United States Region 5
Environmental Protection 77 W. Jackson Blvd. EPA 905-R-10-002
Agency Chicago, Illinois 60604 November 2010
v>EPA Final Report on Acute and
Chronic Toxicity of Nitrate,
Nitrite, Boron, Manganese,
Fluoride, Chloride and
Sulfate to Several Aquatic
Animal Species
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EPA 905-R-10-002
November 2010
Final Report on Acute and Chronic Toxicity of
Nitrate, Nitrite, Boron, Manganese, Fluoride,
Chloride and Sulfate to Several Aquatic Animal Species
Prepared for:
United States Environmental Protection Agency
Office of Science and Technology
Health and Ecological Criteria Division
and
U.S. EPA, Region 5 Water Division
EPA Contract: EP-C-09-001
Work Assignments: B-12 and 1-12
Work Assignment Managers: Tom Poleck and Brian Thompson,
U.S. EPA, Regions
Prepared by:
Great Lakes Environmental Center
739 Hastings Street
Traverse City, Michigan 49686
Contacts: Jamie Saxton and Mailee Garton
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FORWARD
The U.S. Environmental Protection Agency is charged under the Clean Water Act
(§304(a)) with periodically updating water quality criteria (WQC) to protect existing and
designated uses such as aquatic life and human health. Two States in EPA Region 5
identified the need to develop criteria for which no national guidance has been developed
to date. To support these State efforts, EPA Region 5 contracted (Contract EP-C-09-001,
Work Assignments B-12 and 1-12) with Great Lakes Environmental Center (GLEC) and
its subcontractor, the Illinois Natural History Survey (INKS), to test the acute and chronic
toxic effects of nitrate, nitrite, boron, manganese, fluoride, chloride and sulfate on several
aquatic animal species. The results of this Work Assignment are contained in this report.
Any comments on this document should be sent to the Work Assignment Manager,
Thomas Poleck at poleck.thomas@epa.gov or
U.S. EPA Region 5
Water Quality Branch (WQ-16J)
77 W. Jackson Blvd.
Chicago, IL 60604
NOTICE: This document has been reviewed in accordance with U.S. EPA policy. While
every effort was made to ensure the validity of the data in this report, any State, Tribe or
other party should verify for themselves that the data meets all appropriate data quality
requirements. For example, rearing and testing methods for Hyalella have not been
standardized and therefore must be evaluated as to appropriateness in any dataset used for
determining WQC. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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TABLE OF CONTENTS
Executive Summary ix
Introduction 1
Materials and Methods 4
Chemicals Tested and Toxicology Tests Performed 10
Nitrate 1.0
96-hr Toxicity of Nitrate onLampsilis siliquoidea 11
96-hr Toxicity of Nitrate on Megalonaias nervosa 1.5
48-hr Toxicity of Nitrate on Chironomus dilutus 19
96-hr Toxicity of Nitrate on Amphinemura delosa 23
96-hr Toxicity of Nitrate on Sphaerium simile 27
96-hr Toxicity of Nitrate on Hyalella azteca 3.1
96-hr Toxicity of Nitrate on Pimephales promelas 35
32-day Toxicity of Nitrate on Pimephales promelas 39
Nitrite 44
96-hr Toxicity of Nitrite onLampsilis siliquoidea 45
48-hr Toxicity of Nitrite on Chironomus dilutus 49
96-hr Toxicity of Nitrite on Amphinemura delosa 53
96-hr Toxicity of Nitrite on Sphaerium simile 57
96-hr Toxicity of Nitrite on Hyalella azteca 6.1
96-hr Toxicity of Nitrite on Pimephales promelas 65
32-day Toxicity of Nitrite on Pimephales promelas 69
48-hr Toxicity of Nitrite on Ceriodaphnia dubia 75
7-day Toxicity of Nitrite on Ceriodaphnia dubia 79
Boron 83
96-hr Toxicity of Boron onLampsilis siliquoidea 84
96-hr Toxicity of Boron on Megalonaias nervosa 88
96-hr Toxicity of Boron on Ligumia recta_ 92
96-hr Toxicity of Boron on Pimephales promelas 96
32-day Toxicity of Boron on Pimephales promelas 100
48-hr Toxicity of Boron (pH 7.75) on Ceriodaphnia dubia 106
96-hr Toxicity of Boron (pH 6.75) on Pimephales promelas 110
96-hr Toxicity of Boron (pH 7.75) on Pimephales promelas 115
96-hr Toxicity of Boron (pH 8.75) on Pimephales promelas 120
Manganese .125
96-hr Toxicity of Manganese onLampsilis siliquoidea 126
96-hr Toxicity of Manganese on Megalonaias nervosa 130
Fluoride 134
96-hr Toxicity of Fluoride on Sphaerium simile 135
96-hr Toxicity of Fluoride on Hyalella azteca 139
Chloride ZZZZZZZZZIS
96-hr Toxicity of Chloride on Musculium transversum 144
Sulfate 148
96-hr Toxicity of Sulfate on Ligumia recta 149
96-hr Toxicity of Sulfate on Megalonaias nervosa 153
11
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TABLE OF CONTENTS (CONT'D)
References 1.57
Appendix 1: Raw Data for 96-hr Toxicity of Nitrate on Lampsilis siliquoidea
Appendix 2: Raw Data for 96-hr Toxicity of Nitrate on Megalonaias nervosa
Appendix 3: Raw Data for 48-hr Toxicity of Nitrate on Chironomus dilutus
Appendix 4: Raw Data for 96-hr Toxicity of Nitrate on Amphinemura delosa
Appendix 5: Raw Data for 96-hr Toxicity of Nitrate on Sphaerium simile
Appendix 6: Raw Data for 96-hr Toxicity of Nitrate on Hyalella azteca
Appendix 7: Raw Data for 96-hr Toxicity of Nitrate on Pimephales promelas
Appendix 8: Raw Data for 32-day Toxicity of Nitrate on Pimephales promelas
Appendix 9: Raw Data for 96-hr Toxicity of Nitrite on Lampsilis siliquoidea
Appendix 10: Raw Data for 48-hr Toxicity of Nitrite on Chironomus dilutus
Appendix 11: Raw Data for 96-hr Toxicity of Nitrite on Amphinemura delosa
Appendix 12: Raw Data for 96-hr Toxicity of Nitrite on Sphaerium simile
Appendix 13: Raw Data for 96-hr Toxicity of Nitrite on Hyalella azteca
Appendix 14: Raw Data for 96-hr Toxicity of Nitrite on Pimephales promelas
Appendix 15: Raw Data for 32-day Toxicity of Nitrite on Pimephales promelas
Appendix 16: Raw Data for 48-hr Toxicity of Nitrite on Ceriodaphnia dubia
Appendix 17: Raw Data for 7-day Toxicity of Nitrite on Ceriodaphnia dubia
Appendix 18: Raw Data for 96-hr Toxicity of Boron on Lampsilis siliquoidea
Appendix 19: Raw Data for 96-hr Toxicity of Boron on Megalonaias nervosa
Appendix 20: Raw Data for 96-hr Toxicity of Boron on Ligumia recta
Appendix 21: Raw Data for 96-hr Toxicity of Boron on Pimephales promelas
Appendix 22: Raw Data for 32-day Toxicity of Boron on Pimephales promelas
Appendix 23: Raw Data for 48-hr Toxicity of Boron (pH 7.75) on Ceriodaphnia dubia
Appendix 24: Raw Data for 96-hr Toxicity of Boron (pH 6.75) on Pimephales promelas
Appendix 25: Raw Data for 96-hr Toxicity of Boron (pH 7.75) on Pimephales promelas
Appendix 26: Raw Data for 96-hr Toxicity of Boron (pH 8.75) on Pimephales promelas
Appendix 27: Raw Data for 96-hr Toxicity of Manganese on Lampsilis siliquoidea
Appendix 28: Raw Data for 96-hr Toxicity of Manganese on Megalonaias nervosa
Appendix 29: Raw Data for 96-hr Toxicity of Fluoride on Sphaerium simile
Appendix 30: Raw Data for 96-hr Toxicity of Fluoride on Hyalella azteca
Appendix 31: Raw Data for 96-hr Toxicity of Chloride on Musculium transversum
Appendix 32: Raw Data for 96-hr Toxicity of Sulfate on Ligumia recta
Appendix 33: Raw Data for 96-hr Toxicity of Sulfate on Megalonaias nervosa
in
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LIST OF TABLES
Table 1. Experimental matrix for toxicity testing completed by GLEC and INKS.
Table 2. ASTM standard used for each toxicity test completed by GLEC and INHS.
Table 3. Chemical properties of de-chlorinated Lake Michigan (city of Traverse
City) water.
Table 4. Analytical chemistry methods for each toxicity test completed by GLEC
and INHS.
Table 5. LCso estimates for toxicity tests performed using nitrate.
Table 6. Test conditions for 96-hour toxicity test on Lampsilis siliquoidea with
nitrate.
Table 7. Test results for 96-hour toxicity test on Lampsilis siliquoidea with nitrate.
Table 8. Analytical chemistry data for 96-hour toxicity test on Lampsilis
siliquoidea with nitrate.
Table 9. Test conditions for 96-hour toxicity test on Megalonaias nervosa with
nitrate.
Table 10. Test results for 96-hour toxicity test on Megalonaias nervosa with nitrate.
Table 11. Analytical chemistry data for 96-hour toxicity test on Megalonaias
nervosa with nitrate.
Table 12. Test conditions for 48-hour toxicity test on Chironomus dilutus with
nitrate.
Table 13. Test results for 48-hour toxicity test on Chironomus dilutus with nitrate.
Table 14. Analytical chemistry data for 48-hour toxicity test on Chironomus dilutus
with nitrate.
Table 15. Test conditions for 96-hour toxicity test on Amphinemura delosa with
nitrate.
Table 16. Test results for 96-hour toxicity test on Amphinemura delosa with nitrate.
Table 17. Analytical chemistry data for 96-hour toxicity test on Amphinemura
delosa with nitrate.
Table 18. Test conditions for 96-hour toxicity test on Sphaerium simile with nitrate.
Table 19. Test results for 96-hour toxicity test on Sphaerium simile with nitrate.
Table 20. Analytical chemistry data for 96-hour toxicity test on Sphaerium simile
with nitrate.
Table 21. Test conditions for 96-hour toxicity test on Hyalella azteca with nitrate.
Table 22. Test results for 96-hour toxicity test on Hyalella azteca with nitrate.
Table 23. Analytical chemistry data for 96-hour toxicity test on Hyalella azteca with
nitrate.
Table 24. Test conditions for 96-hour toxicity test on Pimephalespromelas with
nitrate.
Table 25. Test results for 96-hour toxicity test on Pimephales promelas with nitrate.
Table 26. Analytical chemistry data for 96-hour toxicity test on Pimephales
promelas with nitrate.
Table 27. Test conditions for 32-day toxicity test on Pimephales promelas with
nitrate.
Table 28. Test results for 32-day toxicity test on Pimephales promelas with nitrate.
IV
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LIST OF TABLES (CONT'D)
Table 29. Survival and growth data for 32-day toxicity test on Pimephalespromelas
with nitrate.
Table 30. Analytical chemistry data for 32-day toxicity test on Pimephales promelas
with nitrate.
Table 31. LCso estimates for toxicity tests performed using nitrite.Table 55. Test
conditions for 48-hour toxicity test on Ceriodaphnia dubia with nitrite.
Table 32. Test conditions for 96-hour toxicity test on Lampsilis siliquoidea with
nitrite.
