A. This Appendix provides procedures to derive standards and guidance values to protect aquatic life from acute and chronic effects. Tier I procedures, sections III through XI, are used where the required data described in section III.B for freshwater species or section III.C for saltwater species are available. Tier II procedures, sections XII through XVI, are used where the data requirements in sections III.B or III.C are not met.

A. These procedures will generally be used to derive statewide standards or guidance values according to the classified uses described in section 702.9 of this Title. Site-specific modifications to such statewide standards or guidance values are required or allowed as described below.

B. Site-specific modifications to chronic or acute aquatic life values may be developed where:

(1) The local water quality characteristics such as pH, hardness, temperature, color, etc., alter the biological availability or toxicity of a pollutant; or

(2) The sensitivity of the aquatic organisms species that occur at the site differs from the species actually tested in developing the criteria. The phrase occur at the site includes the species, genera, families, orders, classes, and phyla that: are usually present at the site; are present at the site only seasonally due to migration; are present intermittently because they periodically return to or extend their ranges into the site; were present at the site in the past, are not currently present at the site due to degraded conditions, and are expected to return to the site when conditions improve; are present in nearby bodies of water, are not currently present at the site due to degraded conditions, and are expected to be present at the site when conditions improve. The taxa that occur at the site cannot be determined merely by sampling downstream and/or upstream of the site at one point in time. Occur at the site does not include taxa that were once present at the site but cannot exist at the site now due to permanent physical alteration of the habitat at the site resulting, for example, from dams, etc.

C. Site-specific modifications also may be developed to acute and chronic aquatic life values to reflect local physical and hydrological conditions.

D. Endangered species considerations.

(1) Any site-specific modifications that result in less stringent values must not be likely to jeopardize the continued existence of endangered or threatened species or result in the destruction or adverse modification of such species' critical habitat.

(2) More stringent modifications shall be developed to protect endangered or threatened species where such modifications are necessary to ensure that water quality is not likely to jeopardize the continued existence of such species or result in the destruction or adverse modification of such species' critical habitat.

A. Certain data should be available to help ensure that each of the major kinds of possible adverse effects receive adequate consideration.

B. To derive an acute or chronic standard or guidance value for freshwater aquatic organisms and their uses, the following must be available:

1. Results of acceptable acute tests (see section IV or VI of this Appendix) with at least one species of freshwater animal in at least eight different families such that all of the following are included:

a. The family Salmonidae in the class Osteichthyes;

b. One other family (preferably a commercially or recreationally important, warmwater species) in the class Osteichthyes (e.g., bluegill, channel catfish);

c. A third family in the phylum Chordata (e.g., fish, amphibian);

d. A planktonic crustacean (e.g., a cladoceran, copepod);

e. A benthic crustacean (e.g., ostracod, isopod, amphipod, crayfish);

f. An insect (e.g., mayfly, dragonfly, damselfly, stonefly, caddisfly, mosquito, midge);

g. A family in a phylum other than Arthropoda or Chordata (e.g., Rotifera, Annelida, Mollusca);

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

2. Acute-chronic ratios (see section VI of this Appendix) with at least one species of aquatic animal in at least three different families provided that of the three species:

a. At least one is a fish;

b. At least one is an invertebrate; and

c. At least one species 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 is desirable but not required for standard or guidance value derivation (see section VIII of this Appendix). 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.

C. To derive a standard or guidance value for saltwater aquatic organisms and their uses, the following must be available:

1. Results of acceptable acute tests (see section IV or VI of this Appendix) with at least one species of saltwater animal in at least eight different families such that all of the following are included:

a. two families in the phylum Chordata;

b. a family in a phylum other than Arthropoda or Chordata;

c. either the Mysidae or Penaeidae family;

d. three other families not in the phylum Chordata (may include Mysida or Penaeidae, whichever was not used above);

e. any other family.

2. Acute-chronic ratios (see section VI of this Appendix) with species of aquatic animals in at least three different families provided that of the three species:

a. at least one is a fish;

b. at least one is an invertebrate; and

c. at least one is an acutely sensitive saltwater species (the other two may be freshwater species).

3. Results of at least one acceptable test with a saltwater alga or vascular plant is desirable but not required for standard or guidance value derivation (see section VIII of this Appendix). 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.

