327 IAC 2-1.5-11 Determination of Tier I aquatic life criteria

Sec. 11. (a) The procedures in this section shall be used to determine acute and chronic Tier I aquatic life criteria. (b) The following considerations regarding the toxic substance shall be considered during the development of Tier I criteria or Tier II values: (1) Each separate chemical that does not ionize substantially in most natural bodies of water should usually be considered a separate substance, except possibly for structurally similar organic compounds that only exist in large quantities as commercial mixtures of the various compounds and apparently have similar biological, chemical, physical, and toxicological properties. (2) For chemicals that ionize substantially in most natural bodies of water, for example: (A) some phenols and organic acids; (B) some salts of phenols and organic acids; and (C) most inorganic salts and coordination complexes of metals and metalloid; all forms that would be in chemical equilibrium should usually be considered one (1) substance. Each different oxidation state of a metal and each different nonionizable covalently bonded organometallic compound should usually be considered a separate substance. (3) The definition of the toxic substance should include an operational analytical component. Identification of a substance simply as sodium, for example, implies total sodium, but leaves room for doubt. If total is meant, it must be explicitly stated. Even total has different operational definitions, some of which do not necessarily measure all that is there in all samples. Thus, it is also necessary to reference or describe the analytical method that is intended. The selection of the operational analytical component should take into account the analytical and environmental chemistry of the material 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 operational analytical considerations shall be as follows: (A) The primary requirements of the operational analytical component shall be as follows: (i) Appropriate for use on samples of receiving water. (ii) Rarely result in underprotection or overprotection of aquatic organisms and their uses. (iii) Compatible with the available toxicity and bioaccumulation data without making extrapolations that are too hypothetical. Toxicity is the property of a substance, or combination of substances, to adversely affect organisms. (B) Because an ideal analytical measurement will rarely be available, an appropriate compromise measurement will usually have to be used. This compromise measurement must fit with the general approach that if an ambient concentration is lower than the criterion or value, unacceptable effects will probably not occur, that is, the compromise measure must not err on the side of underprotection when measurements are made on a surface water. What is an appropriate measurement in one (1) situation might not be appropriate for another. For example, because the chemical and physical properties of an effluent are usually quite different from those of the receiving water, an analytical method that is appropriate for analyzing an effluent might not be appropriate for expressing a criterion or value, and vice versa. A criterion or value should be based on an appropriate analytical measurement, but the criterion or value is not rendered useless if an ideal measurement either is not available or is not feasible. The analytical chemistry of the substance might have to be taken into account when defining the substance or when judging the acceptability of some toxicity tests, but a criterion or value must 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. (4) The use of dissolved metal to set and measure compliance with water quality standards for aquatic life is the recommended approach, because dissolved metal more closely approximates the bioavailable fraction of metal in the water column than does total recoverable metal. Reasons for the consideration of total recoverable metals criteria or values include risk management considerations not covered by evaluation of water column toxicity. The commissioner may, after considering sediment and food chain effects for a particular metal, decide to take a more conservative approach for the metal. This approach could include the expression of aquatic life criteria or values for the metal in the form of total recoverable metal. If the commissioner determines that it is appropriate to express aquatic life criteria or values for a particular metal in the form of dissolved metal, the criteria or values shall be determined as follows: (A) If sufficient toxicological data in the form of dissolved metal are available, these data shall be used in this section and sections 12 and 16 of this rule to derive aquatic life criteria or values directly in the form of dissolved metal. (B) If sufficient toxicological data in the form of dissolved metal are not available, aquatic life criteria or values shall be derived in the form of total recoverable metal using the procedures in this section and sections 12 and 16 of this rule and then multiplied by criteria conversion factors approved by the commissioner to express the criteria or values in the form of dissolved metal. (C) If sufficient toxicological data in the form of dissolved metal are not available and criteria conversion factors for the particular metal have not been approved by the commissioner, aquatic life criteria or values shall be derived in the form of total recoverable metal using the procedures in this section and sections 12 and 16 of this rule and expressed in the form of total recoverable metal. (c) The following data collection