Table 33. Test results for 96-hour toxicity test on Lampsilis siliquoidea with nitrite.
Table 34. Analytical chemistry data for 96-hour toxicity test on Lanpsilis siliquoidea
with nitrite.
Table 35. Test conditions for 48-hour toxicity test on Chironomus dilutus with
nitrite.
Table 36. Test results for 48-hour toxicity test on Chironomus dilutus with nitrite.
Table 37. Analytical chemistry data for 48-hour toxicity test on Chironomus dilutus
with nitrite.
Table 38. Test conditions for 96-hour toxicity test on Amphinemura delosa with
nitrite.
Table 39. Test results for 96-hour toxicity test on Amphinemura delosa with nitrite.
Table 40. Analytical chemistry data for 96-hour toxicity test on Amphinemura
delosa with nitrite.
Table 41. Test conditions for 96-hour toxicity test on Sphaerium simile with nitrite.
Table 42. Test results for 96-hour toxicity test on Sphaerium simile with nitrite.
Table 43. Analytical chemistry data for 96-hour toxicity test on Sphaerium simile
with nitrite.
Table 44. Test conditions for 96-hour toxicity test on Hyalella azteca with nitrite.
Table 45. Test results for 96-hour toxicity test on Hyalella azteca with nitrite.
Table 46. Analytical chemistry data for 96-hour toxicity test on Hyalella azteca with
nitrite.
Table 47. Test conditions for 96-hour toxicity test on Pimephales promelas with
nitrite.
Table 48. Test results for 96-hour toxicity test on Pimephales promelas with nitrite.
Table 49. Analytical chemistry data for 96-hour toxicity test on Pimephales
promelas with nitrite.
Table 50. Test conditions for 32-day toxicity test on Pimephales promelas with
nitrite.
Table 51. Test results for 32-day toxicity test on Pimephales promelas with nitrite.
Table 52. Survival and growth data for 32-day toxicity test on Pimephales promelas
with nitrite.
Table 53. Analytical chemistry data for 32-day toxicity test on Pimephales promelas
with nitrite.
Table 54. Test conditions for 48-hour toxicity test on Ceriodaphnia dubia with
nitrite.
Table 55. Test results for 48-hour toxicity test on Ceriodaphnia dubia with nitrite.
v
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LIST OF TABLES (CONT'D)
Table 56. Analytical chemistry data for 48-hour toxicity test on Ceriodaphnia dubia
with nitrite.
Table 57. Test conditions for 7-day toxicity test on Ceriodaphnia dubia with nitrite.
Table 58. Test results for 7-day toxicity test on Ceriodaphnia dubia with nitrite.
Table 59. Analytical chemistry data for 7-day toxicity test on Ceriodaphnia dubia
with nitrite.
Table 60. LCso estimates for toxicity tests performed using boron.
Table 61. Test conditions for 96-hour toxicity test on Lampsilis siliquoidea with
boron.
Table 62. Test results for 96-hour toxicity test on Lampsilis siliquoidea with boron.
Table 63. Analytical chemistry data for 96-hour toxicity test on Lampsilis
siliquoidea with boron.
Table 64. Test conditions for 96-hour toxicity test on Megalonaias nervosa with
boron.
Table 65. Test results for 96-hour toxicity test on Megalonaias nervosa with boron.
Table 66. Analytical chemistry data for 96-hour toxicity test on Megalonaias
nervosa with boron.
Table 67. Test conditions for 96-hour toxicity test on Ligumia recta with boron.
Table 68. Test results for 96-hour toxicity test on Ligumia recta with boron.
Table 69. Analytical chemistry data for 96-hour toxicity test on Ligumia recta with
boron.
Table 70. Test conditions for 96-hour toxicity test on Pimephalespromelas with
boron.
Table 71. Test results for 96-hour toxicity test on Pimepehalespromelas with boron.
Table 72. Analytical chemistry data for 96-hour toxicity test on Pimephales
promelas with boron.
Table 73. Test conditions for 32-day toxicity test on Pimephales promelas with
boron.
Table 74. Test results for 32-day toxicity test on Pimepehales promelas with boron.
Table 75. Survival and growth data for 32-day toxicity test on Pimephales promelas
with boron.
Table 76. Analytical chemistry data for 32-day toxicity test on Pimephales promelas
with boron.
Table 77. Test conditions for 48-hour toxicity test on Ceriodaphnia dubia with
boron (pH 7.75).
Table 78. Test results for 48-hour toxicity test on Ceriodaphnia dubia with boron at
pH7.75.
Table 79. Analytical chemistry data for 48-hour toxicity test on Ceriodaphnia dubia
with boron at pH 7.75.
Table 80. Test conditions for 96-hour toxicity test on Pimephales promelas with
boron (pH 6.75).
Table 81. Test results for 96-hour toxicity test on Pimephales promelas with boron
at pH 6.75.
VI
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LIST OF TABLES (CONT'D)
Table 82. Analytical chemistry data for 96-hour toxicity test on Pimephales
promelas with boron at pH 6.75.
Table 83. Test conditions for 96-hour toxicity test on Pimephales promelas with
boron (pH 7.75).
Table 84. Test results for 96-hour toxicity test on Pimephales promelas with boron
at pH 7.75.
Table 85. Analytical chemistry data for 96-hour toxicity test on Pimephales
promelas with boron at pH 7.75.
Table 86. Test conditions for 96-hour toxicity test on Pimephales promelas with
boron (pH 8.75).
Table 87. Test results for 96-hour toxicity test on Pimephales promelas with boron
at pH 8.75.
Table 88. Analytical chemistry data for 96-hour toxicity test on Pimephales
promelas with boron at pH 8.75.
Table 89. LCso estimates for toxicity tests performed using manganese.
Table 90. Test conditions for 96-hour toxicity test on Lampsilis siliquoidea with
manganese.
Table 91. Test results for 96-hour toxicity test on Lampsilis siliquoidea with
manganese.
Table 92. Analytical chemistry data for 96-hour toxicity test on Lampsilis
siliquoidea with manganese.
Table 93. Test conditions for 96-hour toxicity test on Megalonaias nervosa with
manganese.
Table 94. Test results for 96-hour toxicity test on Megalonaias nervosa with
manganese.
Table 95. Analytical chemistry data for 96-hour toxicity test on Megalonaias
nervosa with manganese.
Table 96. LCso estimates for toxicity tests performed using fluoride.
Table 97. Test conditions for 96-hour toxicity test on Sphaerium simile with
fluoride.
Table 98. Test results for 96-hour toxicity test on Sphaerium simile with fluoride.
Table 99. Analytical chemistry data for 96-hour toxicity test Sphaerium simile with
fluoride.
Table 100. Test conditions for 96-hour toxicity test on Hyalella azteca with fluoride.
Table 101. Test results for 96-hour toxicity test on Hyalella azteca with fluoride.
Table 102. Analytical chemistry data for 96-hour toxicity test on Hyalella azteca with
fluoride.
Table 103. LCso estimate for toxicity test performed using chloride.
Table 104. Test conditions for 96-hour toxicity test on Musculium transversum with
chloride.
Table 105. Test results for 96-hour toxicity test on Musculium transversum with
chloride.
Table 106. Analytical chemistry data for 96-hour toxicity test on Musculium
transversum with chloride.
vn
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LIST OF TABLES (CONT'D)
Table 107. LCso estimates for toxicity tests performed using sulfate.
Table 108. Test conditions for 96-hour toxicity test on Ligumia recta with sulfate.
Table 109. Test results for 96-hour toxicity test on Ligumia recta with sulfate.
Table 110. Analytical chemistry data for 96-hour toxicity test on Ligumia recta with
sulfate.
Table 111. Test conditions for 96-hour toxicity test on Ligumia recta with sulfate.
Table 112. Test results for 96-hour toxicity test on Megalonaias nervosa with sulfate.
Table 113. Analytical chemistry data for 96-hour toxicity test on Megalonaias
nervosa with sulfate.
Vlll
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EXECUTIVE SUMMARY
EPA guidance (EPA 1985) requires toxicity data from eight taxonomic groups, at a
minimum, to derive a fully-supported water quality criterion. Two States in Region 5 are
in the process of developing criteria for nitrate, boron, manganese and fluoride. This
project was designed to fill existing data gaps by conducting toxicity testing on additional
aquatic species for which data are absent from the literature. In addition, EPA Region 5
included several tests on nitrite, chloride and sulfate to meet other data needs and to
support ongoing consultations with the U.S. Fish and Wildlife Service under the
Endangered Species Act.
Specifically, the toxicity tests that were conducted are listed below along with their
resultant lethal and effective concentrations.
A. Nitrate
1. Lampsilis siliquoidea (96-hour acute test)
[LC50 = 357mg/L]
2. Megalonaias nervosa (96-hour acute test)
[LC50 = 937 mg/L]
3. Chironomus dilutus (48-hour acute test)
[LC50 = 278 mg/L]
4. Amphinemura delosa (96-hour acute test)
[LC50 = 456 mg/L]
5. Sphaerium simile (96-hours acute test)
[LC50 = 371 mg/L]
6. Hyalella azteca (96-hour acute test)
[LC50= 16.4 mg/L]
7. Pimephalespromelas (96-hour acute test)
[LC50 = 415mg/L]
8. Pimephales promelas (32-day chronic test)
[Survival LC50 = 76.8 mg/L, LC25 = 68.2 mg/L, LC20 = 64.6 mg/L,
LCio = 55.5 mg/L, NOEC = 49 mg/L, LOEC = 109 mg/L]
[Growth EC50 = 91.3 mg/L, EC25 = 65.3 mg/L, EC20 = 59.8 mg/L,
ECio = 46.7 mg/L, NOEC = 49 mg/L, LOEC = 109 mg/L]
B. Nitrite
9. Lampsilis siliquoidea (96-hour acute test)
[LC50= 177 mg/L]
10. Chironomus dilutus (48-hour acute test)
[LC50= 15.6 mg/L]
11. Amphinemura delosa (96-hour acute test)
[LC50= 1.0 mg/L]
12. Sphaerium simile (96-hours acute test)
[LC50 = 55.7 mg/L]
13. Hyalella azteca (96-hour acute test)
[LC50= 1.5 mg/L]
IX
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14. Pimephalespromelas (96-hour acute test)
[LC50 = 27.0 mg/L]
15. Pimephales promelas (32-day chronic test)
[Survival LC50 = 11.3 mg/L, LC25 = 7.0 mg/L, LC20 = 5.7 mg/L,
LCio = 2.3 mg/L, NOEC = 6.8 mg/L, LOEC = 15.0 mg/L]
[Growth EC50 = 14.6 mg/L, EC25 = 11.7 mg/L, EC2o = 11.0 mg/L,
ECio = 9.3 mg/L, NOEC = 6.8 mg/L, LOEC = 15.0 mg/L]
16. Ceriodaphnia dubia (48-hour acute test)
[LC50 = 4.6 mg/L]
17. Ceriodaphnia dubia (7-day chronic test)
[Survival LC50 = 4.7 mg/L, LC25 = 3.5 mg/L, LC20 = 3.3 mg/L,
LCio = 2.5 mg/L, NOEC = 3.6 mg/L, LOEC = 7.3 mg/L]
[Reproduction EC50 = 2.4 mg/L, EC25 = 1.9 mg/L, EC20 = 1.7
mg/L, ECio = 1.4 mg/L, NOEC = 0.82 mg/L, LOEC = 1.8 mg/L]
C. Boron
18. Lampsilis siliquoidea (96-hour acute test)
[LC50= 137 mg/L]
19. Megalonaias nervosa (96-hour acute test)
[LC50 = >544 mg/L]
20. Ligumia recta (96-hour acute test)
[LC50 = 147 mg/L]
21. Pimephales promelas (96-hour acute test)
[LC50= 101 mg/L]
22. Pimephales promelas (32-day chronic test)
[Survival LC50 = 28.4 mg/L, LC25 = 20.8 mg/L, LC20 = 18.6 mg/L,
LCio = 12.8 mg/L, NOEC = 12.9 mg/L, LOEC = 27.4 mg/L]
[Growth EC50 = 28.7 mg/L, EC25 = 25.1 mg/L, EC20 = 24.2 mg/L,
ECio = 21.9 mg/L, NOEC = <5.9 mg/L, LOEC = 5.9 mg/L]
23. Ceriodaphnia dubia (48-hour acute test at pH 7.75)
[LC50 = 76.9 mg/L]
24. Pimephales promelas (96-hour acute test at pH 6.75)
[LC50 = 70.6 mg/L]
25. Pimephales promelas (96-hour acute test at pH 7.75)
[LC50= 137 mg/L]
26. Pimephales promelas (96-hour acute test at pH 8.75 )
[LC50= 133 mg/L]
D. Manganese
27. Lampsilis siliquoidea (96-hour acute test)
[LC5o = 43.3mg/L]
28. Megalonaias nervosa (96-hour acute test)
[LC5o = 31.5mg/L]
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E. Fluoride
29. Sphaerium simile (96-hours acute test)
[LC50 = 62.0 mg/L]
30. Hyalella azteca (96-hour acute test)
[LC5o=13.4mg/L]
F. Chloride
31. Musculium tramversum (96-hours acute test)
[LC50= 1,930 mg/L]
G. Sulfate
32. Ligumia recta (96-hour acute test)
[LC50= 1,483 mg/L]
33. Megalonaias nervosa (96-hour acute test)
[LC50 = 3,378 mg/L]
XI
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INTRODUCTION
There are no federal water quality criteria for the protection of aquatic life for nitrate,
nitrite, boron, manganese and fluoride. States in Region 5 of the United States
Environmental Protection Agency (EPA), particularly the states of Minnesota and
Illinois, are in the process of revising or developing water quality criteria for each of
these parameters, with the exception of nitrite. In addition, EPA recently completed a
review of the available data and undertook a draft revision of the chloride criterion.