D. If all required data are available, a numerical standard or guidance value can usually be derived except in special cases. For example, derivation of a chronic standard or guidance value might not be possible if the available ACRs vary by more than a factor of 10 with no apparent pattern. Also, if a standard or guidance value is to be related to a water quality characteristic (see sections V and VII of this Appendix), more data will be required.

E. Confidence in a standard or guidance value usually increases as the amount of available pertinent information increases. Thus, additional data are usually desirable.

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 (FAV). The Final Acute Value is a calculated estimate of the concentration of a test material such that 95 percent of the genera (with which acceptable acute toxicity tests have been conducted on the material) have higher Genus Mean Acute Values (GMAVs). An acute test is a comparative study in which organisms, that are subjected to different treatments, are observed for a short period usually not constituting a substantial portion of their life span. However, in some cases, the Species Mean Acute Value (SMAV) of a commercially or recreationally important species is lower than the calculated FAV, then the SMAV replaces the calculated FAV in order to provide protection for that important species.

B. Acute toxicity tests shall be conducted using acceptable procedures.

C. Except for results with saltwater annelids and mysids, results of acute tests during which the test organisms were fed should not be used, 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, e.g., dilution water in which total organic carbon or particulate matter exceeded five 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 organic carbon or particulate matter, etc., do not affect toxicity.

E. Acute values must be based upon endpoints which reflect the total severe 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 shall be used:

1. Tests with daphnids and other cladocerans must be started with organisms less than 24 hours old and tests with midges must be started with second or third instar larvae. It is preferred that the results should be the 48-hour EC50 based on the total percentage of organism killed and immobilized. If such an EC50 is not available for a test, the 48-hour LC50 should be used in place of the desired 48-hour EC50. An EC50 or LC50 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. An EC50 is a statistically or graphically estimated concentration that is expected to cause one or more specified effects in 50 percent of a group of organisms under specified conditions. An LC50 is a statistically or graphically estimated concentration that is expected to be lethal to 50 percent of a group of organisms under specified conditions.

2. It is preferred that the results of a test with embryos and larvae of barnacles, bivalve molluscs (clams, mussels, oysters and scallops), sea urchins, lobsters, crabs, shrimp and abalones be the 96-hour EC50 based on the percentage of organisms with incompletely developed shells plus the percentage of organisms killed. If such an EC50 is not available from a test, of the values that are available from the test, the lowest of the following should be used in place of the desired 96-hour EC50: 48- to 96-hour EC50s based on percentage of organisms with incompletely developed shells plus percentage of organisms killed, 48- to 96-hour EC50s based on percentage of organisms with incompletely developed shells, and 48-hour to 96-hour LC50s.

3. It is preferred that the result of tests with all other aquatic animal species and older life stages of barnacles, bivalve molluscs (clams, mussels, oysters and scallops), sea urchins, lobsters, crabs, shrimp and abalones be the 96-hour EC50 based on percentage of organisms exhibiting loss of equilibrium plus percentage of organisms immobilized plus percentage of organisms killed. If such an EC50 is not available from a test, of the values that are available from a test the lower of the following should be used in place of the desired 96-hour EC50: the 96-hour EC50 based on percentage of organisms exhibiting loss of equilibrium plus percentage of organisms immobilized and the 96-hour LC50.

4. Tests whose results take into account the number of young produced, such as most tests with protozoans, 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 which are above the solubility of the test material should be used, because rejection of such acute values would bias the Final Acute Value by eliminating acute values for resistant species.

F. If the acute toxicity of the material to aquatic animals has been shown to be related to a water quality characteristic such as hardness or particulate matter for freshwater animals, refer to section V of this Appendix.

G. The agreement of the data within and between species must 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 must not be used. For example, if the acute values available for a species or genus differ by more than a factor of 10, rejection of some or all of the values would be appropriate, absent countervailing circumstances.

H. If the available data indicate that one or more life stages are at least a factor of two more resistant than one or more other life stages of the same species, the data for the more resistant life stages must not be used in the calculation of the SMAV because a species cannot be considered protected from acute toxicity if all of the life stages are not protected.