procedures shall be followed when developing Tier I aquatic life criteria: (1) Collect all data available on the substance concerning toxicity to aquatic animals and plants. (2) All data that are used should be available in typed, dated, and signed hard copy, for example: (A) publication; (B) manuscript; (C) letter; or (D) memorandum; with enough supporting information to indicate that acceptable test procedures were used and that the results are reliable. In some cases, it may be appropriate to obtain written information from the investigator, if possible. Information that is not available for distribution shall not be used. (3) Questionable data, whether published or unpublished, shall not be used. For example, data shall be rejected if they are from tests: (A) that did not contain a control treatment; (B) in which too many organisms in the control treatment died or showed signs of stress or disease; and (C) in which distilled or deionized water was used as the dilution water without the addition of appropriate salts. (4) Data on technical grade materials may be used if appropriate, but data on formulated mixtures and emulsifiable concentrates of the material shall not be used. (5) For some highly volatile, hydrolyzable, or degradable materials, it may be appropriate to use only results of flow-through tests in which the concentrations of test material in test solutions were measured using acceptable analytical methods. A flow-through test is a test with aquatic organisms in which test solutions flow into constant-volume test chambers either intermittently, for example, every few minutes, or continuously, with the excess flowing out. (6) Data shall be rejected if obtained using the following: (A) Brine shrimp, because they usually only occur naturally in water with salinity greater than thirty-five (35) grams per kilogram. (B) Species that do not have reproducing wild populations in North America. (C) Organisms that were previously exposed to substantial concentrations of the test material or other contaminants. (D) Saltwater species except for use in deriving ACR. (7) Questionable data, data on formulated mixtures and emulsifiable concentrates, and data obtained with species nonresident to North America or previously exposed organisms may be used to provide auxiliary information but shall not be used in the derivation of criteria. (d) This subsection establishes the data requirements for the development of Tier I aquatic life criteria as follows: (1) Certain data should be available to help ensure that each of the major kinds of possible adverse effects receives adequate consideration. An adverse effect is a change in an organism that is harmful to the organism. Exposure means contact with a chemical or physical agent. Results of acute and chronic toxicity tests with representative species of aquatic animals are necessary so that data available for tested species can be considered a useful indication of the sensitivities of appropriate untested species. Fewer data concerning toxicity to aquatic plants are usually available because procedures for conducting tests with plants and interpreting the results of such tests are not as well developed. (2) To derive a Tier I criterion for aquatic organisms and their uses, the following must be available: (A) Results of acceptable acute (or chronic) tests (see subsections (e) and (g)) with at least one (1) species of freshwater animal in at least eight (8) different families such that all of the following are included: (i) The family Salmonidae in the class Osteichthyes. (ii) One (1) other family (preferably a commercially or recreationally important, warmwater species) in the class Osteichthyes, for example: (AA) bluegill; or (BB) channel catfish. (iii) A third family in the phylum Chordata, for example: (AA) fish; or (BB) amphibian. (iv) A planktonic crustacean, for example: (AA) a cladoceran; or (BB) copepod. (v) A benthic crustacean, for example: (AA) ostracod; (BB) isopod; (CC) amphipod; or (DD) crayfish. (vi) An insect, for example: (AA) mayfly; (BB) dragonfly; (CC) damselfly; (DD) stonefly; (EE) caddisfly; (FF) mosquito; or (GG) midge. (vii) A family in a phylum other than Arthropoda or Chordata, for example: (AA) Rotifera; (BB) Annelida; or (CC) Mollusca. (viii) A family in any order of insect or any phylum not already represented. (B) Acute-chronic ratios (see subsection (g)) with at least one (1) species of aquatic animal in at least three (3) different families provided that of the three (3) species at least one (1) is: (i) a fish; (ii) an invertebrate; and (iii) an acutely sensitive freshwater species (the other two (2) may be saltwater species). (C) Results of at least one (1) acceptable test with a freshwater algae or vascular plant is desirable but not required for criterion derivation (see subsection (i)). 