Nitrate and Nitrite
Biological field data generated by the State of Minnesota, as well as laboratory-derived
toxicity test data, suggest that nitrate at concentrations similar to those observed in the
surface waters of Minnesota may cause adverse effects to aquatic life. Much of the nitrate
toxicity literature focuses on the effects of nitrate, especially the effects of wastes
associated with fish cultures. More recent literature reports have documented that
amphibians and invertebrates appear to be more sensitive to nitrate than fish.
The state of Minnesota is evaluating the need to develop a nitrate criterion to protect
aquatic life as part of its current triennial Water Quality Standards (WQS) review. Efforts
by Minnesota to develop aquatic life criteria for nitrate have involved a review of all the
pertinent literature to date. The primary sources were the ECOTOX database, as well as a
comprehensive search of the open literature. The scientific studies were each evaluated
based on methodologies in the 1985 guidance of EPA (EPA 1985), and produced two
observations: 1. there is a wide variation in response among aquatic organisms to nitrate
toxicity; and 2. the research has documented the toxicity of nitrate for some aquatic
organisms at environmentally relevant concentrations. In addition, the literature review
found seven acceptable studies that reported acute toxicity values for eleven different
species. Invertebrates (especially aquatic insects) were among the most sensitive taxa.
Associated chronic values from these studies were found to be acceptable for three
separate species, one each for a fish, an amphibian and a cladoceran. Unfortunately, these
data are insufficient to develop Tier I aquatic life criteria for nitrate. Acceptable toxicity
data are lacking for two required taxonomic categories: a benthic invertebrate and one
other invertebrate.
Additional nitrite acute toxicity tests are also required to complete the data set for
calculating a water quality criterion consistent with EPA's 1985 guidelines. Acute tests
with an amphipod (e.g., Hyalella azteca) and a chironomid (e.g., Chironomus dilutus)
would best serve this purpose. In addition, completing acute and chronic (early life stage)
tests with the fathead minnow (Pimephalespromelas) would augment existing data for
fish, and add an additional acute to chronic ratio for calculating a chronic criterion.
Within Region 5, field data have shown elevated levels of nitrate. Given the relationship
between nitrate and nitrite, the potential for nitrite to exist in the aquatic environment
under certain circumstances, the toxic effects of nitrite, and the lack of nitrite data for
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most organisms other than fish, EPA wanted to assess the potential need for a nitrite
criterion by generating additional nitrite toxicity data for several species of organisms.
Boron, Manganese and Fluoride
The state of Illinois is in the process of revising its aquatic life criteria for boron,
manganese and fluoride. The Illinois EPA conducted a literature review and identified the
need for acute and chronic (early life stage) tests with P. promelas to add an additional
acute to chronic ratio for calculating a chronic criterion for boron. Additionally, recent
toxicity data suggest that pH affects the toxicity of boron, which is consistent with the
fact that borate exists in water as a pH-dependent mixture of various borate ions.
Therefore, EPA wanted to assess the effects of pH on the toxicity of boron on
Ceriodaphnia dubia and P. promelas.
For fluoride, the data needs include acute and chronic tests on Hyalella, in order to
develop an additional acute to chronic ratio with an organism other than Pimephales or
Daphnia. The available acute data for an amphipod also need to be supplemented with
tests conducted using a range of test water hardness levels. In order to facilitate the
review of these criteria, and to provide additional support for the database, EPA also
wanted to generate freshwater mussel toxicity data.
Chloride
EPA recently reviewed and developed additional toxicity data for chloride. As part of the
review of that data and subsequent development of a draft revision of the criteria, EPA
determined that additional toxicity information on resident fingernail clams (other than
those from the genus Sphaerium) is desirable. To facilitate the acquisition of these data,
EPA is interested in additional toxicity tests on other resident fingernail clams, such as
Musculium sp.
Sulfate
Additional sulfate toxicity data are required to enhance EPA's basis for making a
determination under the Endangered Species Act (ESA) that the recently adopted sulfate
criteria by Illinois and Indiana are not likely to adversely affect threatened and
endangered species. EPA Region 5 is currently consulting under Section 7 of the ESA on
the Agency's approval of the Illinois and Indiana surface water quality standards. Data on
the sensitivity of mussels are limited and mussels are often one of the most sensitive
species to adverse water quality conditions. Therefore, EPA wanted to generate sulfate
toxicity data for two mussel species (Ligumia recta and Megalonaias nervosa).
Because of the data needs outlined above, Great Lakes Environmental Center (GLEC)
and its subcontractor the Illinois Natural History Survey (INKS) were contracted by the
EPA to augment existing toxicity data through a series of toxicity tests. This document
presents in detail the materials and methods by which GLEC and INKS performed these
-------
toxicity tests with nitrate, nitrite, boron, manganese, fluoride, sulfate and chloride. Also
included are the results of the toxicity tests, including % survival and mortality, growth
and reproduction (where appropriate), water chemistry data, statistical analyses
performed on the data and toxicology endpoint estimates.
-------
MATERIALS AND METHODS
GLEC and INHS performed a total of 33 acute and chronic toxicity tests (Table 1). Tests
on C. dubia, C. dilutus, H. azteca and P. promelas were completed by GLEC; tests on all
other species were completed by INKS. Eight of these tests were designed to determine
acute and/or chronic toxicity of the chemical nitrate to seven different species of aquatic
organisms; nine additional tests were designed to determine acute and/or chronic toxicity
of the chemical nitrite to seven different species of aquatic organisms. Five different
species of aquatic organisms were exposed to boron under acute and/or chronic
conditions during nine separate toxicity tests while two aquatic organisms were exposed
to manganese under acute conditions. Acute toxicity was determined for fluoride,
chloride and sulfate using one of four species of mussels and H. azteca.
Toxicity testing under this Work Assignment was accomplished using the methods
outlined in the ASTM standards (Table 2), GLEC and INHS standard operating
procedures (GLEC 2009 a, GLEC 2009 b, GLEC 2009 c, GLEC 2009 d, GLEC 2009 e,
GLEC 2009 f, GLEC 2009 g and INHS 2009) and the Quality Assurance Project Plan
(GLEC 2009) developed for this Work Assignment. Prior to completing the toxicity test
for each test chemical/organism combination, range-finding toxicity tests were initiated
to determine the appropriate test chemical concentrations that would "bracket" the LCso
estimates. The range of concentrations for these tests was determined either from the
literature or from laboratory experiences with similar organisms. Once the appropriate
range in test chemical concentrations was defined for each test chemical/organism
combination, definitive toxicity testing was completed.
Dilution water used for the tests consisted of either EPA Moderately Hard Reconstituted
Water (MHRW) (EPA 2002a and EPA 2002 b), EPA Dilute Mineral Water (DMW)
(EPA 2002 a and EPA 2002 b) or de-chlorinated Lake Michigan water (City of Traverse
City, Michigan water passed through an activated carbon filter). The chemical properties
of the de-chlorinated Lake Michigan water are presented in Table 3; these data were
generated both before and after the P. promelas flow-through test with boron at pH 8.75.
Water quality parameters (water temperature, pH, dissolved oxygen (DO), conductivity,
hardness, alkalinity and ammonia) were measured throughout the tests at pre-determined,
test-specific intervals. Water temperature, pH, DO and conductivity were measured in
situ using calibrated meters. Hardness, alkalinity and ammonia were measured in water
collected from test chambers using methods outlined in SM 2340 C (APHA et al. 2006
a), SM 2320 B (APHA et al. 2006 b) and SM 4500-NH3 (APHA et al. 2006 c),
respectively. Test concentrations of the target test chemicals were also verified using the
analytical chemistry methods outlined in Table 4 and laboratory-specific standard
operating procedures (GLEC 2008, GLEC 2009 h, Simmerman 2008, UL 2008 a, UL
2008 b, UL 2009 a and UL 2009 b). For toxicity testing completed by GLEC, target test
chemicals were verified by the GLEC analytical chemistry laboratory (nitrate and nitrite)
or SOS Analytical (boron and fluoride). Target test chemical sample analysis for toxicity
testing completed by INHS was completed by the INHS analytical laboratory (nitrate and
nitrite) or Underwriters Laboratories (boron, fluoride, chloride, sulfate and manganese).
-------
Once the toxicity tests were complete, statistical analysis of the mortality (and where
appropriate in chronic tests, growth or reproduction) data was completed following
guidance outlined in EPA (2002 a), EPA (2002 b) and the ASTM methods (Table 2).
Specifically, data were analyzed using either TOXSTAT software (Gulley 1996) or
EPA's Toxicity Relationship Analysis Program (TRAP, Version 1.00). Where
appropriate, data were transformed to meet the assumptions of normality and
homogeneity of variance. Analysis of variance (ANOVA), parametric (e.g. Probit) or
non-parametric (e.g. Spearman-Karber) tests and mathematical models (TRAP) were then
used to determine appropriate toxicology endpoints, including: LCso.LCso, LC25, LC20
and LCio estimates; ECso, £€25, EC20, ECio estimates for growth or reproduction; and No
Observed Effects Concentration (NOEC) and Lowest Observed Effects Concentration
(LOEC) estimates.
-------
Table 1. Experimental matrix for toxicity testing completed by GLEC and INKS.
