronic ratios for species whose Species Mean Acute Values areclose to the Final Acute Value. 2. If no major trend is apparent, and the acute-chronic ratios for a number of species are within a factor of IO, the Fii Acute-Chronic Ratio should be calculated as the geometric mean of all the Species Mean Acute-Chronic Ratios available for both freshwater and saltwater species. 3. For acute tests conducted on metals and possibly other substances with embryos and larvae of barnacles, bivalve molluscs, sea urchins, lobsters, crabs, shrimp, and abalones (see section IV.E2), it is probably appropriate to assume that theacute-chronic ratio is 2. Chronic 

### testsare very difficult to conduct with most such species,but the sensitivities of embryos and larvae would likely determine the results of life cycle tests. Thus, if the lowest available Species Mean Acute Values were determined with embryos and larvae of such species, the Final Acute-Chronic Ratio should probably be assumed to be 2,so that the Final Chronic Value is equal to the Criterion Maximum Concentration (seesection XIB) 4. If the most appropriate Species Mean Acute-Chronic Ratios are less than 2.0, and especially if they are less than 1.0, acclimation has probably occurred during the chronic test. Because continuous exposure and acclimation cannot be assured to provide adequate protection in field situations, the Final Acute-Chronic Ratio should be assumed to be2,sothat the Final Chronic Value isequal to the Criterion Maximum Concentration (seesection XLB). If the available SpeciesMean Acute-Chronic Ratios do not fit one of these cases,a Final Acute-Chronic Ratio probably cannot beobtained, and aFinal Chronic Value probably cannot becalculated. L. Calculate the Final Chronic Value by dividing the Final Acute Value by the Final Acute-Chronic Ratio. If there was a Final Acute Equation rather than a Final Acute Value, seealso section W.A. M. If the SpeciesMean Chronic Value of acommercially or recreationally important species is lower than the calculated Final Chronic Value, then that Species Mean Chronic Value should be used asthe Final Chronic Value instead ofthe calculated Final Chronic Value. N. Go tosection VIII.

VII. Final Chronic Equation
A. A Final Chronic Equation can be derived in two ways. The procedure described here will result in the chronic slope being the same asthe acute slope. The procedure described in steps B through N usually will result in the chronic slope being different from the acute slope. 1. If acute-chronic ratios are available for enough species at enough values of the water quality characteristic to indicate that the acute-chronic ratio is probably the same for ail species and is probably independent of the water quahty characteristic, calculate the Final Acute-Chronic Ratio asthe geometric mean of the available Species Mean Acute-Chronic Ratios. 2. Calculate the Final Chronic Value at the selected value Z of the water quality characteristic by dividing the Final Acute Value at Z (seesection V.M) by the Final Acute-Chronic Ratio. 3. UseV =pooled acute slope (seesection V.M) asL =pooled chronic slope. 4. Go to section W.M.
B. When enough data are available to show that chronic toxicity to at least one species is related to awater quality characteristic, the relationship should be taken into account as described in steps B through G or using analysis of covariance. The two methods are equivalent and produce identical 

### results.The manual method descriid in the next paragraph provides an understanding of this application of covariance analysis, but computerized versions of covariance analysis are much more convenient for analyzing large data sets.If two or more factors affect toxicity, multiple regression analysis should be used. C. For eachspecies for which comparable chronic toxicity values areavailable at two or more different values of the water quality characteristic, perform a least squa_m regression ofD. the chronic toxicity values on the corresponding values of the water quality characteristic