I. For each species for which at least one acute value is available, the SMAV shall be calculated as the geometric mean of the results of all acceptable flow-through acute toxicity tests in which the concentrations of test material were measured with the most sensitive tested life stage of the species. For a species for which no such result is available, the SMAV shall be calculated as the geometric mean of all acceptable acute toxicity tests with the most sensitive tested life stage, 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 (nominal concentrations are acceptable for most test materials if measured concentrations are not available) of test material. A renewal test is a test with aquatic organisms in which either the test solution in a test chamber is removed and replaced at least once during the test or the test organisms are transferred into a new test solution of the same composition at least once during the test. A static test is a test with aquatic organisms in which the solution and organisms that are in a test chamber at the beginning of the test remain in the chamber until the end of the test, except for removal of dead test organisms.

Note 1: Data reported by original investigators must not be rounded off. Results of all intermediate calculations must not be rounded off to fewer than four significant digits.

Note 2: 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 numbers is the square root of the product of the two numbers, and the geometric mean of one number is that number. Either natural (base e) 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 logarithms used.

Note 3: Geometric means, rather than arithmetic means, are used here because the distributions of sensitivities of individual organisms in toxicity tests on most materials and the distributions of sensitivities of species within a genus are more likely to be lognormal than normal. Similarly, geometric means are used for ACRs 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 denominators will result in the geometric mean of the set of corresponding quotients.

J. For each genus for which one or more SMAVs are available, the GMAV shall be calculated as the geometric mean of the SMAVs available for the genus.

K. Order the GMAVs from high to low.

L. Assign ranks, R, to the GMAVs from “1” for the lowest to “N” for the highest. If two or more GMAVs are identical, assign them successive ranks.

M. Calculate the cumulative probability, P, for each GMAV as R/ (N + 1).

N. Select the four GMAVs which have cumulative probabilities closest to 0.05 (if there are fewer than 59 GMAVs, these will always be the four lowest GMAVs)

O. Using the four selected GMAVs, and Ps, calculate

P. 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 (FAV), then that geometric mean must be used as the FAV instead of the calculated FAV.

Q. See section VI of this Appendix.

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 shall be taken into account as described in sections V.B through V.G of this Appendix or using analysis of covariance. 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 sets. If two or more factors affect toxicity, multiple regression analysis shall 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 is that between hardness and acute toxicity of metals in fresh water and a log-log relationship fits these data, 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, no transformation or a different transformation might fit the data better, and appropriate changes will be necessary throughout this section.

C. Decide whether the data for each species are relevant, 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 it is based 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 it it is 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 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, rejection of some or all of the values would be appropriate, absent countervailing justification. If useful slopes are not available for at least one fish and one invertebrate or if the available slopes are 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 IV.G of this Appendix, using the results of tests conducted under conditions and in waters similar to those commonly used for toxicity tests with the species.

D. For each species, calculate the geometric mean of the available acute values and then divide each of the acute values for the species by the geometric mean for the species. This normalizes the acute values so that the geometric mean of the normalized values for each species individually and for any combination of species is 1.0.

E. Similarly normalize the values of the water quality characteristic for each species individually using the same procedure as above.

F. Individually for each species perform a least squares regression of the normalized acute values of the water quality characteristic. The resulting slopes and 95 percent confidence limits will be identical to those obtained in section V.B. of this Appendix. If, however, the data are actually plotted, the line of best fit for each individual species will go through the point 1,1 in the center of the graph.

G. Treat all of the normalized data as if they were all for the same species and perform a least squares regression of all of the normalized acute values on the corresponding normalized values of the water quality characteristic to obtain the pooled acute slope, V, and its 95 percent confidence limits. If all of the normalized data are actually plotted, the line of best fit will go through the point 1,1 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 quality characteristic. (These were calculated in sections V.D and V.E of this Appendix).

I. For each species, calculate the logarithm, Y, of the SMAV at a selected value, Z, of the water quality characteristic using the equation:

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

Note: Alternatively, the SMAVs at Z can be obtained by skipping step H above, 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 species individually. This alternative procedure allows an examination of the range of the adjusted acute values for each species.

K. Obtain the FAV at Z by using the procedure described in sections IV.J through IV.O of this Appendix.

L. If, for a commercially or recreationally important species the geometric mean of the acute values at Z from flow-through tests in which the concentrations of the test material were measured is lower than the FAV at Z, then the geometric mean must be used as the FAV instead of the FAV.