Test Species and Duration
Ceriodaphnia dubia - 48 hr
Lampsilis siliquoidea 96 hr
Chironomus dilutus - 48 hr
Amphinemura delosa - 96 hr
Sphaerium simile - 96 hr
Hyalella azteca - 96 hr
Pimephales promelas - 96 hr
Pimephales promelas - 32 day
Ceriodaphnia dubia - 1 day
Ceriodaphnia dubia - 48 hr (pH 7.75)
Pimephales promelas - 96 hr (pH 6.75)
Pimephales promelas - 96 hr (pH 7.75)
Pimephales promelas - 96 hr (pH 8.75)
Ligumia recta - 96 hr
Megalonaias nervosa - 96 hr
Nitrate
X
X
X
X
X
X
X
X
Nitrite
X
X
X
X
X
X
X
X
X
Boron
X
X
X
X
X
X
X
X
X
Manganese
X
X
Test Species and Duration
Sphaerium simile - 96 hr
Hyalella azteca - 96 hr
Musculium transversum - 96 hr
Ligumia recta - 96 hr
Megalonaias nervosa - 96 hr
Fluoride
X
X
Chloride
X
Sulfate
X
X
-------
Table 2. ASTM standard used for each toxicity test completed by GLEC and INKS.
Test Species and Duration
Ceriodaphnia dubia - 48 hr
Lampsilis siliquoidea 96 hr
Chironomus dilutus - 48 hr
Amphinemura delosa - 96 hr
Sphaerium simile - 96 hr
Hyalella azteca - 96 hr
Pimephales promelas - 96 hr
Pimephales promelas - 32 day
Ceriodaphnia dubia - 7 day
Ceriodaphnia dubia - 48 hr (pH 7.75)
Pimephales promelas - 96 hr (pH 6.75)
Pimephales promelas - 96 hr (pH 7.75)
Pimephales promelas - 96 hr (pH 8.75)
Ligumia recta - 96 hr
Megalonaias nervosa - 96 hr
Nitrate
ASTM 2455-06
ASTM E729
ASTM E729
ASTM E729
ASTM E729
ASTM E729
ASTME1241
ASTM 2455-06
Nitrite
ASTME729
ASTM 2455-06
ASTM E729
ASTM E729
ASTM E729
ASTM E729
ASTM E729
ASTM El 241
ASTM El 295
Boron
ASTM 2455 -06
ASTM E729
ASTM El 241
ASTM E729
ASTM E729
ASTM E729
ASTM E729
ASTM 2455 -06
ASTM 2455 -06
Test Species and Duration
Sphaerium simile - 96 hr
Lampsilis siliquoidea 96 hr
Hyalella azteca - 96 hr
Musculium transversum - 96 hr
Megalonaias nervosa - 96 hr
Manganese
ASTM 2455-06
ASTM 2455-06
Fluoride
ASTM E729
ASTM E729
Chloride
,
ASTM E729
Test Species and Duration
Ligumia recta - 96 hr
Megalonaias nervosa - 96 hr
Sulfate
ASTM 2455-06
ASTM 2455-06
-------
Table 3. Chemical properties of de-chlorinated Lake Michigan (city of Traverse
City) water.
Parameter
Total Aluminum
Dissolved Aluminum
Total Antimony
Total Arsenic
Total Barium
Total Beryllium
Total Cadmium
Total Calcium
Chloride *
Total Chromium
Total Cobalt
Total Copper
Total Iron
Total Lead
Total Magnesium
Total Manganese
Total Mercury
Total Nickel
Total Potassium
Total Selenium
Total Silver
Total Sodium
Sulfate *
Total Thallium
Total Vanadium
Total Zinc
Level of Detection
(mg/L)
0.05
0.05
0.002
0.002
0.05
0.001
0.001
0.1
1
0.005
0.02
0.004
0.05
0.002
5.0
0.05
0.0005
0.05
0.1
0.005
0.001
0.5
2
0.002
0.004
0.05
2/12/2010 Result
(mg/L)
ND
ND
ND
0.003
ND
ND
ND
42.6
14
ND
ND
ND
ND
ND
13.7
ND
ND
ND
1.42
0.006
ND
8.83
29
ND
ND
ND
2/16/2010 Result
(mg/L)
0.05
ND
ND
ND
ND
ND
ND
32.8
ND
ND
ND
ND
ND
12.3
ND
ND
ND
1.33
ND
ND
7.75
ND
ND
ND
ND = Below level of detection.
* Sample collected on November 11, 2010.
-------
Table 4. Analytical chemistry methods for each toxicity test completed by GLEC
and INKS.
Test Species and Duration
Ceriodaphnia dubia - 48 hr
Lampsilis siliquoidea 96 hr
Chironomus dilutus - 48 hr
Amphinemura delosa - 96 hr
Sphaerium simile - 96 hr
Hyalella azteca - 96 hr
Pimephales promelas - 96 hr
Pimephales promelas - 32 day
Ceriodaphnia dubia - 1 day
Megalonaias nervosa - 96 hr
Nitrate
EPA 353.1 and EPA 354.1
SM 4500 NO3 and SM 4500 NO2
EPA 353.1 and EPA 354.1
EPA 353.1 and EPA 354.1
SM 4500 N03 and SM 4500 NO2
SM 4500 NO3 and SM 4500 NO2
SM 4500 N03 and SM 4500 NO2
EPA 353.1 and EPA 354.1
Nitrite
SM 4500 NO3 and SM 4500 NO2
EPA 353.1 and EPA 354.1
SM 4500 NO3 and SM 4500 NO2
EPA 353.1 and EPA 354.1
EPA 353.1 and EPA 354.1
SM 4500 N03 and SM 4500 NO2
SM 4500 NO3 and SM 4500 NO2
SM 4500 N03 and SM 4500 NO2
SM 4500 NO3 and SM 4500 NO2
Test Species and Duration
Ceriodaphnia dubia - 48 hr
Lampsilis siliquoidea 96 hr
Pimephales promelas - 96 hr
Pimephales promelas - 32 day
Ceriodaphnia dubia - 48 hr (pH 7.75)
Pimephales promelas - 96 hr (pH 6.75)
Pimephales promelas - 96 hr (pH 7.75)
Pimephales promelas - 96 hr (pH 8.75)
Ligumia recta - 96 hr
Megalonaias nervosa - 96 hr
Boron
ICPMS 200.8
EPA 200.8
EPA 200.8
EPA 200. 8
EPA 200.8
EPA 200.8
EPA 200. 8
ICPMS 200.8
ICPMS 200.8
Manganese
ICPMS 200.7
ICPMS 200.7
Test Species and Duration
Sphaerium simile - 96 hr
Hyalella azteca - 96 hr
Musculium transversum - 96 hr
Fluoride
EPA 300.0
EPA 300.0
Chloride
EPA 300.0
Test Species and Duration
Ligumia recta - 96 hr
Megalonaias nervosa - 96 hr
Sulfate
EPA 300.0
EPA 300.0
-------
CHEMICALS TESTED AND TOXICITY TESTS PERFORMED
Nitrate
Table 5 provides a summary of estimated LCso values for the eight toxicity tests
performed using nitrate. LCso values ranged between 16.4 and 937 mg NOs-N/L.
Table 5. LCso estimates for toxicity tests performed using nitrate.
Test Species and Duration
Lampsilis siliquoidea 96 hr
Chironomus dilutus - 48 hr
Amphinemura delosa - 96 hr
Sphaerium simile - 96 hr
Hyalella azteca - 96 hr
Pimephales promelas - 96 hr
Pimephales promelas - 32 day
Megalonaias nervosa - 96 hr
LC50(mgNO
3-N/L)
357
278
456
371
16.4
415
76.8
937
For each of the acute toxicity tests completed using nitrate, two tables were generated:
the first summarizes the test results for each toxicity test, including nominal and
analytical test concentration and LCso estimates with confidence intervals; the second
table summarizes analytical chemistry data collected throughout the toxicity tests. The
results of chronic tests performed with nitrate were summarized in three tables: the first
summarizes nominal and analytical test concentrations, LCso estimates with confidence
intervals, NOEC and LOEC estimates, mean survival and mean biomass; the second table
summarizes replicate-specific survival and growth data and the third table summarizes
analytical chemistry data collected throughout the toxicity tests. Also discussed, if
applicable, are deviations from the guidance provided in the ASTM method used to
complete the toxicity testing.
Nitrate and nitrite concentration data generated by the GLEC analytical chemistry
laboratory were reported as nitrate (mg NOs/L) and nitrite (mg NCh/L). For consistency
(i.e. so that the data correspond to the nitrate and nitrogen data generated by the
analytical chemistry laboratory used by the INKS), the GLEC data were converted to
nitrate-nitrogen (N-NCVL) and nitrite-nitrogen (N-NCVL) for LCso determinations and
reporting purposes. The data as reported by the GLEC analytical laboratory are available
in the appendices.
10
-------
96-hr Toxicity of Nitrate on Lampsilis siliquoidea
The 96-hr test to determine the toxicity of nitrate on L. siliquoidea was completed by
INHS. Test organisms, < 5-day old juveniles collected from the Missouri State University
laboratory culture, were acclimated to the dilution water (MHRW), test temperature and
other test conditions prior to test initiation. Once acclimated, test organisms were
examined for any disease, stress, parasites, etc. If free from ailments, test organisms were
randomly assigned to the test chambers (which were randomly assigned to testing
locations); four replicates were used per treatment with five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 1,129, 564, 282, 141, and 70
mg N-NO3/L.
Testing was conducted at 20 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 6; test results are provided in Table 7. Analytical chemistry data are provided in
Table 8. Accompanying information, including raw laboratory data, analytical chemistry
data and statistical analyses, is provided in Appendix 1.
11
-------
Table 6. Test conditions for 96-hour toxicity test on Lampsilis siliquoidea with
nitrate.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg N-NO3/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination- mg
N-N03/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Lampsilis siliquoidea, juveniles <5 days old, Missouri State
University
Static, 96 hours
June 03 - 07, 2009
20 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
50 mL beaker
40 mL
5
4
20
1,129, 564,282, 141, and 70
1,144, 580, 291, 143, and 74
None
USEPA MHRW
Sodium Nitrate: Sigma Aldrich, > 99.0%, ACS Reagent,
Cas. No. 7631-99-4, Lot # B0110978
None
None
Mortality (LC50)
12
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Table 7. Test results for 96-hour toxicity test on Lampsttis siliquoidea with nitrate.
Results of a Lampsilis siliauoidea 96-Hour Static Acute Toxicitv Test
Conducted 06/03/09 - 06/07/09 Using: Sodium Nitrate: Sigma Aldrich Cas. No. 7631-99-4
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
70 (74) mg/L
141(143)mg/L
2 82 (291) mg/L
564 (580) mg/L
1129 (1144) mg/L
Cumulative Percent Affected a
24-Hr
5
0
0
5
15
85
48-Hr
10
0
5
10
30
100
72-Hr
10
15
5
20
55
100
96-Hr
10
25
25
35
75
100
LC50 Values* (mg/L)
24-Hr 48-Hr
72-Hr 96-Hr
815 598 491 357
96-Hour LC50* = 357 mg/L
LC50 95% Confidence Limits
24-Hr 48-Hr
LL 730 498
UL909 717
72-Hr 96-Hr
383 250
631 509
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 Confidence
Limit Values: Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ and EC™ values are determined based on measured concentrations.
13
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Table 8. Analytical chemistry data for 96-hour toxicity test on Lampsttis siliquoidea
with nitrate.