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. (3) If all required data are available, a numerical criterion can usually be derived except in special cases. For example, derivation of a chronic criterion might not be possible if the available ACRs vary by more than a factor of ten (10) with no apparent pattern. Also, if a criterion is to be related to a water quality characteristic (see subsections (f) and (h)), more data will be required. (4) Confidence in a criterion usually increases as the amount of available pertinent information increases. Thus, additional data are usually desirable. (e) The following procedures shall be used to calculate an FAV: (1) Appropriate measures of the acute (short term) toxicity of the material to a variety of species of aquatic animals are used to calculate the FAV. The calculated FAV is a calculated estimate of the concentration of a test material such that ninety-five percent (95%) of the genera (with which acceptable acute toxicity tests have been conducted on the material) have higher 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 SMAV of a commercially or recreationally important species of the Great Lakes system is lower than the calculated FAV, then the SMAV replaces the calculated FAV in order to provide protection for that important species. (2) Acute toxicity tests shall be conducted in accordance with this subsection. (3) 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. (If the minimum acute-chronic ratio data requirements (as described in subsection (d)(2)(B)) are not met with freshwater data alone, saltwater data may be used.) (4) Results of acute tests conducted in unusual dilution water, for example, dilution water in which total organic carbon or particulate matter exceeded five (5) milligrams per liter, shall 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 do not affect toxicity. (5) Acute values must be based upon endpoints that 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: (A) Tests with daphnids and other cladocerans must be started with organisms less than twenty-four (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 forty-eight (48) hour EC50 based on the total percentage of organisms killed and immobilized. If such an EC50 is not available for a test, the forty-eight (48) hour LC50 should be used in place of the desired forty-eight (48) hour EC50. An EC50 or LC50 of longer than forty-eight (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. (B) 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 ninety-six (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 ninety-six (96) hour EC50: (i) Forty-eight (48) hour to ninety-six (96) hour EC50s based on percentage of organisms with incompletely developed shells plus percentage of organisms killed. (ii) Forty-eight (48) hour to ninety-six (96) hour EC50s based upon percentage of organisms with incompletely developed shells. (iii) Forty-eight (48) hour to ninety-six (96) hour LC50s. If the minimum acute-chronic ratio data requirements (as described in subsection (d)(2)(B)) are not met with freshwater data alone, saltwater data may be used. (C) 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 ninety-six (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 ninety-six (96) hour EC50: (i) The ninety-six (96) hour EC50 based on percentage of organisms exhibiting loss of equilibrium plus percentage of organisms immobilized. (ii) The ninety-six (96) hour LC50. (D) Tests results that take into account the number of young produced, such as most tests with protozoans, are not considered acute tests, even if the duration was ninety-six (96) hours or less. (E) If the tests were conducted properly, acute values reported as greater than values and those that 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. (6) 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 subsection (f). (7) 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 ten (10), rejection of some or all of the values would be appropriate, absent countervailing circumstances. (8) If the available data indicate that one (1) or more life stages are at least a factor of two (2) more resistant than one (1) or more other life stages of the same species, the data for the more resistant life stages shall 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. (9) For each species for which at least one (1) 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, for example, 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. The following conditions are applicable to this calculation: (A) Data reported by original investigators must not be rounded off. Results of all intermediate calculations must not be rounded off to fewer than four (4) significant digits. (B) 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 (2) numbers is the square root of the product of the two (2) numbers, and the geometric mean of one (1) 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, for example, the antilog used must match the logarithms used. (C) 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. (10) For each genus for which one (1) or more SMAVs are available, the GMAV shall be calculated as the geometric mean of the SMAVs available for the genus. (11) Order the GMAVs from high to low. (12) Assign ranks, R, to the GMAVs from "1" for the lowest to "N" for the highest. If two (2) or more GMAVs are identical, assign them successive ranks. (13) Calculate the cumulative probability, P, for each GMAV as R/(N + 1). (14) Select the four (4) GMAVs that have cumulative probabilities closest to five-hundredths (0.05) (if there are fewer than fifty-nine (59) GMAVs, these will always be the four (4) lowest GMAVs). (15) Using the four (4) selected GMAVs and Ps, calculate: (A) (B) (C) (D) (16) If, for a commercially or recreationally important species of the Great Lakes system, 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 FAV, then that geometric mean must be used as the FAV instead of the calculated FAV. (f) When enough data are available to show that acute toxicity to two (2) or more species is similarly related to a water quality characteristic, the relationship shall be taken into account as described in subdivisions (1) through (6) or using analysis of covariance. The two (2) methods are equivalent and produce identical results. The manual method described in this subsection 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 (2) or more factors affect toxicity, multiple