M. The Final Acute Equation is written as:

A. There are two methods for calculating a Final Chronic Value (FCV). Selection of the appropriate methodology is dependent upon the availability of chronic toxicity data. If chronic toxicity data for the species described in section III.B.1 for freshwater species or section III.C.1 for saltwater species are available, the FCV can be calculated in the same manner as the FAV. Otherwise, the FCV can be calculated by dividing the FAV by the Final Acute-Chronic Ratio (FACR). The data requirements for calculating the FACR are identified in sections III.B.2 and III.C.2 for freshwater and saltwater species respectively. In some cases, it might not be possible to calculate a FCV. The FCV is (a) a calculated estimate of the concentration of a test material such that 95 percent of the genera (with which acceptable chronic toxicity tests have been conducted on the material) have higher GMCVs or (b) the quotient of an FAV divided by an appropriate ACR, or (c) the SMCV of a commercially or recreationally important species, if the SMCV is lower than the calculated estimate or the quotient, whichever is applicable.

Note: As the name implies, the ACR is a way of relating acute and chronic toxicities.

B. A chronic standard or guidance value shall be based on results of flow-through (except renewal is acceptable for daphnids) chronic tests in which the concentrations of test material in the test solutions were properly measured at appropriate times during the test. A chronic test is a comparative study in which organisms, that are subjected to different treatments, are observed for a long period or a substantial portion of their life span.

C. Results of chronic tests in which survival, growth, or reproduction in the control treatment was unacceptably low shall not be used. The limits of acceptability will depend on the species.

D. Results of chronic tests conducted in unusual dilution water, e.g., dilution water in which total organic carbon or particulate matter exceeded five 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, etc., do not affect toxicity.

E. Chronic values must be based on endpoints and lengths of exposure appropriate to the species. Therefore, only results of the following kinds of chronic toxicity tests shall be used:

1. Life-cycle toxicity tests consisting of exposures of each of two or more groups of individuals of a species 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 fish should begin with embryos or newly hatched young less than 48 hours old, continue through maturation and reproduction, and should 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, and for ceriodaphnids not less than seven 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. Partial life-cycle toxicity tests consist of exposures of each of two more groups of individuals of a species of fish to a different concentration of the test material through most portions of a life cycle. Partial life-cycle 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. A life-cycle test is a comparative study in which organisms, that are subjected to different treatments, are observed at least from a life stage in one generation to the same life-stage in the next generation. 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.

3. Early life-stage toxicity tests consisting of 28- to 32-day (60 days post hatch for salmonids) exposures of the early life stages of a species of fish 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 life-cycle and partial life-cycle tests with the same species. Therefore, when results of a life-cycle or partial life-cycle test are available, results of an early life-stage test with the same species should not be used. Also, results of early life-stage tests in which the incidence of mortalities or abnormalities increased substantially near the end of the test shall not be used because the results of such tests are possibly not good predictions of comparable life-cycle or partial life-cycle tests.

F. A chronic value may 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.

1. A lower chronic limit is the highest tested concentration:

a. In an acceptable chronic test;

b. Which did not cause an unacceptable amount of adverse effect on any of the specified biological measurements; and

c. Below which no tested concentration caused an unacceptable effect.

2. An upper chronic limit is the lowest tested concentration:

a. In an acceptable chronic test;

b. Which did cause an unacceptable amount of adverse effect on one or more of the specified biological measurements; and,

c. Above which all tested concentrations also caused such an effect.

Note: Because various authors have used a variety of terms and definitions to interpret and report results 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 reduction (e.g., three percent) might be considered acceptable even if it is statistically significantly different 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 has been shown to be related to a water quality characteristic such as hardness or particulate matter for freshwater animals, refer to section VII of this Appendix.

H. If chronic values are available for the species in eight families as described in section III.B.1 or section III.C.1 of this Appendix, respective SMCVs shall be calculated for each species for which at least one chronic value is available by calculating the geometric mean of the results of all acceptable life-cycle and partial life-cycle toxicity tests with the species; for a species of fish for which no such result is available, the SMCV is the geometric mean of all acceptable early life-stage tests. Appropriate GMCVs shall also be calculated. A GMCV is the geometric mean of the SMCVs for the genus. The FCV shall be obtained using the procedure described in sections IV.J through IV.O of this Appendix, substituting SMCV and GMCV for SMAV and GMAV respectively. See section VI.M of this Appendix.