Nominal (Measured) Test
Concentration
N-NO2" N-NO3"'b
(mg/L) (mg/L)
Temperature pH D.O. Conductivity Alkalinity Hardness
(°C) (s.u.) (mg/L) (umhos) (mg/L) (mg/L)
Dilution water/Control
70 (74) mg/L
141 (143) mg/L
282 (291) mg/L
564 (580) mg/L
1129 (1144) mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
na
73
75
74
144
142
148
143
283
299
291
582
579
580
1158
1131
1144
20.1
19.9
20.0
20.1
19.9
20.1
19.9
20.0
20.0
19.9
20.1
19.8
20.1
20.1
19.9
20.0
19.8
20.0
20.1
19.9
20.0
19.8
20.0
20.0
20.0
20.0
19.9
20.0
20.1
19.9
8.0
8.0
7.9
8.0
7.9
8.0
7.9
8.0
7.9
7.9
7.9
7.9
7.81
7.77
8.01
7.82
8.00
7.85
8.05
7.72
8.10
7.86
8.12
7.81
305
310
912
920
1484
1500
2610
2620
4760
4770
8720
8720
60
62
60
62
60
62
60
62
60
62
60
62
90
92
90
92
90
92
90
92
90
92
90
92
"Nitrite Analysis Method 354.1
"Nitrite + Nitrate Analysis Method 353.1
na = not applicable
14
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96-hr Toxicity of Nitrate on Megalonaias nervosa
The 96-hr test to determine the toxicity of nitrate onM nervosa was completed by INHS.
Test organisms, < 5-day old juveniles collected from the Genoa National Fish Hatchery
culture, were acclimated to the dilution water (MHRW), test temperature and other test
conditions prior to test initiation. Once acclimated, test organisms were examined for any
disease, stress, parasites, etc. If free from ailments, test organisms were randomly
assigned to the test chambers (which were randomly assigned to testing locations); four
replicates were used per treatment with five organisms per replicate. In one replicate of
the 282 mg/L treatment, a test organism was inadvertently crushed, but this was
accounted for in the LCso calculation.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 2,258, 1,129, 564, 282, and
141mgN-NO3/L.
Testing was conducted at 20 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 9; test results are provided in Table 10. Analytical chemistry data are provided in
Table 11. Accompanying information, including raw laboratory data, analytical chemistry
data and statistical analyses, is provided in Appendix 2.
15
-------
Table 9. Test conditions for 96-hour toxicity test on Megalonaias nervosa with
nitrate.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg N-NO3/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination- N-
NO3/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Megalonaias nervosa, juveniles <5 days old (Genoa
National Fish Hatchery)
Static, 96 hours
October 23 - 27, 2009
20 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
50 mL beaker
40 mL
5
4
20
2,258, 1,129, 564, 282, and 141
2,002, 1,108, 533, 279, and 138
None
USEPA MHRW
Sodium Nitrate: Sigma Aldrich, > 99.0%, ACS Reagent,
Cas. No. 7631-99-4, Lot # B0110978
None
None
Mortality (LC50)
16
-------
Table 10. Test results for 96-hour toxicity test on Megalonaias nervosa with nitrate.
Results of a Mesalonaias nervosa 96-Hour Static Acute Toxicitv Test
Conducted 06/03/09 - 06/07/09 Using: Sodium Nitrate: Sigma Aldrich Cas. No. 7631-99-4
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
141(138)mg/L
282 (279) mg/L
564 (533) mg/L
1129 (1108) mg/L
2258 (2002) mg/L
Cumulative Percent Affected a
24-Hr
5
0
0
0
40
90
48-Hr
5
0
0
0
55
100
72-Hr
5
0
0
0
65
100
96-Hr
5
0
0
0
70
100
LCSO Values* (mg/L)
24-Hr
1,203
48-Hr 72-
Hr 96-Hr
1,035 969 937
96-Hour LC5n* = 937 mg/L
LC50 95% Confidence Limits
24-Hr
LL 991
UL 1,460
48-Hr 72-
Hr 96-Hr
893 841 818
1,199 1,116 1,073
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not
Method(s) Used to Determine LC5
Limit Values: Spearman-Karber
reliable
o Confidence
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ and EC™ values are determined based on measured concentrations.
17
-------
Table 11. Analytical chemistry data for 96-hour toxicity test on Megalonaias nervosa
with nitrate.
Nominal (Measured) Test
Concentration
Dilution water/Control
141 (138)mg/L
282 (279) mg/L
564 (533) mg/L
112 (1108) mg/L
225 8 (2002) mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
N-NO2" N-NO3"'" Temp. pH D.O. Cond. Alkalinity Hardness Ammonia
(mg/L) (mg/L) (°C) (s.u.) (mg/L) (inmos) (mg/L) (mg/L) (mg/L)
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.07
<0.07
na
132.6
143.4
138
275.6
283.4
279
543.3
523.7
533
1189.4
1032.8
1108
2011.8
1992.2
2002
20.9
20.9
20.8
20.9
20.8
20.8
20.9
20.8
20.9
20.9
20.8
20.9
20.8
20.9
20.9
20.9
20.9
20.8
20.9
20.9
20.9
20.9
20.8
20.9
20.9
20.9
20.9
20.8
20.9
20.9
8.0
8.0
8.1
8.0
8.0
8.2
8.1
8.1
7.8
8.0
8.2
8.1
8.1
7.9
8.0
8.2
8.1
8.1
7.9
8.0
8.1
8.1
8.0
8.0
8.0
8.1
8.1
8.0
8.0
8.0
8.34
8.13
8.26
8.36
8.08
8.34
8.06
8.34
8.34
7.92
8.35
8.15
8.25
8.34
7.95
8.35
8.08
8.16
8.32
7.92
8.39
8.15
8.24
8.30
7.96
8.43
8.16
8.16
8.25
7.90
303
304
306
305
315
1454
1421
1383
1370
1361
2550
2520
2470
2480
2480
4680
4610
4560
4560
4610
8710
8650
8530
8440
8540
16240
16000
15690
15810
15910
60
60
60
60
60
62
60
62
60
62
60
64
90
92
90
90
90
90
90
92
90
92
90
92
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
"Nitrite Analysis Method 354.1
"Nitrite + Nitrate Analysis Method 353.1
na = not applicable
temp. = temperature; Cond. = conductivity
18
-------
48-hr Toxicity of Nitrate on Chironomus dilutus
The 48-hr test to determine the toxicity of nitrate on C. dilutus was completed by GLEC.
Test organisms, 10-days old collected from the GLEC laboratory culture, were
acclimated to the dilution water (MHRW), test temperature and other test conditions prior
to test initiation. Once acclimated, test organisms were examined for any disease, stress,
parasites, etc. If free from ailments, test organisms were randomly assigned to the test
chambers (which were randomly assigned to testing locations); four replicates were used
per treatment with five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 88, 146, 244, 406, and 678
mg N-NO3/L.
Testing was conducted at 22 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Probit method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 12; test results are provided in Table 13. Analytical chemistry data are provided
in Table 14. Accompanying information, including raw laboratory data, analytical
chemistry data, reference toxicant data and statistical analyses, is provided in Appendix
3.
19
-------
Table 12. Test conditions for 48-hour toxicity test on Chironomus dilutus with
nitrate.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg N-
N03/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination- mg N-
N03/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Chironomus dilutus, 10 days old, GLEC culture
Static, 48 hours
July 23-July 25, 2009
22 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
150 mL beaker
100 mL
5
4
20
678, 406, 244, 146, and 88
621,372,255, 166, and 91
None
USEPA MHRW
Sodium Nitrate: Sigma Aldrich, > 99.0%, ACS Reagent
Cas. No. 7631-99-4, Batch # 66696LJ
None
None
Mortality (LC50)
20
-------
Table 13. Test results for 48-hour toxicity test on Chironomus dilutus with nitrate.
Results of a Chironomus dilutus (tentans) 48-Hour Static Acute Toxicitv Test
Conducted 07/23/09 - 07/25/09 Using: Nitrate (Sodium Nitrate: Sigma Aldrich Cas. No. 7631-99-4)
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
88 (91) mg/L
146(166)mg/L
244 (255) mg/L
407 (372) mg/L
678 (621) mg/L
Cumulative Percent Affected a
24-Hr
0
(0)
0
(0)
0
(0)
15
(15)
20
(20)
25
(25)
48-Hr
0
(0)
0
(0)
15
(15)
45
(45)
65
(65)
100
(100)
72-Hr
96-Hr
LCSO* Values (mg/L)
24-Hr 48-Hr
72-Hr 96-Hr
>621 278 NA NA
48-Hour LC50* = 278 mg/L
LC50* 95% Confidence Limits
24-Hr 48-Hr
LL NA 239
UL NA 325
72-Hr 96-Hr
NA NA
NA NA
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 Confidence
Limit Values: Probit
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ values are determined based on the measured concentrations.
21
-------
Table 14. Analytical chemistry data for 48-hour toxicity test on Chironomus dilutus
with nitrate.
Nominal (Measured) Test
Concentration
Dilution
water/Control
DayO
Day1
Day 2
Nitrate"
(mg NO3-N/L)
<0.0012
<0.0012
<0.0012
0.0
Nitrite9
(mg NO2-N/L)
<0.0011
0.0084
Temperature
(°C)
22.3
21.3
22.4
PH
(s.u.)
7.98
7.94
7.84
D.O.
(mg/L)
7.4
7.7
7.6
Conductivity
(mmhos)
292
290
Alkalinity
(mg/L)
60
90
Hardness
(mg/L)
84
124
Ammonia
(mg/L)
ND
88 (91)mg/L
DayO
Day1
Day 2
83.6
98.3
90.9
0.0011
0.0150
22.1
21.3
22.4
7.99
7.90
7.87
7.5
7.7
7.7
854
860
146(166) mg/L
DayO
Day1
Day 2
187.9
143.7
165.8
<0.0011
0.0072
22.1
21.3
22.3
7.98
7.90
7.89
7.6
7.7
7.5
1368
1304
244(255) mg/L
DayO
Day1
Day 2
255.3
255.9
255.6
0.0016
0.0013
0.0110
22.6
21.4
22.3
7.95
7.86
7.94
7.6
7.7
7.5
2030
2000
407 (372) mg/L
DayO
Day1
Day 2
325.3
419.0
372.1
0.0024
0.0112
22.7
21.4
22.3
7.96
7.85
7.93
7.7
7.8
7.7
2860
2740
678(621) mg/L
DayO
Day1
Day 2
607.6
634.7
621.2
0.0025
0.0173
22.2
21.4
22.3
7.95
7.8
7.93
7.9
8.0
7.5
4900
4870
60
64
120
136
ND
" Nitrite Analysis Method SM 4500 NO2
" Nitrate Analysis Method SM 4500 NO3
ND Not Detect; below detection limit
22
-------
96-hr Toxicity of Nitrate on Amphinemura delosa
The 96-hr test to determine the toxicity of nitrate on A delosa was completed by INKS.
Test organisms, field collected nymphs, were acclimated to the dilution water (MHRW),
test temperature and other test conditions prior to test initiation. Once acclimated, test
organisms were examined for any disease, stress, parasites, etc. If free from ailments, test
organisms were randomly assigned to the test chambers (which were randomly assigned
to testing locations); four replicates were used per treatment with four organisms per
replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 2,258, 1,129, 564, 282, and
141mgN-NO3/L.
Testing was conducted at 12 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 15; test results are provided in Table 16. Analytical chemistry data are provided
in Table 17. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 4.