regression analysis shall be used. An acute criterion based on a water quality characteristic shall be determined as follows: (1) For each species for which comparable acute toxicity values are available at two (2) 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 ninety-five percent (95%) confidence limits for each species. (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 or temperature, no transformation or a different transformation might fit the data better, and appropriate changes will be necessary throughout this section.) (2) 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 (6) 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 (2) data points, however, might be useful if it is consistent with other information and if the two (2) 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 ten (10), rejection of some or all of the values would be appropriate, absent countervailing justification. Return to subsection (e)(7), using the results of tests conducted under conditions and in waters similar to those commonly used for toxicity tests with the species if any of the following occur: (A) Useful slopes are not available for at least one (1) fish and one (1) invertebrate. (B) The available slopes are too dissimilar. (C) Too few data are available to adequately define the relationship between acute toxicity and the water quality characteristic. (3) 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 one (1.0). (4) Similarly normalize the values of the water quality characteristic for each species individually using the procedure in subdivisions (1) through (3). (5) Individually for each species perform a least squares regression of the normalized acute values of the water quality characteristic. The resulting slopes and ninety-five percent (95%) confidence limits will be identical to those obtained in subdivision (1). 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. (6) 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 ninety-five percent (95%) 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. (7) 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 subdivisions (3) and (4)). (8) For each species, calculate the logarithm, Y, of the SMAV at a selected value, Z, of the water quality characteristic using the equation: Y = ln W - V(ln X - ln Z) (9) For each species calculate the SMAV at Z using the equation: SMAV = eY (10) Alternatively, the SMAVs at Z can be obtained by skipping the step in subdivision (7), using the equations in subdivisions (8) and (9) 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. (11) Obtain the FAV at Z by using the procedure described in subsection (e)(10) through (e)(15). (12) If, for a commercially or recreationally important species of the Great Lakes system, 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 calculated in subdivision (11). (13) The final acute equation is written as: (FAV) = e(V[ln(water quality characteristic)] + A - V[ln Z]) Where: V = pooled acute slope. A = ln(FAV at Z). Because V, A, and Z are known, the FAV can be calculated for any selected value of the water quality characteristic. (g) The following procedures shall be used to calculate an FCV: (1) Depending on the data that are available concerning chronic toxicity to aquatic animals, the FCV can be calculated in the same manner as the FAV or by dividing the FAV by the final acute-chronic ratio (FACR). In some cases, it might not be possible to calculate an FCV. The FCV is one (1) of the following as applicable: (A) A calculated estimate of the concentration of a test material such that ninety-five percent (95%) of the genera (with which acceptable chronic toxicity tests have been conducted on the material) have higher GMCVs. (B) The quotient of an FAV divided by an appropriate ACR (ACR is a way of relating acute and chronic toxicities). (C) The SMCV of an important or critical species, if the SMCV is lower than the calculated estimate or the quotient. (2) Chronic values 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. (3) 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. (4) Results of chronic tests conducted in unusual dilution water, for example, dilution water in which total organic carbon or particulate matter exceeded five (5) milligrams per liter, should not be used unless: (A) a relationship is developed between chronic toxicity and organic carbon or particulate matter; or (B) data show that the organic carbon or particulate matter do not affect toxicity. (5) 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: (A) Life-cycle toxicity tests consisting of exposures of each of two (2) 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, the following procedures shall be followed: (i) Tests with fish should: (AA) begin with embryos or newly hatched young less than forty-eight (48) hours old; (BB) continue through maturation and reproduction; and (CC) end not less than twenty-four (24) days (ninety (90) days for salmonids) after the hatching of the next generation. 