Note: Section VI.I through VI.L are for use when chronic values are not available for freshwater species in eight taxonomic families as described in section III.B.1 of this Appendix, or for saltwater species in eight taxonomic families as described in section III.C.1 of this Appendix.

I. For each chronic value for which at least one corresponding appropriate acute value is available, calculate an ACR, using for the numerator the geometric mean of the results of all acceptable flow-through (except static is acceptable for daphnids and midges) acute tests in the same dilution water in which the concentrations are measured. For fish, the acute test(s) should be conducted with juveniles. The acute test(s) should be part of the same study as the chronic test. If acute tests were not conducted as part of the same study, but were conducted as part of a different study in the same laboratory and dilution water, then they 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 ACR shall not be calculated.

J. For each species, calculate the SMACR as the geometric mean of all ACRs available for that species. If the minimum ACR data requirements for calculation of a freshwater chronic standard or guidance value (as described in section III.B.2 of this Appendix are not met with freshwater data alone, saltwater data may be used along with the freshwater data. Conversely, if the minimum ACR data requirements for calculation of a saltwater chronic standard or guidance value (as described in section III.C.2 of this Appendix) are not met with saltwater data alone, freshwater data may be used along with the saltwater data.

K. For some materials, the ACR seems to be the same for all species, but for other materials the ratio seems to increase or decrease as the SMAV increases. Thus the FACR can be obtained in three ways, depending on the data available:

1. If the species mean ACR seems to increase or decrease as the SMAVs increase, the FACR shall be calculated as the geometric mean of the ACRs for species whose SMAVs are close to the FAV.

2. If no major trend is apparent and the ACRs for all species are within a factor of ten, the FACR shall be calculated as the geometric mean of all of the SMACRs.

3. If the most appropriate SMACRs are less than 2.0, and especially if they are less than 1.0, acclimation has probably occurred during the chronic test. In this situation, because continuous exposure and acclimation cannot be assured to provide adequate protection in field situations, the FACR should be assumed to be two, so that the FCV is equal to the Aquatic (Acute) value A(A). (See section X.B of this Appendix.) If the available SMACRs do not fit one of these cases, a FACR may not be obtained and a Tier I FCV probably cannot be calculated.

L. Calculate the FCV by dividing the FAV by the FACR.

FCV = FAV ÷ FACR

If there is a Final Acute Equation rather than a FAV, see also section V of this Appendix.

M. If the SMCV of a commercially or recreationally important species is lower than the calculated FCV, then that SMCV must be used as the FCV instead of the calculated FCV.

N. See section VIII of this Appendix.

A. A Final Chronic Equation can be derived in two ways. The procedure described in section VII.A of this Appendix will result in the chronic slope being the same as the acute slope. The procedure described in sections VII.B through N of this Appendix will usually result in the chronic slope being different from the acute slope.

1. If ACRs are available for enough species at enough values of the water quality characteristic to indicate that the ACR appears to be the same for all species and appears to be independent of the water quality characteristic, calculate the FACR as the geometric mean of the available SMACRs.

2. Calculate the FCV at the selected value Z of the water quality characteristic by dividing the FAV at Z (see section V.M of this Appendix) by the FACR.

3. Use V = pooled acute slope (see section V.M of this Appendix), and L = pooled chronic slope.

4. See section VII.M of this Appendix.

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 sections C through G below or using analysis of covariance. 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 sets. If two or more factors affect toxicity, multiple regression analysis shall 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 squares regression of 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 best documented relationship is that between hardness and acute toxicity of metals in fresh water and a log-log relationship fits these data, 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, no transformation or a different transformation might fit the data better, and appropriate changes will be necessary throughout this section. It is probably preferable, but not necessary, to use the same transformation that was used with the acute values in section V of this Appendix.

