Appendix J. Aquatic Ecological Effects Data This appendix is a summary of the data being evaluated for ecological effects on aquatic organisms. Each section consists of several sets of data. Tables presenting the data submitted with the application for registration are presented first. The lowest value from this table is used to set a “benchmark” that is used in evaluating the data from the open literature—from EPA’s ECOTOX database. Only the studies associated with the ECOTOX data that have values less than these benchmarks are examined further. If any of these latter values are acceptable, then that value becomes the final value used in the risk assessment for that particular taxonomic group. Because the stability of endosulfan TGAI in aquatic tests is a concern due to its volatility, preference was given to those studies that quantified endosulfan exposure via analytical measurements and where appropriate, used a flow-through exposure regime. In some cases, however, these data were lacking (e.g., aquatic nonvascular plants), and toxicity values based on nominal concentrations were used to fulfill a data gap. In general, studies from the open literature do not provide sufficient documentation for evaluating all or most of the OPPTS Guideline evaluation criteria. Consistent with the Overview Document (USEPA 2004), if sufficient documentation was provided to judge that the open literature studies were scientifically valid and acceptable endpoints were determined that were below the ‘benchmark’ values, they were typically classified as supplemental-- but acceptable for quantitative use. If sufficient documentation was not provided to comprehensively judge the scientific validity of the study but no information existed that suggested the study would be invalid, these studies were typically classified as supplemental--but acceptable for qualitative use. This classification was often applied to studies with nominal exposure concentrations. I. Acute Toxicity to Freshwater Fish Submitted Data for Freshwater Fish Acute Values Two freshwater fish toxicity studies using the TGAI are required to establish the toxicity of endosulfan to fish. The preferred test species are rainbow trout (a coldwater fish) and bluegill sunfish (a warmwater fish). Rainbow trout were the most sensitive (LC50 = 0.83 μg/L) species tested (Table J-1). Since the LC50 value was less than 0.1 mg/L, endosulfan is categorized as very highly toxic to freshwater fish on an acute exposure basis. The freshwater fish acute toxicity testing requirement (Guideline 72-1) is fulfilled (MRID 38806, 40094602, 40098001, 136999, 05008271). Endosulfan Sulfate Toxicity Acute toxicity of endosulfan sulfate on freshwater fish was tested using bluegill sunfish. The 96-hour LC50 of endosulfan sulfate on bluegill was 3.8 μg/L and is considered very highly toxic (MRID 46382604). Acute toxicity testing of endosulfan sulfate (99.9% radiopurity) was also tested with common carp (Gries and van der Kolk, 2000a; MRID 45421402), which also indicated that the degradate is very highly toxic (96-hr LC50 = 2.2 μg/L) to freshwater fish. The study was classified as supplemental since it failed to comply with EPA’s recommended species and testing conditions. These results are consistent with studies of parent endosulfan where 96-hr LC50 values ranged from 0.37 to 3.3 μg/L. Formulated End Use Product The acute toxicity of endosulfan to freshwater fish was tested using technical end product. There was considerable variability for LC50 values within formulations tested; toxicity estimates ranged from 0.47 μg/L to 5.6 μg/L for the 50 WP and 33.7% formulations, respectively (Table J-2). Similar to the results on technical grade active ingredient, rainbow trout were the most sensitive species tested using technical end product. Of the formulated products tested, the 4% formulation was the least toxic with an LC50 estimate of 28 μg/L. Based on the test results, the formulation of endosulfan is classified as being very highly toxic to freshwater fish species. The freshwater fish acute toxicity testing requirement using technical end product (Guideline 72-1) is fulfilled (MRID 00128655, BA007902, 250401A). Additional Open Literature Information (ECOTOX) for Freshwater Fish Acute Values All of the open literature acute values from EPA’s ECOTOX database for freshwater fish are presented in Figure J-1. The vertical line represents the benchmark from the registrant’s data (0.83 ug/L). These data are primarily for an exposure duration of 4 days; however, other durations were included (e.g., 1 or 2 day exposures) if there was no 4 day LC50 for a particular test. Tables J-3 and J-4 contains the acute LC50 values that are less than this benchmark. When LC50 values for multiple exposure durations from the same test were available, only the value for 4 day exposure was included. There were 150 LC50 values from 