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. (ii) Tests with daphnids should begin with young less than twenty-four (24) hours old and last for not less than twenty-one (21) days, and for ceriodaphnids not less than seven (7) days. For daphnids, data should be obtained and analyzed on survival and young per female. (iii) Tests with mysids should begin with young less than twenty-four (24) hours old and continue until seven (7) days past the median time of first brood release in the controls. For mysids, data should be obtained and analyzed on survival, growth, and young per female. (B) Partial life-cycle toxicity tests consist of exposures of each of two (2) or 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 fifteen (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 (1) generation to the same life stage in the next generation. Exposure to the test material should: (i) begin with immature juveniles at least two (2) months prior to active gonad development; (ii) continue through maturation and reproduction; and (iii) end not less than twenty-four (24) days (ninety (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. (C) Early life-stage toxicity tests consisting of twenty-eight (28) to thirty-two (32) day (sixty (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.) (6) A chronic value may be obtained by analyzing chronic data using regression analysis or by calculating the geometric mean of the lower and upper chronic limits from a chronic test as follows: (A) A lower chronic limit is the highest tested concentration: (i) in an acceptable chronic test; (ii) that did not cause an unacceptable amount of adverse effect on any of the specified biological measurements; and (iii) below which no tested concentration caused an unacceptable effect. (B) An upper chronic limit is the lowest tested concentration: (i) in an acceptable chronic test; (ii) that did cause an unacceptable amount of adverse effect on one (1) or more of the specified biological measurements; and (iii) above which all tested concentrations also caused such an effect. (C) 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 (for example, three percent (3%)) might be considered acceptable even if it is statistically significantly different from the control, whereas a large percent reduction (for example, thirty percent (30%)) might be considered unacceptable even if it is not statistically significant. (7) 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 subsection (h). (8) If chronic values are available for species in eight (8) families as described in subsection (d)(2)(A), an SMCV shall be calculated for each species for which at least one (1) 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 subsection (e)(10) through (e)(15), substituting SMCV and GMCV for SMAV and GMAV, respectively. See subdivision (10). (9) The following procedures are for use when chronic values are not available for species in eight (8) taxonomic families as described in subsection (d)(2)(A): (A) For each chronic value for which at least one (1) 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 tests should be conducted with juveniles. The acute tests 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. (B) For each species, calculate the SMACR as the geometric mean of all ACRs available for that species. If the minimum ACR data requirements (as described in subsection (d)(2)(B)) are not met with freshwater data alone, saltwater data may be used along with the freshwater data. (C) 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 the following three (3) ways, depending on the data available (If the available SMACRs do not fit one (1) of these cases, a FACR may not be obtained and a Tier I FCV probably cannot be calculated.): (i) 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. (ii) If no major trend is apparent and the ACRs for all species are within a factor of ten (10), the FACR shall be calculated as the geometric mean of all of the SMACRs. (iii) If the most appropriate SMACRs are less than two (2.0), and especially if they are less than one (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 (2), so that the FCV is equal to the CMC. (See subsection (k)(1).) (D) Calculate the FCV by dividing the FAV by the FACR. FCV = FAV ÷ FACR. If there is a final acute equation rather than an FAV, see also subsection (f). (10) If the SMCV of a commercially or recreationally important species of the Great Lakes system is lower than the calculated FCV, then that SMCV must be used as the FCV instead of the calculated FCV. (h) When enough data are available to show that toxicity to two (2) or more species is similarly related to a water quality characteristic, the relationship shall be taken into account as described in this subsection. A final chronic equation can be derived in two (2) ways. The procedure described in subdivision (1) will result in the chronic slope being the same as the acute slope. The procedure described in subdivision (2) will usually result in the chronic slope being different from the acute slope. A chronic criterion based on a water quality characteristic shall be determined as follows: (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, then: (A) calculate the FACR as the geometric mean of the available SMACRs; (B) calculate the FCV at the selected value Z of the water quality characteristic by dividing the FAV at Z (see subsection (f)(11)) by the FACR; and (C) use V = pooled acute slope (see subsection (f)(6)), and L = pooled chronic slope (see subdivision (2)(F)). (2) When enough data are available to show that chronic toxicity to at least one (1) species is related to a water quality characteristic, the relationship should be taken into account as described in clauses (A) through (E) or using analysis of covariance. The two (2) methods are equivalent and produce identical results. The manual method described in this subdivision 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 (2) or more factors affect toxicity, multiple regression analysis shall be used. The manual method for taking into account the relationship of chronic toxicity to a water quality characteristic is the following: (A) For each species for which comparable chronic toxicity values are available at two (2) 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 