to obtain the slope and its 95percent confidence limits for eachspecies. NOTE: becausethebestdocumented relationship fitting thesedataisthatbetweenhardness and acutetoxicity of metab in freshwater and a log-log relationship, geometic meansand natural logarithms ofboth toxicity and water quality areusedin the restof this section.For relationships basedonotherwaterquality characteristicssuchaspH,temperature,orsalinity, no tmnsforrnation or a different transformation might fit the data better, and appropriate changeswill benecessary throughout thissection.It isprobably prefetable,but not necessary, tousethesametransformation thatwasus4 with theacutevaluesinsectionV. Decide whether the data for each species are useful, taking into account the range and number of the tested values of the water quality characteristic and the degree of avent within and between species. For example, a slope based on six data points might beof limited value if founded only on data for avery narrow range of values of the water quality characteristic. A slope based on only two data points, however, might be useful ifit is consistent with other information and if the two points cover abroad enough range of the water quality characteristic. In addition, chronic values that appear to be questionable in comparison with other acute and chronic data available for the same species and for other species in the samegenus probably should not be used. For example, if after adjustment for the water quality characteristic the chronic values available for a species or genus differ by more than a factor of 10, probably some or all of the values should be rejected. If a useful chronic slope is not available for at least one species, or if the available slopes are too dissimiIar, or if too few data are available to adequately define the relationship between chronic toxicity and the water quality characteristic, the chronic slope is probably the same as the acute slope, which is equivalent to assuming that the acute-chronic ratio is independent of the water quality characteristic. Alternatively, return tosection VII-I, using the results oftestsconducted under conditions and in waters similar to those commonly used for toxicity testswith the species. E. Individually for eachspecies, calculate the geometric mean of the available chronic values and then divide each chronic value for aspecies by its mean. This normalizes the chronic values sothat the geometric mean of the normalized values for eachspecies individually, and for any combination of species, is 1.O. F. Similarly normalize the values of the water quality characteristic for each species, individually. G. Individually for eachspecies, perform aleast squares regression ofthe normalized chronic toxicity values on the corresponding normalized values ofthe water quality characteristic. The resulting slopes and the 95 percent confidence limits will be identical to those obtained in section B.Now, however, if the data areactually plotted, the line of best fit for each individual species will go through the point 1,l in the center of the graph. H. Treat all the normalized data as if they were all for the same species and perform a least squares regression of alI the normalized chronic values on the corresponding normalized values of the water quaIity characteristic to obtain the pooled chronic slope, L, and ik 95 percent con6dence limits. If alI the normalized data areactually plotted, the line of best fit will go through the point 1,l in the center of the graph. I. For eachspecies, calculate the geometric mean, M, of the toxicity values and the geometric mean, P,of the values of the water quality characteristic, (These were calculated in steps E and F.) J. For each species, calculate the logarithm, Q, of the Species Mean Chronic Value at a selected value, Z of the water quality characteristic using the equation: Q=lnM-L(lnP-lnZ). NOTE: Although it is not necessary, it wilI usually bebestto usethesamevalue ofthewater quality chrrcteristic hereaswasusedinsectionV.I.K. For eachspecies, calculate aSpeciesMean Chronic Value atZ using the equation: SMCV =e*. NOTE: Alternatively, theSpecies MeanChronic ValuesatZcan beobtained byskipping stepJ, using the equations in stepsJand K to adjust eachacutevalue individually to Z and then calculating the geometric meansof the adjusted values for eachspeciesindividually. This alternative procedure allows anexamination of the rangeof theadjusted chronic values for eachspecies. L. Obtain the Final Chronic Value atZ by using the procedure described in section IV,stepsJ through 0. M. Lf the Species Mean Chronic Value at Z of a commercially or recreationally important species is lower than the calculated Final Chronic Value at Z, then that Species Mean Chronic Value should be used asthe Final Chronic Value at Z instead of the calculated Final Chronic Value. N. The Final Chronic Equation iswritten as:

Final chronic Value = e(l[ht(wattr quality d\xtietitic)] + h s - L[tn a) where L =pooled chronic slope S= Final Chronic Value atZ. Because L S,and Z are known, the Final Chronic Value can be calculated for any selected value of the water quality characteristic.

VIILFinal Plant Value A. Appropriate measuresofthe toxicity ofthe material toaquatic plants areused tocornparr the relative sensitivities of aquatic plants and animals. Although procedures for conducting and interpreting the results oftoxicity testswith plants ZLIP not well developed, results oftestswith plants usually indicate that criteria which adequately protect aquatic animals and their uses will probably alsoprotect aquatic plants and their uses. B. A plant value is the result of a 96-hour test conducted with an alga, or a chronic test conducted with an aquatic vascular plant. NOTE: Atestof the toxicity of a metalto aplant usually should not beused if the medium contained an excessiveamount of acomplexing agent,such asEDTA,that might affectthe toxicity of the metal. Concentrations of EDTA above about 200pg/L should probably be consideredexcessive. C. The Final Plant Value should beobtained by selecting the lowest result ,ftom atestwith an important aquatic plant species in which the concentrations of test material were measured, and the endpoint was biologically important.

IX. Final Residue Value A. The Final Residue Value is intended to prevent concentrations in commercially or recreationally important aquatic speciesfrom affecting marketability because they exceed applicable FDA action levels and to protect wildlife (including fishes and birds) that consume aquatic organisms from demonstrated unacceptable effects. The Final Residue Value is the lowest of the residue values that are obtained by dividing maximum permissible tissue concentrations by appropriate bioconcentration or bioaccumulation factors. A maximum permissible tissue concentration is either (a) an FDA action level (Compliance Policy Guide, U.S. Food & Drug Admin. 1981)for fish oil or for the edible portion of fish or shellfish, or amaximum acceptable dietary intake based on observations on survival, growth, or reproduction in a chronic wildlife feeding study or a long-term wildlife field study. If no maximum permissible tissue concentration is available, go to section Xbecause no Final Residue Value canbederived.8. Bioconcentration Factors (BCFs) and bioaccumulation factors (BAFs) are quotients of the