D. Decide whether the data for each species are relevant, 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 it is based 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 more useful if it is consistent with other information and if the two points cover a broad 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 in most cases 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, rejection of some or all of the values is, in most cases, absent countervailing circumstances, appropriate. If a useful chronic slope is not available for at least one species or if the available slopes are too dissimilar or if too few data are available to adequately define the relationship between chronic toxicity and the water quality characteristic, it might be appropriate to assume that the chronic slope is the same as the acute slope, which is equivalent to assuming that the ACR is independent of the water quality characteristic. Alternatively, return to section VI.H of this Appendix, 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 the mean for the species. 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 1.0.

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 VII.B of this Appendix. Now, however, if the data are actually plotted, the line of best fit for each individual species will go through the point 1,1 in the center of the graph.

H. Treat all of the normalized data as if they were all the same species and perform a least squares regression of all of the normalized chronic values on the corresponding normalized values of the water quality characteristic to obtain the pooled chronic slope, L, and its 95 percent confidence limits.

If all normalized data are actually plotted, the line of best fit will go through the point 1,1 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 are calculated in sections VII.E and F of this Appendix.)

J. For each species, calculate the logarithm, Q, of the SMCV at a selected value, Z, of the water quality characteristic using the equation:

Q = ln M − L(ln P − ln Z)

Note: Although it is not necessary, it is recommended that the same value of the water quality characteristic be used here as was used in section V of this Appendix.

K. For each species, calculate a SMCV at Z using the equation:

SMCV = exp(Q)

Note: Alternatively, the SMCV at Z can be obtained by skipping section VII.J of this Appendix, using the equations in sections VII.J and K of this Appendix to adjust each chronic 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 FCV at Z by using the procedure described in sections IV.J through O of this Appendix.

M. If the SMCV at Z of a commercially or recreationally important species is lower than the calculated FCV at Z, then that SMCV shall be used as the FCV at Z instead of the calculated FCV.

N. The Final Chronic Equation is written as:

FCV = exp (L[ln(water quality characteristic)] + lnS − L[lnZ])

where:

L = pooled chronic slope and S = FCV at Z.

Because L, S, and Z are known, the FCV can be calculated for any selected value of the water quality characteristic.

A. A Final Plant Value (FPV) is the lowest plant value that was obtained with an important aquatic plant species in an acceptable toxicity test for which the concentrations of the test material were measured and the adverse effect was biologically important. Appropriate measures of the toxicity of the material to aquatic plants are used to compare the relative sensitivities of aquatic plants and animals. Although procedures for conducting and interpreting the results of toxicity tests with plants are not well-developed, results of tests with plants usually indicate that criteria which adequately protect aquatic animals and their uses will, in most cases, 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 shall 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 200 ug/L should be considered excessive.

C. The FPV shall be obtained by selecting the lowest result from a test with an important aquatic plant species in which the concentrations of test material are measured and the endpoint is biologically important.

Pertinent information that could not be used in earlier sections might be available concerning adverse effects on aquatic organisms. The most important of these are data on cumulative and delayed toxicity, reduction in survival, growth, or reproduction, or any other adverse effect that has been shown to be biologically important. Delayed toxicity is an adverse effect to an organism that results from, and occurs after the end of, its exposure to one or more test materials. 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 sections IV.D and VI.D of this Appendix), from chronic tests in which the concentrations were not measured (see section VI.B of this Appendix), from tests with previously exposed organisms, and from tests on formulated mixtures or emulsifiable concentrates. Such data might affect a criterion if the data were obtained with a commercially or recreationally important species, the test concentrations were measured, and the endpoint was biologically important.

A. Standards or guidance values to protect aquatic life include: the Aquatic (Acute) or A(A) and the Aquatic (Chronic) or A(C).

B. The A(A) is equal to one-half the FAV. The A(A) is an estimate of the highest concentration of a material in the water column to which an aquatic community can be exposed briefly without resulting in an unacceptable effect.

C. The A(C) is equal to the lowest of the FCV or the FPV (if available) unless other data (see section IX of this Appendix) show that a lower value should be used. The A(C) is an estimate of the highest concentration of a material in the water column to which an aquatic community can be exposed indefinitely without resulting in an unacceptable effect. If toxicity is related to a water quality characteristic, the A(C) is obtained from the Final Chronic Equation or FPV (if available) that results in the lowest concentrations in the usual range of the water quality characteristic, unless other data (see section IX) show that a lower value should be used.

