APPENDIX H 

Derivation of the 1985 

Aquatic Life Criteria 

WATER QUALITY STANDARDS HANDBOOK 

SECOND EDITION 
Derivation of the 1985 

Aquatic Life Criteria 

The following is a summary of the Guidelines for Derivation of Criteria for Aquatic Life. The complete text is found in "Guidelines for 

Deriving Numerical National Water Quality Criteria for the Protection of Aquatic Organisms and Their Uses” available from National 

Technical Information Service - PB85-227049. 

Derivation of numerical national water quality criteria for the protection of aquatic organisms and 

their uses is a complex process that uses information from many areas of aquatic toxicology. When a 

national criterion is needed for a particular 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 are available, they are used to estimate 

the highest one-hour average concentration that should not result in unacceptable effects on 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 a water 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 

effects that might be biologically important. 

If a thorough review of the pertinent information indicates that enough acceptable data exists, 

numerical national water quality criteria are derived for fresh water or salt water or both to protect 

aquatic organisms and their uses from unacceptable effects due 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 a separate 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 a separate 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 are transported to a laboratory. 

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 be available, a compromise 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 a surface water). Because the 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 is to measure the 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 some toxicity tests, but a criterion should 

not be based 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 be data from tests that did not contain a control 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 of the 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 35 g/kg; 

• Species that 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 

tests with aquatic plants and interpreting the results are not as well 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. To derive a criterion for freshwater aquatic organisms and their uses, the following should 

be available: 

1. Results of acceptable acute tests (see section 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 class Osteichthyes. 

• A second family in the class Osteichthyes, preferably a commercially or 

recreationally important warmwater species, such as bluegill or channel catfish. 

• A third family in the phylum Chordata (may be in the class Osteichthyes or may 

be an amphibian etc.). 

• A planktonic crustacean such as a cladoceran or copepod. 

• A benthic crustacean (ostracod, isopod, amphipod, crayfish, etc.). 

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

• A family in a phylum other than Arthropoda or Chordata, such as Rotifera, 

Annelida, Mollusca. 

• A family in any order of insect or any phylum not already represented. 

2. Acute-chronic ratios (see section VI) with species of aquatic animals in at least three 

different families, provided that: 

• At least one is a fish; 

• 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, test data on a plant in another phylum (division) should also be available. 
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 a criterion for saltwater aquatic organisms and their uses, the following should 

be available: 

1. Results of acceptable acute tests (see section 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 a phylum 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 (see s&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 be an 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 a test with a plant in another phylum (division) should also be available. 

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 a criterion might not be possible if the available 

acute-chronic ratios vary by more than a factor of 10 with no apparent pattern. Also, if a 

criterion is to be related to a water quality characteristic T (see sections 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 be possrble to derive a criterion 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 are usually 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 E 729 and 724). 

C. Except for tests with saltwater annelids and mysids, do not use results of acute tests 

during which test organisms 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. Tests with daphnids and other cladocerans should be started with organisms less than 

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 of organisms lolled. If such an ECs,-, is not available from 

a test, the 48-hour LCw should be used in place of the desired 48-hour ECm. An ECw or 

LCw of longer than 48 hours can be used as long as the animals were not fed and the 

control animals were acceptable at the 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 EC50 based 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 EC 50. If the duration of the test was between 48 and % 

hours, the ECw or LCm at the end of the test should be used. 

3. The acute values from tests with all other freshwater and saltwater animal species and 

older life stages of barnacles, bivalve molluscs, sea urchins, lobsters, crabs, shrimps, 

and abalones should be the %-hour ECw based on the percentage of organisms 

exhibiting loss of equilibrium, plus the percentage of organisms immobilized, plus the 

percentage of organisms killed. If such an EC% is not available from a test, the %-hour 

LCm should be used in place of the desired %-hour ECm. 

4. Tests with singlecelled organisms are not considered acute tests, even if the duration 

was 96 hours 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 be derived based on that water quality characteristic. (Co to section V.) 