concentration of a material in one or more tissues of an aquatic organism, divided by the average concentration in the solution in which the organism had been living. A BCF is intended to account only for net uptake dimctly from water and thus almost must be measured in a laboratory test. Someuptake during the bioconcentration test might not be directly from water if the food sorbs some of the test material before it is eaten by the test organisms. A BAF is intended to account for net uptake from both food and water in a real-world situation. A BAF almost must be measured in a field situation in which predators accumulate the material directly from water and by consuming prey that could have accumulated the material from both food and water. The BCF and BAF are probably similar for a material with a low BCF,but the BAF is probably higher than the BCF for materials with high BCFs. Although BCFs are not too difficult to determine, very few BAFs have been measured acceptably because adequate measurements must be made of the material’s concentration in water to ascertain if it was reasonably constant for a long enough time over the range of territory inhabited by the organisms. Because so few acceptable BAFs are available, only BCFs will be discussed further. However, ifan acceptable BAF isavailable for amaterial, it should be used instead of any available BCFs. C. If a maximum permissible tissue concentration is available for a substance (e.g., parent material, parent material plus metabolites, etc.), the tissue concentration used in the calculation of the BCF should be for the same substance. Otherwise, the tissue concentration used in the calculation of the BCF should derive from the material and its metabolites that arestructurally similar and are not much more soluble in water than the parent material. 1. A BCFshould beused only if the testwas flow-through, the BCFwas calculated based on measured concentrations of the test material in tissue and in the test solution, and the exposure continued at least until either apparent steady state or 28 days was reached. Steady state is reached when the BCF does not change significantly over a period of time, such as2days or 16percent of the length of the exposure, whichever is longer. The BCF used from a test should be the highest of the apparent steady-state BCF, if apparent steady state was reached; the highest BCF obtained, if apparent steady state was not reached; and the projected steady state BCF,if calculated. 2. Whenever aBCF is determined for alipophilic material, the percent lipids should also bedetermined in the tissue(s) for which the BCFwas calculated. 3. A BCF obtained from an exposure that adversely affected the test organisms may be used only if it is similar to a BCF obtained with unaffected organisms of the same species at lower concentrations that did not causeadverse effects. 4. Because maximum permissible tissue concentrations are almost never based on dry weights, a BCF calculated using dry tissue weights must be converted to awet tissue weight basis. If no conversion factor is reported with the BCF,multiply the dry weight BCFby 0.1for plankton and by 0.2for individual speciesof fishes and invertebrates. 5. If more than one acceptable BCF is available for aspecies, the geometric mean of the available values should be used; however, the BCFs are from different lengths of exposure and the BCF increases with length of exposure, then the BCF for the longest exposure should be used.
E. If enough pertinent data exists, several residue values can be calculated by dividing maximum permissible tissue concentrations by appropriate BCFs: 1. For each available maximum acceptable dietary intake derived from achronic feeding study or along-term field study with wildlife (including birds and aquatic organisms), the appropriate BCF is based on the whole body of aquatic species that constitutes or represents amajor portion of the diet of the tested wildlife species.X. 2. For an FDA action level for fish or shellfish, the appropriate BCF is the highest geometric mean species BCF for the edible portion (muscle for decapods, muscle with or without skin for fishes, adductor muscle for scallops, and total soft tissue for other bivalve molluscs) of aconsumed species.The highest species BCF is used because FDA action levels areapplied on aspecies-by-species basis. F. For lipophilic materials, calculating additional residue values is possible. because the steady-state BCF for a lipophilic material seemsto be proportional to percent lipids from one tissue to another and from one species to another, extrapolations can be made from tested tissues, or species to untested tissues, or specieson the basis of percent lipids. 1. For each BCF for which the percent lipids is known for the same tissue for which the BCF was measured, normalize the BCF to a 1percent lipid basis by dividing it by the percent lipids. This adjustment to a 1percent lipid basis is intended to make all the measured BCFs for a material comparable regardless of the species or tissue with which the BCFwas measured. 2. Calculate the geometric mean-normalized BCF. Data for both saltwater and freshwater speciesshould be used to determine the mean-normalized BCFunless they show that the normalized BCFsareprobably not similar. 3. Calculate all possible residue values by dividing the available maximum permissible tissue concentrations by the mean-normalized BCF and by the percent lipids values appropriate to the maximum permissible tissue concentrations, i.e., (maximum permissible tissue concentration) Resiobe va’“e 1 (mean normalized BCF)(appropriate percent lipids)

l For an FDA action level for fish oil, the appropriate percent lipids value is 100.

l For an FDA action level for fish, the appropriate percent lipids value is 11for freshwater criteria and 10for saltwater criteria because FDA action levels are applied species-by-species to commonly consumed species.The highest lipid contents in the edible portions of important consumed species areabout 11 percent for both the freshwater chinook salmon and lake trout and about 10 percent for the saltwater Atlantic herring.