23
-------
Table 15. Test conditions for 96-hour toxicity test on Amphinemura delosa with
nitrate.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg N-NO3/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination- mg
N-NO3/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Amphinemura delosa, field-collected nymphs
Static, 96 hours
May 14- 18,2009
12 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
250 mL beaker
200 mL
4
4
16
2,258, 1,129, 564, 282, and 141
2,286, 1,230, 596, 274, and 152
None
USPEA MHRW
Sodium Nitrate: Sigma Aldrich, > 99.0%, ACS Reagent,
Cas. No. 7631-99-4, Lot # B0110978
None
None
Mortality (LC50)
24
-------
Table 16. Test results for 96-hour toxicity test on Amphinemura delosa with nitrate.
Results of a Amphinemura delosa 96-Hour Static Acute Toxicitv Test
Conducted 05/14/09 - 05/18/09 Using: Nitrate (Sodium Nitrate: Sigma Aldrich Cas. No. 7631-99-4)
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
141(152)mg/L
282 (274) mg/L
564 (596) mg/L
1 129 (1230) mg/L
2258 (2286) mg/L
Cumulative Percent Affected a
24-Hr
0
12.5
6.25
6.25
25
68.75
48-Hr
0
12.5
31.25
37.5
81.25
100
72-Hr
0
12.5
37.5
43.75
87.5
100
96-Hr
0
12.5
37.5
50
93.75
100
LC50 Values* (mg/L)
24-Hr
48-Hr 72-
Hr 96-Hr
1753 584 499 456
96-Hour LC50* = 456 mg/L
LC50 95% Confidence Limits
24-Hr
LL 1394
UL2204
48-Hr 72-
Hr 96-Hr
409 352 325
834 706 642
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not
reliable
Method(s) Used to Determine LC50 Confidence
Limit Values: Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC50 and EC50 values are determined based on measured concentrations.
25
-------
Table 17. Analytical chemistry data for 96-hour toxicity test on Amphinemura delosa
with nitrate.
Nominal (Measured) Test
Concentration
N-NO2" N-NO3"'b
(mg/L) (mg/L)
Temperature pH D.O. Conductivity Alkalinity Hardness
(°C) (s.u.) (mg/L) (umhos) (mg/L) (mg/L)
Dilution water/Control
141 (152) mg/L
282 (274) mg/L
564 (596) mg/L
1129 (1230) mg/L
225 8 (2286) mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
<0.08
<0.07
<0.08
<0.08
<0.08
<0.07
<0.08
<0.07
<0.08
<0.07
<0.08
<0.07
<0.08
<0.07
na
161
143
152
274
274
274
599
593
596
1260
1200
1230
2200
2375
2286
12.5
12.3
12.4
12.5
12.8
12.5
12.4
12.4
12.5
12.8
12.5
12.3
12.4
12.5
12.8
12.5
11.9
12.4
12.5
12.8
12.5
11.9
12.4
12.5
12.8
12.5
12.0
12.4
12.5
12.8
7.9
7.9
7.9
7.8
7.8
8.0
7.9
7.9
7.9
7.9
8.0
7.9
7.9
7.9
7.9
8.0
7.9
7.9
7.9
7.9
8.0
7.9
7.9
7.9
7.9
7.9
7.9
7.9
7.9
7.9
8.80
9.85
8.80
9.57
9.09
9.92
9.09
9.93
9.11
9.92
9.08
9.97
298
297
297
300
298
1440
1455
1458
1460
1441
2540
2560
2560
2560
2540
4690
4690
4700
4710
4650
8720
8770
8780
8790
8670
16260
16270
16270
16280
16230
60
60
60
60
60
62
60
60
60
62
60
60
90
88
92
92
92
92
92
92
92
92
92
92
* Nitrite Analysis Method 354.1
"Nitrite + Nitrate Analysis Method 353.1
na = not applicable
26
-------
96-hr Toxicity of Nitrate on Sphaerium simile
The 96-hr test to determine the toxicity of nitrate on S. simile was completed by INHS.
Test organisms, juveniles released from field-collected adults, were acclimated to the
dilution water (MHRW), test temperature and other test conditions prior to test initiation.
Once acclimated, test organisms were examined for any disease, stress, parasites, etc. If
free from ailments, test organisms were randomly assigned to the test chambers (which
were randomly assigned to testing locations); four replicates were used per treatment with
five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 1,129, 564, 282, 141, and 71
mg N-NO3/L.
Testing was conducted at 22 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 18; test results are provided in Table 19. Analytical chemistry data are provided
in Table 20. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 5.
27
-------
Table 18. Test conditions for 96-hour toxicity test on Sphaerium simile with nitrate.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg N-NO3/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination- mg
N-NO3/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Sphaerium simile, juveniles (released from field-collected
adults)
Static, 96 hours
July 21-25, 2009
22 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
150 mL beaker
120 mL
5
4
20
1,129, 564,282, 141, and 71
1,125, 569,272, 132, and 65
None
USPEA MHRW
Sodium Nitrate: Sigma Aldrich, > 99.0%, ACS Reagent,
Cas. No. 7631-99-4, Lot # B0110978
None
None
Mortality (LC50)
28
-------
Table 19. Test results for 96-hour toxicity test on Sphaerium simile with nitrate.
Results of a Sphaerium simile 96-Hour Static Acute Toxicitv Test
Conducted 07/21/09 - 07/25/09 Using: Sodium Nitrate: Sigma Aldrich Cas. No
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
7 1(65) mg/L
141 (132) mg/L
282 (272) mg/L
564 (569) mg/L
1129 (1125) mg/L
Cumulative Percent Affected a
24-Hr
0
0
0
0
0
0
48-Hr
0
0
0
0
40
85
72-Hr
0
5
5
5
100
100
96-Hr
0
5
10
5
100
100
7631-99-4
LC50 Values* (mg/L)
24-Hr
48-Hr 72-
Hr 96-Hr
>1125 652 386 371
96-Hour LC50* = 371 mg/L
LC50 95% Confidence Limits
24-Hr
LLNR
ULNR
48-Hr 72-
Hr 96-Hr
508 NR 323
838 NR 426
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not
Method(s) Used to Determine LC5
Limit Values: Spearman-Karber
reliable
o Confidence
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ and EC™ values are determined based on measured concentrations.
29
-------
Table 20. Analytical chemistry data for 96-hour toxicity test on Sphaerium simile
with nitrate.
Nominal (Measured) Test
Concentration
N-NO2" N-NO3"'b
(mg/L) (mg/L)
Temperature pH D.O. Conductivity Alkalinity Hardness
(°C) (s.u.) (mg/L) (umhos) (mg/L) (mg/L)
Dilution water/Control
71 (65) mg/L
141 (132) mg/L
282 (272) mg/L
564 (569) mg/L
1129 (1125) mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
-------
96-hr Toxicity of Nitrate on Hyalella azteca
The 96-hr test to determine the toxicity of nitrate on H. azteca was completed by GLEC.
H. azteca were collected from GLEC's laboratory culture. These organisms are
maintained in 10 gallon glass aquaria; plastic artificial turf and screen mesh serve as a
substrate for the culture. The tanks are filled with de-chlorinated Lake Michigan water
(City of Traverse City, Michigan water passed through an activated carbon filter).
Cultures are fed 50 mL of 4 g/L Tetrafin slurry daily. When visible algae are not
observed within the glass aquaria, algae (Selenastrum sp.) are used as a supplement to the
Tetrafin slurry. Additionally, on occasion, dried Aspen (Populus sp.) leaves are prepared
as a food supplement. The culture is maintained in a 16-hour light: 8-hour dark
photoperiod at a temperature between 23 and 26 °C.
Test organisms were acclimated to the dilution water (MHRW), test temperature and
other test conditions prior to test initiation. Once acclimated, test organisms were
examined for any disease, stress, parasites, etc. If free from ailments, test organisms were
randomly assigned to the test chambers (which were randomly assigned to testing
locations); four replicates were used per treatment with five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 1.8, 2.9, 4.9, 8.1, 13.6, and
22.6 mg N-NO3/L.
Testing was conducted at 22 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso was with the Probit
(48 and 72-hour) and Trimmed Spearman-Karber (96-hour) methods.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 21; test results are provided in Table 22. Analytical chemistry data are provided
in Table 23. Accompanying information, including raw laboratory data, analytical
chemistry data, reference toxicant data and statistical analyses, is provided in Appendix
6.
31
-------
Table 21. Test conditions for 96-hour toxicity test on Hyalella azteca with nitrate.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg N-
NO3/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination- mg N-
NO3/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Hyalella azteca, 10 days old, GLEC culture
Static, 96 hours
August 6-August 10,2009
22 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
150 mL beaker
100 mL
5
4
20
22.6,13.6, 8.1, 4.9, 2.9, and 1.8
25.8, 15.2,9.1,5.7, 3.2, and 2.1
None
USPEA MHRW
Sodium Nitrate: Sigma Aldrich, > 99.0%, ACS Reagent
Cas. No. 7631-99-4, Batch # 66696LJ
None
None
Mortality (LC50)
32
-------
Table 22. Test results for 96-hour toxicity test on Hyalella azteca with nitrate.
Results of a Hvalella azteca 96-Hour Static Acute Toxicitv Test
Conducted 08/06/09 - 08/10/09 Using: Nitrate (Sodium Nitrate: Sigma Aldrich Cas. No. 7631-99-4)
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
1.8(2.1)mg/L
2.9 (3.2) mg/L
4.9 (5.7) mg/L
8. 1(9.1) mg/L
13.6 (15.2) mg/L
22.6 (25.8) mg/L
Cumulative Percent Affected a
24-Hr
0
(0)
0
(0)
0
(0)
0
(5)
0
(10)
10
(15)
30
(60)
48-Hr
0
(0)
10
(10)
0
(0)
0
(0)
15
(15)
10
(10)
50
(75)
72-Hr
0
(0)
10
(10)
5
(5)
0
(0)
15
(15)
20
(20)
65
(65)
96-Hr
0
(0)
10
(10)
10
(10)
15
(15)
20
(20)
25
(25)
90
(90)
LCSO *Values (mg/L)
24-Hr 48-Hr 72-Hr 96-Hr
>25.8 28 23 16.4
96-Hour LC50* = 16.4 mg/L
LCSO 95% Confidence Limits
24-Hr 48-Hr 72-Hr 96-Hr
LL NA 20.7 17.3 13.3
ULNA 56.2 38.9 20.0
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 Confidence
Limit Values: Trimmed Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ values are determined based on measured concentrations.
33
-------
Table 23. Analytical chemistry data for 96-hour toxicity test on Hyalella azteca with
nitrate.
Nominal (and M
Test Concent
Dilution
water/Control
easured)
rations
DayO
Day1
Day 2
Day3
Day 4
Nitrate"
(mg N03-N/L)
<0.0011
0.0443
0.0560
0.050
Nitrite"
(mg N02-N/L)
<0.0011
0.0201
0.0208
Temperature
(°C)
22.0
22.0
22.7
22.3
21.9
PH
(S.U.)
8.00
7.98
8.26
8.07
7.89
D.O.