ninety-five percent (95%) confidence limits for each species. (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 or 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 subsection (f).) (B) 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 (6) 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 (2) data points, however, might be more useful if it is consistent with other information and if the two (2) 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 ten (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 (1) 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 subsection (g)(8), using the results of tests conducted under conditions and in waters similar to those commonly used for toxicity tests with the species. (C) 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 one (1.0). (D) Similarly, normalize the values of the water quality characteristic for each species individually. (E) 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 ninety-five percent (95%) confidence limits will be identical to those obtained in this subdivision. 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. (F) 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 ninety-five percent (95%) 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. (G) 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 clauses (C) and (D).) (H) 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) (Although it is not necessary, it is recommended that the same value of the water quality characteristic be used here as was used in subsection (f).) (I) For each species, calculate an SMCV at Z using the equation: SMCV = eQ (Alternatively, the SMCV at Z can be obtained by skipping clause (G), using the equations in clause (H) and this clause 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.) (J) Obtain the FCV at Z by using the procedure described in subsection (e)(10) through (e)(15). (3) If the SMCV at Z of a commercially or recreationally important species of the Great Lakes system is lower than the calculated FCV at Z, then that SMCV shall be used as the FCV at Z instead of the calculated FCV. (4) The final chronic equation is written as: FCV = e(L[ln(water quality characteristic)] + lnS- L[lnZ]) Where: L = pooled chronic slope. 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. (i) An 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 that adequately protect aquatic animals and their uses will, in most cases, also protect aquatic plants and their uses. When developing an FPV, the following apply: (1) A plant value is the result of a ninety-six (96) hour test conducted with an alga or a chronic test conducted with an aquatic vascular plant. (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 two hundred (200) μg/L should be considered excessive.) (2) 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. (j) Pertinent information that could not be used in earlier subsections may be available concerning adverse effects on aquatic organisms. The following are data that may affect a criterion if the data were obtained with an important species, the test concentrations were measured, and the endpoint was biologically important: (1) 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 (1) or more test materials. (2) Species for which no other data are available. (3) Behavioral, biochemical, physiological, microcosm, and field studies. (4) Tests conducted in unusual dilution water (see subsections (e)(4) and (g)(4)). (5) Chronic tests in which the concentrations were not measured (see subsection (g)(2)). (6) Tests with previously exposed organisms (see subsection (c)(6)(C)). (7) Tests on formulated mixtures or emulsifiable concentrates (see subsection (c)(4)). (k) A criterion consists of two (2) concentrations, the CMC and the CCC, determined as follows: (1) The CMC is equal to one-half (1/2) the FAV. The CMC 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. (2) The CCC is equal to the lowest of the FCV or the FPV (if available) unless other data (see subsection (j)) show that a lower value should be used. The CCC 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 CCC 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 subsection (j)) show that a lower value should be used. (3) Round both the CMC and the CCC to two (2) significant digits. (4) The criterion is stated as follows: (A) The procedures described in the Tier I methodology indicate that, except possibly where a commercially or recreationally important species is very sensitive, aquatic organisms should not be affected unacceptably if the four (4) day average concentration of (insert name of substance) does not exceed (insert the CCC for the substance) μg/L more than once every three (3) years on the average and if the one (1) hour average concentration does not exceed (insert the CMC for the substance) μg/L more than once every three (3) years on the average. (B) If the CMC averaging period of one (1) hour or the CCC averaging period of four (4) days is inappropriate for the pollutant, or if the once-in-three-year allowable excursion frequency is inappropriate for the pollutant or for the sites to which a criterion is applied, then the commissioner may specify alternative averaging periods or frequencies. The choice of an alternative averaging period or frequency shall be justified by a scientifically defensible analysis demonstrating that the alternative values will protect the aquatic life uses of the water. Appropriate laboratory data or well-designed field biological surveys shall be submitted to the U.S. EPA as justification for differing averaging periods or frequencies of exceedance.