l For amaximum acceptable dietary intake derived from achronic feeding study or along-term field study with wildlife, the appropriate percent lipids is that of an aquatic speciesor group of aquatic species that constitute amajor portion of the diet of the wildlife species. G. The Final Residue Value isobtained by selecting the lowest of the available residue values. NOlE In somecases,

the Final ResidueValuewill notbelow enough. Forexample,aresidue value calculated from aFDA action level will probably result in anaverageconcenkation in the edible portion of a fatty speciesat the action level. Someindividual organisms and possibly somespecieswill have residue concentrations higher than the meanvalue, but no mechanism has been devised to provide appropriate additional protection. Also, some chronic feedingstudiesand long-term field studieswith wildlife identify concentrations that causeadverse effectsbut do not identify concentrations that do not causeadverseeffects; again, no mechanism hasbeendevised to provide appropriate additional protection. These aresomeofthespeciesand usesthatarenotprotectedatall timesin all places.
Other Data Pertinent information that could not be used in earlier sections might be available concerning adverse effects on aquatic organisms and their uses.The most important of these are data on cumulative and delayed toxicity, flavor impairment, reduction in survival, growth, or reproduction, or any other adverse effect shown to be biologically important. Especially important are data for species for which no other data are available. Data from behavioral, biochemical, physiological, microcosm, and field studies might alsobeavailable. Data might be available from testsconducted in unusual dilution water (seeIV.D and VLD), from chronic testsin which the concentrations were not measured (seeVIB), from testswith previously exposed

organisms (seeBF), and from tests on formulated mixtures or emulsifiable concentrates (see IID). Suchdata might affect acriterion if they were obtained with an important species,the test concentrations were measured, and the endpoint was biologically important.

XI. Criterion A. A criterion consists of two concentrations: the Criterion Maximum Concentration and the Criterion Continuous Concentration. B. The Criterion Maximum Concentration (CMC) isequal to one-half the Final Acute Value. C. The Criterion Continuous Concentration (CCC) is equal to the lowest of the Final Chronic Value, the Final Plant Value, and the Final Residue Value, unless other data (seesection X) show that alower value should be used. If toxicity is related to awater quality charactekstic, the Criteria Continuous Concentration is obtained from the Final Chronic Equation the Final Plant Value, and the Final Residue Value by selecting the one,or the combination, that IeHllt3inthelowest amcentrations in the usual range of the water quality characteristic, unlessotherdata(seesectionX)showthatalowffvalwshouMbeused. D. Round both the Criterion Maximum Concentration and the Criterion Continuous Concentration to two significant digits.
E. The criterion isstated asfollows: The procedures described in the ‘Guidelines for Deriving Numerical National Water Quality Criteria for the Protection of Aquatic Organisms and Their Uses” indicate that, except possibly where alocally important species isvery sensitive, (1) aquatic organisms and their uses should not be affected unacceptably if the four-day average concentration of (2)does not exceed (3) c(g/L more than once every three years on the average, and if the one-hour average concentration does not exceed (4) pg/L more than once every three years on the average. *where (1) = insert freshwater or saltwater

(2) = insert name of material (3) = insert the Criterion Continuous Concentration (4) =insert the Criterion Maximum Concentration.

XII. Find Review A. The derivation of the criterion should becarefully reviewed by rechecking eachstep of the guidelines. Items that should beespecially checked are 1. If unpublished data areused, arethey well documented? 2 Are all required data available? 3. Isthe range of acute values for any species greater than afactor of lo? 4. Isthe range of SpeciesMean Acute Values for any genus greater than afactor of 101 5. Isthere more than afactor of 10difference between the four lowest Genus Mean Acute Values? 6. Are any of the four lowest Genus Mean Acute Values questionable? 7. Isthe Final Acute Value reasonable in comparison with the SpeciesMean Acute Values and Genus Mean Acute Values? 8. For any commexially or mcreationally important species, isthe geometric mean ofthe acute values from flow-through testsin which the concentrations oftest material were measured lower than the Final Acute Value?9. Are any of the chronic values questionable? 10. Are chronic values available for acutely sensitive species? 11. Isthe range of acute-chronic ratios greater than afactor of 107 12. Is the Final Chronic Value reasonable in comparison with the available acute and chronic data? 13. Is the measured or predicted chronic value for any commercially or recreationally important speciesbelow the Final Chronic Value? 14. Are any of the other data important? 15. Do any data looklike they might beoutliers? 16. Are there any deviations from the guidelines? Are they acceptable?
B. On the basis of all available pertinent laboratory and field information, determine if the criterion is consistent with sound scientific evidence. If not, another criterion - either higher or lower - should bederived using appropriate modifications of these guidelines.