(mg/L)
8.4
8.5
8.2
8.2
8.0
Conductivity
(mmhos)
300
290
Alkalinity
(mg/L)
60
60
Hardness
(mg/L)
80
80
Ammonia
(mg/L)
ND
1.8 (2.1) mg/L
DayO
Day1
Day 2
Day3
Day 4
2.13
2.16
2.15
2.1
<0.0011
0.0062
22.0
22.0
22.7
22.5
22.1
8.06
7.99
8.25
8.02
7.84
8.4
8.6
8.7
8.2
8.0
352
327
2.9 (3.2) mg/L
DayO
Day 1
Day 2
DayS
Day 4
2.93
3.40
3.2
<0.0011
0.0067
22.0
22.1
22.7
22.8
22.2
8.08
7.95
8.10
8.04
7.84
8.4
8.6
8.7
8.3
8.0
362
330
4.9 (5.7) mg/L
DayO
Day1
Day 2
DayS
Day 4
5.68
5.77
5.7
<0.0036
0.0051
22.0
22.1
22.7
22.9
22.2
8.10
7.97
8.09
8.09
7.80
8.4
8.6
8.8
8.5
8.0
377
356
8.1 (9.1) mg/L
DayO
Day1
Day 2
DavS
Day 4
9.76
8.52
9.1
<0.0011
0.0062
22.0
22.1
22.7
22.9
22.2
8.07
8.00
8.08
8.11
7.80
8.4
8.6
8.9
8.7
8.0
402
390
13.6(15.2) mg/L
DayO
Day 1
Day 2
DayS
Day 4
15.52
14.89
15.2
<0.0011
0.4104
22.0
22.1
22.7
22.9
22.2
8.07
8.02
8.09
8.07
7.80
8.4
8.5
9.0
8.7
8.0
443
400
22 6 (25. 8) mg/L
DayO
Day1
Day 2
DayS
Day 4
27.45
24.22
25.8
<0.0011
0.0133
22.0
22.1
22.7
22.9
22.1
8.04
8.01
8.06
8.06
7.84
8.4
8.5
9.0
8.9
8.0
513
500
60
60
80
84
ND
a Nitrite Analysis Method SM 4500 NO2
" Nitrate Analysis Method SM 4500 NO3
ND Not Detect; below detection limit
34
-------
96-hr Toxicity of Nitrate on Pimephales promelas
The 96-hr test to determine the toxicity of nitrate on P. promelas was completed by
GLEC. Test organisms, collected from the GLEC laboratory culture, were acclimated to
the dilution water (de-chlorinated Lake Michigan water), test temperature and other test
conditions prior to test initiation. Once acclimated, test organisms were examined for any
disease, stress, parasites, etc. If free from ailments, test organisms were randomly
assigned to the test chambers (which were randomly assigned to testing locations); two
replicates were used per treatment with ten organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 87.9, 146, 244, 407 and 678
mg N-NO3/L.
Testing was conducted at 25 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Probit method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 24; test results are provided in Table 25. Analytical chemistry data are provided
in Table 26. Accompanying information, including raw laboratory data, analytical
chemistry data, reference toxicant data and statistical analyses, is provided in Appendix
7.
35
-------
Table 10. Test conditions for 96-hour toxicity test on Pimephales promelas with
nitrate.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg N-
NO3/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination- mg N-
NO3/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Pimephales promelas, (weight 0.11 g and length 16 mm),
GLEC culture
Static, 96 hours
August 6-August 10,2009
25 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
4000 mL beaker
3500 mL
10
2
20
678, 407, 244, 146, and 87.9
720, 406, 262, 160, and 98
None
De-chlorinated Lake Michigan Water
Sodium Nitrate: Sigma Aldrich, > 99.0%, ACS Reagent
Cas. No. 7631-99-4, Batch # 66696LJ
None
None
Mortality (LC50)
36
-------
Table 25. Test results for 96-hour toxicity test on Pimephales promelas with nitrate.
Results of a Pimephales promelas 96-Hour Static Acute Toxicitv Test
Conducted 08/06/09 - 08/10/09 Using: Nitrate (Sodium Nitrate: Sigma Aldrich Cas. No. 7631-99-4)
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
88 (98) mg/L
146(160)mg/L
244 (262) mg/L
407 (406) mg/L
678 (720) mg/L
Cumulative Percent Affected a
24-Hr
0
(0)
0
(0)
0
(0)
0
(0)
0
(0)
35
(35)
48-Hr
0
(0)
0
(0)
0
(0)
20
(20)
20
(20)
65
(65)
72-Hr
0
(0)
0
(0)
0
(0)
20
(20)
25
(25)
70
(70)
96-Hr
0
(0)
0
(0)
5
(5)
25
(25)
50
(50)
80
(80)
LCSO *Values (mg/L)
24-Hr 48-Hr 72-Hr 96-Hr
>720 580 536 415
96-Hour LC50* = 415 mg/L
LC50* 95% Confidence Limits
24-Hr 48-Hr 72-Hr 96-Hr
LL NA 465 437 343
ULNA 841 730 526
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 Confidence
Limit Values: Probit
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ values are determined based on measured concentrations.
37
-------
Table 26. Analytical chemistry data for 96-hour toxicity test on Pimephales promelas
with nitrate.
Nominal (and IVI
Test Concen
Dilution
water/Control
easured)
ration
DayO
Day 1
Day 2
Day3
Day 4
Nitrate"
(mg NO3-N/L)
0.2528
0.2460
0.2345
0.2444
Nitrite1
(mg NO2-N/L)
<0.0011
0.0028
Temperature
(°C)
25
24.6
24.3
24.4
24.4
24.4
pH
(s.u.)
8.04
7.71
7.81
8.28
8.00
8.04
D.O.
(mg/L)
8.4
5.9
6.6
6.7
7.0
8.4
6.0
7.4
6.9
7.0
Conductivity
(mmhos)
310
302
Alkalinity
(mg/L)
96
100
Hardness
(mg/L)
136
140
Ammonia
(mg/L)
ND
88 (98) mg/L
DayO
Day 1
Day 2
Day3
Day 4
97.5830
97.5830
97.5830
<0.0011
0.0113
25.0
24.6
24.2
24.3
24.4
24.3
8.10
7.68
7.77
8.11
8.12
8.14
8.4
5.9
7.7
6.7
6.8
8.4
5.7
6.3
6.9
7.0
1030
979
146(160) mg/L
DayO
Day 1
Day 2
DayS
Day 4
1 62. 1 866
156.3135
160.6054
159.7018
<0.0011
0.0218
0.0205
25.0
24.6
24.2
24.4
24.3
24.4
8.09
7.68
7.75
8.11
8.10
8.14
8.4
5.8
7.9
6.9
6.6
8.4
5.8
6.6
6.8
6.7
1483
1429
244 (262) mg/L
DayO
Day 1
Day 2
DayS
Day 4
266.7721
256.3813
261.5767
0.0026
0.0883
25.0
24.6
24.3
24.4
24.4
24.4
8.07
7.68
7.65
8.10
8.05
8.03
8.4
5.8
5.9
6.5
6.7
8.4
5.7
5.4
6.4
6.9
2241
2170
407 (406) mg/L
DayO
Dav1
Day 2
DayS
Day 4
410.2101
402.5299
406.3700
0.0026
0.1096
25.0
24.6
24.4
24.3
24.4
24.4
8.07
7.57
7.47
8.00
7.94
8.02
8.4
5.9
6.7
6.2
6.4
8.4
5.8
6.0
6.0
6.6
3361
3250
678 (720) mg/L
DayO
Day 1
Day 2
DavS
Day 4
754.0095
685.5658
719.6747
<0.0011
<0.0011
0.1428
25.0
24.6
24.4
24.3
24.4
24.4
8.05
7.47
7.42
7.88
8.03
7.94
8.4
5.9
5.9
6.6
6.5
8.4
5.6
5.9
6.7
6.6
5550
5240
100
104
136
140
ND
1 Nitrite Analysis Method SM 4500 NO2
b Nitrate Analysis Method SM 4500 NO3
ND Not Detect; below detection limit
38
-------
32-day Toxicity of Nitrate on Pimephales promelas
The 32-day test to determine the toxicity of nitrate on P. promelas was completed by
GLEC. The fish were continuously exposed for 32 days to five concentrations of nitrate
(nominal concentrations of 28.2, 56.5, 113, 226 and 452 mg N-NCVL) and to a dilution
water control using a continuous flow-through system (Benoit et al. 1982). The
temperature-controlled test concentration solutions were supplied to each test chamber
via the continuous flow-through system at a rate of approximately four turnovers a day.
There were four replicate test chambers for each treatment. The flow through test was
conducted at 25 ± 1 °C with a photoperiod of 16 hr light and 8 hr darkness (ambient
laboratory light).
After test concentrations had achieved steady state in the flow through system, the test
was initiated with < 24 hour old fertilized embryos. The embryos were randomly
assigned to incubation cups until each incubation cup contained 30 embryos. The
incubation cups were randomly assigned to the 2.5 L glass test chambers (1 cup per
chamber) and suspended in the test solutions from a rocker arm assembly. The rocker
arm assembly moves the incubation cups in a reciprocal motion within each test chamber.
Embryos were inspected on a daily basis and the number of live, hatched and dead
embryos was recorded. On Day 5 of the test (three days after first hatch), the surviving
fish were randomly thinned to achieve 20 fish in each test chamber. The remainder of the
surviving fish was discarded. The number of surviving fish was recorded at test
termination (32 days). In addition, the wet weights were recorded for each fish at test
termination. Because of the size range offish in each test chamber, all of the fish from
each test chamber were weighed together to determine average dry weight.
There were several instances throughout the test when instantaneous water temperature
measurements made within individual replicates exceeded those allowed in the toxicity
testing method. However, incidences of temperature exceedance only occurred
intermittingly and the average water temperatures (across the duration of the test) in each
replicate were always ± 0.4 °C of the target test temperature (25 °C) in all treatments. In
addition, the water temperature as recorded by the continuous temperature logger was
never outside the range outlined in the method. Therefore, these water temperature
exceedances likely had no effect on the results of this toxicity test.
In addition, there was an instance of low DO (4.3 mg/L) in one of the replicates at test
termination. Because this incident occurred on the last day of the test and similarly low
DO concentrations were never observed on any other day of the test, this low DO was an
anomaly and likely had no effect on the results of this toxicity test.
Once the test was complete, the LCso, NOEC, and LOEC values were determined using
the average measured concentrations with the Spearman Karber and ANOVA methods.
LC25, LC2o, LCio, ECso, £€25, EC20 and ECio values were estimated using EPA's TRAP.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 27; test results are provided in Table 28. Survival and growth data are provided
39
-------
in Table 29 and analytical chemistry data are provided in Table 30. Accompanying
information, including raw laboratory data, analytical chemistry data and statistical
analyses, is provided in Appendix 8.
Table 27. Test conditions for 32-day toxicity test on Pimephales promelas with
nitrate.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg N-
N03/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination- mg N-
N03/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Pimephales promelas, (<24 hour fertilized embryos ),
GLEC culture
Continuous flow-through; 32 days
July 24-August 25,2009
25 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
Live Brine Shrimp (Artemia nauplii) Twice daily
2.5 Liter glass Tank
2,000 mL
30 eggs, thinned to 20 larvae after hatch
4
120 eggs, thinned to 80 larvae after hatch
452,226, 113, 56.5, and 28.2
441,226, 109,48.6, and 21.7
Continuous flow through, 4 turnovers per day
De-chlorinated Lake Michigan Water
Sodium Nitrate: Sigma Aldrich, > 99.0%, ACS Reagent
Cas. No. 7631-99-4, Batch # 66696LJ
None
None
Survival (LC50, LC25)LC20 and LC10,NOEC and LOEC) and
Growth (EC50, EC25> EC20 and EC10, NOEC and LOEC)
40
-------
Table 28. Test results for 32-day toxicity test on Pimephales promelas with nitrate.