G. If the available data indicate that one or more life stages are at least a factor of 2 more resistant 

than one or more other life stages of the same species, the data for the more resistant life stages 

should not be used in the calculation of the Species Mean Acute Value because a species can be 

considered protected from acute toxicity only if all life stages are protected. 

H. The agreement of the data within and between species should be considered. 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 in 
I. 

calculation of a Species Mean 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 be calculated as the geometric mean of the results of all flow-through tests in 

which the concentrations of test material were measured. For a species for which no such 

result is available, the Species Mean Acute Value should be calculated as the 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. Results of all 

intermediate calculations should be rounded to four significant digits. 

NOTE The geometric mean of N numbers is the 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 the sum by N, and taking the antilog of the quotient. The geometric mean 

of two numkn is the square root of the product of the two numbers, and the geometric mean 

of one number is that number. Either natural (base 0) or common (base 10) logarithms can be 

used to calculate geometric means as long as they are used consistently within each set of data 

(i.e., the antilog used must match the logarithm used). 

NOTE: Geometric means rather than arithmetic means are used here because the distributions 

of individual organisms’ sensitivities in toxicity tests on most materials, and the distributions 

of species’ sensitivities within a genus, are more likely to be lognormal than normal. Similarly, 

geometric means ate used for acutethronic ratios and bioconcentration factors because 

quotients are likely to be closer to 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 mean of the set of corresponding 

quotients. 

J. The Genus Mean Acute Value should be calculated as the geometric mean of the Species 

Mean Acute Values available for each genus. 

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 each Genus Mean Acute Value as R/ (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 

(See original document, referenced at beginning of this appendix, for development of the 

calculation procedure and Appendix 2 for example calculation and computer program.) 

NOTE: Natural logarithms (logarithms to base e, denoted as In) are used herein merely 

because they are easier to use on some hand calculators and computen than common (base 10) 

logarithms. Consistent use of either will produce the same result. 
F’. If for a commercially or recreationally important species the geometric mean of the acute 

values from flow-through tests in which the concentrations of test material were 

measured is lower than the calculated Final Acute Value, then that geometric mean should 

be used as the Final Acute Value instead of the calculated Final Acute Value. 

Q. Go to section VI. 

V. Final Acute Equation 

A. When enough data are available to show that acute toxicity to two or more species is similarly 

related to a water quality characteristic, the relationship should be taken into account as 

described in @on IV, steps B through G, or using analysis of covaxiance. The two methods 

are equivalent and produce identical results. The manual method described below provides 

an understanding of this application of covariance analysis, but computerized versions of 

covariance analysis are much more convenient for analyzing large data tests. If two or more 

factors affect toxicity, multiple regression analysis should be used. 

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 95 percent confidence limits for each species. 

NOTE: Because the best documented relationship fitting these data is that between hardness 

and acute toxicity of metals in freshwater and a log-log relationship, geometric means and 

natural logarithms of both toxicity and water quality are used in the rest of this section. For 

relationships based on other water quality characteristics such as pH, temperature, or salinity, 

no transformation or a different transformation might fit the data better, and appropriate 

changes will be necessary. 

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 a very 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 a broad 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 a species or genus differ by more than a factor 

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 tests with the species. 

D. Individually for each species, calculate the geometric mean of the available acute values 

and then divide each of these acute values by the mean for the species. This normalizes the 

values so that the geometric mean of the normalized values for each species, 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 of the water quality characteristic. 

The resulting slopes and 95 percent confidence limits will be identical to those obtained in 
step B. However, now, if the data are actually plotted, the line of best fit for each individual 

specie will go thmugh the point 1,l in the center of the graph. 

G. Treat normalized data as if they were all for the same species and perform a least 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, of the values of the water quahty characteristic. (These were calculated 

in steps D and E.) 

I. For each species, calculate the logarithm, Y, of the Species Mean Acute Value at a selected 

value, Z of the water quality characteristic using the equation: 

Y=lnW-V(lnX-1nZ). 

J. For each species, calculate the SMAV at 2 using the equation: 

SMAV = e”. 