D. Round both the A(A) and the A(C) to two significant digits.

A. The derivation of the standard or guidance value should be carefully reviewed by rechecking each step of the guidance in this part. 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 10?

4. Is the range of SMAVs for any genus greater than a factor of 10?

5. Is there more than a factor of 10 difference between the four lowest GMAVs?

6. Are any of the lowest GMAVs questionable?

7. Is the FAV reasonable in comparison with the SMAVs and GMAVs?

8. For any commercially or recreationally 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 FAV?

9. Are any of the chronic values used questionable?

10. Are any chronic values available for acutely sensitive species?

11. Is the range of acute-chronic ratios greater than a factor of 10?

12. Is the FCV 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 FCV?

14. Are any of the other data important?

15. Do any data look like they might be outliers?

16. Are there any deviations from the guidance in this part? Are they acceptable?

B. On the basis of all available pertinent laboratory and field information, determine if the standard or guidance value is consistent with sound scientific evidence. If it is not, another standard or guidance value, either higher or lower, shall be derived consistent with the guidance in this part.

If all eight minimum data requirements for calculating an FAV using Tier I are not met, a Secondary Acute Value (SAV) shall be calculated for a chemical as follows:

To calculate a SAV, the lowest GMAV in the database is divided by the Secondary Acute Factor (SAF) (Table 1 of this Appendix) corresponding to the number of satisfied minimum data requirements listed in the Tier I methodology (section III.B.1 of this Appendix for freshwater species and section III.C.1 for saltwater species). Data requirements contained in sections I, II, and IV shall be applied to calculation of a SAV. If all eight minimum data requirements are satisfied, a Tier I value calculation may be possible. In order to calculate a freshwater SAV, the database must contain, at a minimum, a genus mean acute value (GMAV) for one of the following three genera in the family Daphnidae - Ceriodaphnia sp., Daphnia sp., or Simocephalus sp. In order to calculate a saltwater SAV, it would be desirable if the database contained, at a minimum: a genus mean acute value (GMAV) for a species or genus in one of the following families - Mysidae or Penaeidae; and a GMAV for a saltwater fish.

If appropriate, the SAV shall be made a function of a water quality characteristic in a manner similar to that described in Tier I.

If three or more experimentally determined ACRs, meeting the data collection and review requirements of section VI of this Appendix, are available for the chemical, determine the FACR using the procedure described in section VI. If fewer than three acceptable experimentally determined ACRs are available, use enough assumed ACRs of 18 so that the total number of ACRs equals three. Calculate the Secondary Acute-Chronic Ratio (SACR) as the geometric mean of the three ACRs. Thus, if no experimentally determined ACRs are available, the SACR is 18.

Calculate the Secondary Chronic Value (SCV) using one of the following:

A. SCV = FAV ÷ SACR (use FAV from Tier I)

B. SCV = SAV ÷ FACR

C. SCV = SAV ÷ SACR

If appropriate, the SCV will be made a function of a water quality characteristic in a manner similar to that described in Tier I.

If for a commercially or recreationally important species the geometric mean of the acute values or chronic values from flow-through tests in which the concentrations of the test materials were measured is lower than the calculated SAV or SCV, then that geometric mean must be used as the SAV or SCV instead of the calculated SAV or SCV.

A. Standards or guidance values to protect aquatic life shall include: the Aquatic (Acute) or A(A) value and the Aquatic (Chronic) or A(C) value.

B. The A(A) is equal to one-half of the SAV.

C. The A(C) is equal to the lowest of the SCV or the Final Plant Value, if available, unless other data (see section IX of this Appendix) show that a lower value should be used.

If toxicity is related to a water quality characteristic, the A(C) is obtained from the Secondary Chronic Equation or FPV, if available, that results in the lowest concentrations in the usual range of the water quality characteristic, unless other data (see section IX of this Appendix) show that a lower value should be used.

D. Round both the A(A) and the A(C) to two significant digits.

On the basis of all available pertinent laboratory and field information, determine if the Tier II value is consistent with sound scientific evidence. If it is not, another value, either higher or lower, shall be derived consistent with the guidance in this Appendix.