35 open literature studies representing 31 freshwater fish species. Forty-five of these values were below the freshwater fish benchmark (0.83 μg/L) that was based on the information provided by the registrant. These data are presented in nine different studies and represent 13 species. Endosulfan: Freshwater Fish LC50s n = 150 1.0 0.9 0.8 0.7 0.6 0.5 Probability Rank Probability 0.4 0.3 0.2 0.1 0.0 0.1 1 10 100 1000 10000 Concentration (ug/L) Figure J-1. Plot of the freshwater fish endosulfan acute data. Forty-eight 96 hr LC50 values are available from an interlaboratory comparison of acute tests for large subadults of the fathead minnow Pimephales promelas and fingerlings of the rainbow trout Oncorhynchus mykiss (Lemke1981—ECOTOX reference # 9479). All Page J-2 of the laboratories used the same technical grade endosulfan. For each fish species there are 24 LC50 values, 12 from static tests and 12 from flow-through tests. The static test values for fathead minnows ranged from 0.97 to 3.45 μg/L (all values are above the benchmark). The 12 flow-through tests for this species had LC50s ranging from 0.29 to 1.91 μg/L (seven of these values were below the benchmark). The static tests for rainbow trout ranged from 0.49 to 2.43 μg/L (six of these 12 values were below the benchmark) and the flow-through tests had values ranging from 0.17 to 0.75 μg/L (all 12 values below the benchmark). And additional set of fourteen 96 hr LC50s are presented in Sunderam et al. (1992— ECOTOX reference # 5850). These data are for eight species of fish from Australia. Six of the eight species had at least one LC50 values less than the benchmark. These are the golden perch Macquaria ambigua, the common carp Cyprinus carpio, the eastern rainbow fish Melanotaenia duboulayi, the bony bream Nematolosa erebi, the harlequinfish Rasbora sp. and the rainbow trout Oncorhynchus mykiss. The endosulfan used was technical grade, and the common carp was the most sensitive species in this study and was the most sensitive freshwater fish overall with an LC50 of 0.1 μg/L. Seven tests in 1988 and 1989 used the striped bass Morone saxatilis and yielded similar LC50 values, ranging from >0.22 to 0.43 μg/L (Fujimura et al. 1991—ECOTOX reference # 15472). All of the tests used larval fish, ranging in age from 6 to 45 days post hatch, and were conducted as flow-through tests. Adult Asian swamp eels, Monopterus albus, were tested using a commercial endosulfan end product (33% purity—mixture of α and β endosulfans). The 96 hr LC50 obtained in this study was 0.42 μg endosulfan/L (Hii et al. 2007—ECOTOX reference # 100663). Another study using males and females of Jenynsia multidentata—also with a commercial preparation (35% purity)—demonstrated that males may be more sensitive than females; the 96 hr LC50s were 0.719 and 1.317 μg endosulfan/L, respectively (Ballesteros et al. 2007—ECOTOX reference # 103207). Three different studies compared various forms of endosulfan; α and β endosulfans, technical grade mixture of the two, as well as commercial sources of powders and emulsified concentrates (Table J-5). In each case the α-endosulfan was the most toxic form, and the only form with an LC50 less than the benchmark. Juveniles of Labeo rohita had an LC50 of 0.33 μg/L for the α form; compared with 7.1 for the β-endosulfan and 1.0 to 1.25 for the technical grade and commercial preparations (Rao et al. 1980—ECOTOX reference # 468). The snake-head catfish, Channa punctata had an LC50 of 0.16 μg/L for α-endosulfan, and the other forms of endosulfan all had LC50s from 2.5 to 16 μg/L (Devi, et al. 1981—ECOTOX reference # 15157). Finally, the 96 hr LC50 for α- endosulfan for the rainbow trout Oncorhynchus mykiss (Wan et al. 2005—ECOTOX reference # 87973) is 0.5 μg/L, in addition, a 60/40 mixture of α- and β-endosulfans resulted in an LC50 of 0.7 μg/L. The other forms of endosulfan had LC50s greater than the benchmark. This included the only freshwater fish acute test from the ECOTOX data set that used endosulfan sulfate (LC50 of 1.4 μg/L). In almost all cases the commercial end product was less toxic (based on active ingredient) than the technical grade. Page J-3 Table J-1. Summary of freshwater fish acute toxicity data submitted by registrant for endosulfan Species/Flow- 96-hour Toxicity MRID No. Study through or Static % ai LC50 (μg/L) Category Author/Year Classification Bluegill sunfish very highly BA007903/ (Lepomis 96 2.08 supplemental toxic EPA 1976 macrochirus) Bluegill sunfish very highly 38806/ (Lepomis 100 1.7 acceptable toxic Buccafusco 1976 macrochirus) Bluegill sunfish very highly 05014941/Pickering (Lepomis 96.6 3.3 supplemental toxic &Henderson 1966 macrochirus) Bluegill sunfish very highly 40094602/ (Lepomis 96 1.2 supplemental toxic Johnson&Finley 1980 macrochirus) static Rainbow trout a b very highly 05003107/ (Oncorhynchus tech 1.5 supplemental toxic Macek et al.