NOTE: Alternatively, the Species Mean Acute Values at Z can be obtained by skipping step H 

using the equations in steps I and J to adjust each acute value individually to Z, and then 

calculating the geometric mean of the adjusted values for each specks individually. 

This akrnative procedure allows an examination of the range of the adjusted acute 

values for each species. 

K. Obtain the Final Acute Value at Z by using the procedure described in section IV steps J 

through 0. 

L. If the Species Mean Acute Value at Z of a comrnercialIy or recreationally important species 

is lower than the calculated Final Acute Value at Z, then that Species Mean Acute Value 

should be used as the Final Acute Value at Z instead of the calculated Final Acute Value. 

M. The Final Acute Equation is written as: 

Final Acute Value = e(V[lll(Watcf qldity chanctcrlstic)] + hl A - V[hl ZD 

where 

V = pooled acute slope 

A = Final Acute Value at Z. 

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 as the 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 a Final Chronic Value. 

NOTE: As the name implies, the Acute-Chronic Ratio is a way of relating acute and chronic 

toxkitks. The Acute-Chronic Ratio is basically the inverse of the application factor, but this 

new name is better Krause it is more descriptive and should help prevent confusion between 

‘applkation factors’ and ‘safety factors.’ Acute-Chronic Ratios and application factom are 

ways of relating the acute and 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 of aquatic organisms. Another advantage of the AcuteChronic Ratio is that it will 

usually be pater than 1; this should avoid the confusion as to whether a large l pplkation 

factor &s one that is cloee to unity or one that has a denominator that is much greater than the 

numerator. 
B. Chronic values should be based on results of flow- through chronic tests in which the 

concentrations of test material in the test solutions were properly measured at appropriate 

times during the test. (Exception: renewal, which is acceptable for daphnids.) 

C. Results of chronic tests in 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 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 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 be based 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 a specks to a different 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 less than 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 less than 24-hours old and last for not less than 21 days. Tests with 

mysids should begin with young less than 24-hours old and continue until seven days 

past the median time of first brood release in the controls. 

For fish, data should be obtained 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. For daphnids, data should be obtained 

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 tests consisting of exposures of each of two or more groups of 

individuals in a fish species to a concentration of the test material through most 

portions of a life cycle. Partial lifecycle tests are allowed with fish species that require 

more than a year to reach sexual maturity so that all major life stages can be exposed to 

the test material in less than 15 months. 

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 less than 24 days (90 days for salmonids) after the 

hatching of the next generation. Data should be obtained 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 

be obtained and analyzed on survival and growth. 

NOTE: Results of an early life stage test are used as predictions of results of lifecycle and 

partial lifeqck tests with the same species. Therefore, when results of a total or partial 

lifecycle test are available, results of an early life stage test with the same specks should 

not be used. Also, results of early life stage tests in which the incidence of mortalities or 

abnormalities increased substantially near the end should not be used because these 

results are possibly not good predictions of the results of comparable total or partial life 

cycle cx partial life cycle tests. 
F. A chronic value can be obtained by calculating the geometric mean of the lower and upper 

chronic limits from a chronic test or by analyzing chronic data using regression analysis. A 

lower chronic limit is the 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 an effect. 

NOTE Because various authm have used a variety of terms and definitions to interpret and 

report resulb of chronic tests, reported results should be reviewed carefully. The amount of 

effect that is considered unacceptable is often based on a statistical hypothesis test but might 

also be defined in terms of a specified percent reduction from the controls. A small percent 

rtduction (e.g., 3 percent) might be considered acceptable even if it is statistically significantly 

diffeerent from the control whereas a large percent reduction (e.g., 30 percent) might be 

considered unacceptable even if it is not statistically 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 be derived based on that water quaI.@ characteristic. Go to section VII. 

H. If chronic values are available for species in eight families as described in sections BIB.1 or 

1II.C. 1, a Species Mean Chronic Value should also be calculated for each species 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, steps J through 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 is acceptable for daphnids.) 

For fish, the acute test(s) should have been conducted with juveniles and should have 

been part of the same study as the chronic test. If acute tests were not conducted as part 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 tests are available, an acute-chronic ratio should not be calculated. 

J. For each species, cakulate the species mean acute-chronic ratio as the 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 seems to increase or decrease as the Species Mean Acute Value 

increases. Thus the Final Acute-Chronic Ratio can be obtained in four ways, depending on 

the data available: 

1. If the Species Mean Acute-Chronic ratio seems to increase or decrease as the Species 

Mean Acute Value increases, the Final Acute-Chronic Ratio should be calculated as the 

geometric mean of the acute-chronic ratios for species whose Species Mean Acute 

Values are close 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 the 
acute-chronic ratio is 2. Chronic tests are 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 (see section 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 be 2, so that the Final Chronic Value is equal to the Criterion Maximum 

Concentration (see section XLB). 

If the available Species Mean Acute-Chronic Ratios do not fit one of these cases, a 

Final Acute-Chronic Ratio probably cannot be obtained, and a Final Chronic Value 

probably cannot be calculated. 

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, 

see also section W.A. 

M. If the Species Mean Chronic Value of a commercially or recreationally important species is 

lower than the calculated Final Chronic Value, then that Species Mean Chronic Value 

should be used as the Final Chronic Value instead of the calculated Final Chronic Value. 

N. Go to section 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 as the 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 as the 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 (see section V.M) by the Final 

Acute-Chronic Ratio. 

3. Use V = pooled acute slope (see section V.M) as L = 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 a water 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 each species for which comparable chronic toxicity values are available at two or more 

different values of the water quality characteristic, perform a least squa_m regression of 
D. 

the chronic toxicity values on the corresponding values of the water quality characteristic 

to obtain the slope and its 95 percent confidence limits for each species. 

NOTE: because the bestdocumented relationship fitting these data is that between hardness 

and acute toxicity of metab in fresh water and a log-log relationship, geometic means and 

natural logarithms of both toxicity and water quality are used in the rest of this section. For 

relationships based on other water quality characteristics such as pH, temperature, or salinity, 

no tmnsforrnation or a different transformation might fit the data better, and appropriate 

changes will be necessary throughout this section. It is probably prefetable, but not necessary, 

to use the same transformation that was us4 with the acute values in section V. 

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 be of limited value if founded only on data for a very 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 a broad 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 same genus 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 

to section VII-I, using the results of tests conducted under conditions and in waters similar 

to those commonly used for toxicity tests with the species. 

E. Individually for each species, calculate the geometric mean of the available chronic values 

and then divide each chronic value for a species by its mean. This normalizes the chronic 

values so that the geometric mean of the normalized values for each species 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 each species, perform a least squares regression of the normalized chronic 

toxicity values on the corresponding normalized values of the 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 are actually 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 are actually plotted, the line of best fit 

will go through the point 1,l in the center of the graph. 

I. For each species, 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 be best to use the same value of the water 

quality chrrcteristic here as was used in section V.I. 
K. For each species, calculate a Species Mean Chronic Value at Z using the equation: 

SMCV = e*. 

NOTE: Alternatively, the Species Mean Chronic Values at Zcan be obtained by skipping step J, 

using the equations in steps J and K to adjust each acute value individually to Z and then 

calculating the geometric means of the adjusted values for each species individually. This 

alternative procedure allows an examination of the range of the adjusted chronic values for 

each species. 

L. Obtain the Final Chronic Value at Z by using the procedure described in section IV, steps J 

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 as the Final Chronic Value at Z instead of the calculated 

Final Chronic Value. 

N. The Final Chronic Equation is written 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 at Z. 

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 measures of the toxicity of the material to aquatic plants are used to cornparr the 

relative sensitivities of aquatic plants and animals. Although procedures for conducting and 

interpreting the results of toxicity tests with plants ZLIP not well developed, results of tests with 

plants usually indicate that criteria which adequately protect aquatic animals and their uses 

will probably also protect 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: A test of the toxicity of a metal to a plant usually should not be used if the medium 

contained an excessive amount of a complexing agent, such as EDTA, that might affect the 

toxicity of the metal. Concentrations of EDTA above about 200 pg/L should probably be 

considered excessive. 

C. The Final Plant Value should be obtained by selecting the lowest result ,ftom a test with 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 species from 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 a maximum 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 can be derived. 
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. Some uptake 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 is available for a material, 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 are structurally similar and are not much more soluble in water than the 

parent material. 

1. A BCF should be used only if the test was flow-through, the BCF was 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 as 2 days or 16 percent 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 a BCF is determined for a lipophilic material, the percent lipids should also 

be determined in the tissue(s) for which the BCF was 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 cause adverse 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 a wet tissue 

weight basis. If no conversion factor is reported with the BCF, multiply the dry weight 

BCF by 0.1 for plankton and by 0.2 for individual species of fishes and invertebrates. 

5. If more than one acceptable BCF is available for a species, 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 a chronic feeding 

study or a long-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 a major 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 a consumed species. The highest species BCF is used because FDA 

action levels are applied on a species-by-species basis. 

F. For lipophilic materials, calculating additional residue values is possible. because the 

steady-state BCF for a lipophilic material seems to 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 species on 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 1 percent lipid basis by dividing it by the 

percent lipids. This adjustment to a 1 percent lipid basis is intended to make all the 

measured BCFs for a material comparable regardless of the species or tissue with 

which the BCF was measured. 

2. Calculate the geometric mean-normalized BCF. Data for both saltwater and 

freshwater species should be used to determine the mean-normalized BCF unless they 

show that the normalized BCFs are probably 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 11 for 

freshwater criteria and 10 for 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 are about 11 

percent for both the freshwater chinook salmon and lake trout and about 10 

percent for the saltwater Atlantic herring. 

l For a maximum acceptable dietary intake derived from a chronic feeding study or 

a long-term field study with wildlife, the appropriate percent lipids is that of an 

aquatic species or group of aquatic species that constitute a major portion of the 

diet of the wildlife species. 

G. The Final Residue Value is obtained by selecting the lowest of the available residue values. 

NOlE In some cases, the Final Residue Value will not be low enough. For example, a residue 

value calculated from a FDA action level will probably result in an average concenkation in 

the edible portion of a fatty species at the action level. Some individual organisms and 

possibly some species will have residue concentrations higher than the mean value, but no 

mechanism has been devised to provide appropriate additional protection. Also, some 

chronic feeding studies and long-term field studies with wildlife identify concentrations that 

cause adverse effects but do not identify concentrations that do not cause adverse effects; 

again, no mechanism has been devised to provide appropriate additional protection. These 

are some of the species and uses that are not protected at all times in 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 also be available. Data might be 

available from tests conducted in unusual dilution water (see IV.D and VLD), from chronic tests 
in which the concentrations were not measured (see VIB), from tests with previously exposed 

organisms (see BF), and from tests on formulated mixtures or emulsifiable concentrates (see 

IID). Such data might affect a criterion 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) is equal 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 (see section X) 

show that a lower value should be used. If toxicity is related to a water 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 is stated as follows: 

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 a locally important species is very 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 be carefully reviewed by rechecking each step of the 

guidelines. Items that should be especially checked are 

1. If unpublished data are used, are they well documented? 

2 Are all required data available? 

3. Is the range of acute values for any species greater than a factor of lo? 

4. Is the range of Species Mean Acute Values for any genus greater than a factor of 101 

5. Is there more than a factor of 10 difference between the four lowest Genus Mean Acute 

Values? 

6. Are any of the four lowest Genus Mean Acute Values questionable? 

7. Is the Final Acute Value reasonable in comparison with the Species Mean Acute Values 

and Genus Mean Acute Values? 

8. For any commexially or mcreationally important species, is the geometric mean of the 

acute values from flow-through tests in which the concentrations of test 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. Is the range of acute-chronic ratios greater than a factor 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 species below the Final Chronic Value? 

14. Are any of the other data important? 

15. Do any data looklike they might be outliers? 

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 be derived using appropriate modifications of these guidelines.