Results of a Pimephales promelas 32-Dav Continuous Flow Chronic Toxicitv Test
Conducted 07/24/09 - 08/25/09
Usins: Nitrate (Sodium Nitrate: Sigma Aldrich Cas. No. 7631-99-4)
Test Solution Concentrations
Measured
Embryo Percent Hatch (%)
32-Day Mean Survival (%)
32-Day Average Biomass1 (mg)
Primary Control/
Dilution Water
100
88.8
5.44
22 mg/L
100
86.3
5.72
49 mg/L
100
92.5
5.54
109 mg/L
99
16.3a
1.91"
226 mg/L
98
10.0a
0.84a
441 mg/L
99
Oa
Oa
Embryo Percent Hatch NOEC:
32-Day LC50*:
32-Day Survival NOEC:
32-Day Survival LOEC:
32-Day Growth NOEC:
32-Day Growth LOEC:
32-Day LC25:
441mg/L 32-Day LC20:
76.8 mg/L (70 mg/L-84 mg/L) 32-Day LC10:
49 mg/L 32-Day EC50:
109 mg/L 32-Day EC25:
49 mg/L 32-Day EC20:
109 mg/L 32-Day EC10:
68.2 mg/L (48.8 mg/L - 95.4 mg/L)
64.6 mg/L (44.7 mg/L - 93.5 mg/L)
55.5 mg/L (34.7 mg/L - 88.7 mg/L)
91.3 mg/L (72.9 mg/L - 114.3 mg/L)
65.3 mg/L (44.4 mg/L - 96.1 mg/L)
59.8 mg/L (38.7 mg/L - 92.4 mg/L)
46.7 mg/L (26.2 mg/L - 83.4 mg/L)
a: Significantly different from dilution water control (p< 0.05)
*: All LC, EC, NOEC and LOEC values are determined based on the average measured nitrate concentration.
NOEC: No-Observed-Effect-Concentration
LOEC: Lowest-Observed-Effect-Concentration
'Biomass: Biomass is the average dry weight of the four replicates calculated by the total dry weight of surviving
organisms divided by the initial number of organisms (20).
41
-------
Table 29. Survival and growth data for 32-day toxicity test on Pimephales promelas
with nitrate.
Nominal (and Measured) Test
Concentration
Dilution
water/Control
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
Number of
Eggs at Test
Initiation
30
30
30
30
Number of
Hatched
Larvae
30
30
30
30
Number of
Dead Eggs
0
0
0
0
Percent
Hatched
Larvae
100.0
100.0
100.0
100.0
Number of Larvae
at Test
Termination
18
18
20
15
Percent Survival at Test
Termination*
90.0
90.0
100.0
75.0
Biomass1 (mg)
5.34
5.94
5.65
4.79
Average 100.0 88.8 5.43
28 (22) mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
30
30
30
30
0
0
0
0
100.0
100.0
100.0
100.0
18
15
18
18
90.0
75.0
90.0
90.0
6.62
5.67
5.34
5.26
Average 100.0 86.3 5.72
56 (49) mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
30
30
30
30
0
0
0
0
100.0
100.0
100.0
100.0
19
20
20
15
95.0
100.0
100.0
75.0
5.45
5.98
5.25
5.46
Average 100.0 92.5 5.54
11 3 (109) mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
30
29
30
30
0
1
0
0
100.0
96.7
100.0
100.0
3
4
4
2
15.0
20.0
20.0
10.0
1.78
1.84
2.79
1.25
Average 99.2 16.3 1.91
226 (226) mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
29
30
29
30
1
0
1
0
96.7
100.0
96.7
100.0
1
4
2
1
5.0
20.0
10.0
5.0
0.20
2.02
1.04
0.10
Average 98.3 10.0 0.84
452 (441) mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
29
30
30
30
1
0
0
0
96.7
100.0
100.0
100.0
0
0
0
0
0.0
0.0
0.0
0.0
0.00
0.00
0.00
0.00
Average 99.2 0.0 0.00
* On Day 5 of the test, (three days after first hatch) the surviving fish were randomly thinned to 20 fish in each test chamber. Percent surval at test
termination is the number of surviving at test termination divided by 20.
1 Biomass: Biomass is the total dry weight of surviving organisms divided by the initial number of organisms (20)
42
-------
Table 30. Analytical chemistry data for 32-day toxicity test on Pimephales promelas
with nitrate.
Nominal (and Measured) Test
Conent rations
Dilution
Water/Control
Average
Replicate #
1
Replicate #
2
Replicate #
Replicate #
4
N03
(mg NO3-
N/L)
0.17
(0.005-0.258)
0.26
(0.221-0.307)
0.26
(0.226-0.289)
0.24
(0.210-0.271)
0.23
NO2
(mg NO2
N/L)
0.006
0.003
0.003
0.003
Temperature
(°Q
25.3
(24.0-26.3)
25.0
(24.2-26.0)
25.0
(24.1-25.6)
25.0
(24.2-26.1)
pH
(s.u.)
7.97
8.13
8.17
8.24
DO
(mg/L)
7.4
(7.2-7.8)
7.6
(7.2-8.2)
7.7
(7.3-8.2)
7.7
(7.2-8.1)
SC
(mmos)
298
296
295
296
Hardness
(mg/L)
180
Alkalinity
(mg/L)
93
Ammonia
(mg/L)
ND
ND
ND
ND
28ing/L
(22 mg/L)
Average
Replicate #
1
Replicate #
2
Replicate #
3
Replicate #
4
20
(12-40)
22
(13-41)
21
(14-41)
24
(13-41)
22
0.006
0.003
0.006
0.006
24.9
(24.0-25.8)
24.9
(24.1-26.0)
25.0
(24.2-25.7)
25.1
(24.2-25.7)
8.21
8.22
8.27
8.32
7.6
(6.7-8.4)
7.7
(7.1-8.2)
7.8
(7.3-9.2)
7.9
(7.2-9.2)
442
483
426
418
154
104
ND
ND
ND
ND
56ing/L
(49mg/L)
Average
Replicate #
1
Replicate #
2
Replicate #
Replicate #
4
48
(38-65)
48
(37-67)
51
(38-69)
47
(39-70)
49
0.009
0.006
0.006
0.006
25.0
(24.2-26.0)
24.9
(24.1-25.8)
25.1
(24.3-26.1)
25.1
(24.2-26.0)
8.23
8.22
8.16
8.23
7.6
(6.6-9.1)
7.8
(7.4-8.1)
7.6
(7.1-8.0)
7.8
(7.0-9.0)
626
689
654
667
140
107
ND
ND
ND
ND
113 mg/L
(109 mg/L)
Average
Replicate #
1
Replicate #
2
Replicate #
Replicate #
4
107
(90-146)
112
(95-141)
107
(88-137)
109
(92-139)
109
0.009
0.003
0.003
0.006
24.8
(24.0-26.0)
24.6
(23.7-26.0)
24.9
(24.2-25.6)
24.8
(23.7-25.5)
8.27
8.24
8.26
8.26
7.6
(6.8-8.2)
7.7
(7.0-8.3)
7.9
(7.4-9.4)
7.8
(7.1-9.3)
1045
1083
984
958
164
101
ND
ND
ND
ND
226 mg/L
(226 mg/L)
Average
Replicate #
1
Replicate #
2
Replicate #
3
Replicate #
4
220
(157-285)
232
(168-300)
225
(172-297)
227
(158-297)
226
0.006
0.225
0.006
0.006
24.9
(24.2-26.0)
24.7
(23.6-25.9)
25.0
(24.2-25.8)
25.0
(24.0-25.9)
8.24
8.10
8.25
8.26
7.6
(6.9-8.0)
7.2
(4.3-8.0)
7.7
(6.6-9.4)
7.8
(6.8-9.4)
2449
2206
2137
2240
132
104
ND
ND
ND
ND
452 mg/L
(441 mg/L)
Average
Replicate #
1
Replicate #
2
Replicate #
Replicate #
4
453
(389-485)
421
(341-471)
452
(411-493)
437
(411-463)
441
0.006
0.006
0.033
0.009
25.1
(24.2-26.0)
24.8
(23.9-26.0)
24.8
(24.0-25.7)
25.0
(24.1-25.8)
8.22
8.23
8.15
8.19
7.5
(6.4-8.1)
7.7
(6.9-8.3)
7.5
(6.8-9.4)
7.8
(6.2-9.4)
3820
3763
4001
3610
146
101
ND
ND
ND
ND
DO: Dissolved Oxygen
SC: Specific Conductance
ND: Not detected
43
-------
Nitrite
Table 31 provides a summary of estimated LCso values for the nine toxicity tests
performed using nitrite. LCso values ranged between 1.0 and 177 mgNOs-N/L.
Table 31. LCso estimates for toxicity tests performed using nitrite.
Test Species and Duration
Ceriodaphnia dubia - 48 hr
Lampsilis siliquoidea 96 hr
Chironomus dilutus - 48 hr
Amphinemura delosa - 96 hr
Sphaerium simile - 96 hr
Hyalella azteca - 96 hr
Pimephales promelas - 96 hr
Pimephales promelas - 3 2 day
Ceriodaphnia dubia - 1 day
LC50(mgNO
2-N/L)
4.6
177
15.6
1.0
55.7
1.5
27.0
11.3
4.7
For each of the acute toxicity tests completed using nitrite, two tables were generated: the
first summarizes the test results for each toxicity test, including nominal and analytical
test concentration and LCso estimates with confidence intervals; the second table
summarizes analytical chemistry data collected throughout the toxicity tests. The results
of chronic tests performed with nitrite were summarized in three tables: the first
summarizes nominal and analytical test concentrations, LCso estimates with confidence
intervals, NOEC and LOEC estimates, mean survival and mean biomass; the second table
summarizes replicate-specific survival and growth data and the third table summarizes
analytical chemistry data collected throughout the toxicity tests. Also discussed, if
applicable, are deviations from the guidance provided in the ASTM method used to
complete the toxicity testing.
Nitrate and nitrite concentration data generated by the GLEC analytical chemistry
laboratory were reported as nitrate (mg NOs/L) and nitrite (mg NCh/L). For consistency
(i.e. so that the data correspond to the nitrate and nitrogen data generated by the
analytical chemistry laboratory used by the INKS), the GLEC data were converted to
nitrate-nitrogen (N-NCb/L) and nitrite-nitrogen (N-NCh/L) for LCso determinations and
reporting purposes. The data as reported by the GLEC analytical laboratory are available
in the appendices.
44
-------
96-hr Toxicity of Nitrite on Lampsilis siliquoidea
The 96-hr test to determine the toxicity of nitrite on L. siliquoidea was completed by
INHS. Test organisms, < 5-day old juveniles collected from the Missouri State University
laboratory culture, were acclimated to the dilution water (MHRW), test temperature and
other test conditions prior to test initiation. Once acclimated, test organisms were
examined for any disease, stress, parasites, etc. If free from ailments, test organisms were
randomly assigned to the test chambers (which were randomly assigned to testing
locations); four replicates were used per treatment with five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 304, 152, 76, 38, 19, and 9.5
mg N-NO2/L.
Testing was conducted at 20 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 32; test results are provided in Table 33. Analytical chemistry data are provided
in Table 34. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 9.
45
-------
Table 32. Test conditions for 96-hour toxicity test on Lampsilis siliquoidea with
nitrite.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume and Depth of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg N-NO2/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination- mg
N-NO2/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Lampsilis siliquoidea, juveniles <5 days old, Missouri State
University
Static, 96 hours
September 08 - 12,2009
20 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
50 mL beaker
40 mL
5
4
20
304, 152, 76, 38, 19, and 9.5
312.8, 158.1, 82.0, 39.1, 19.6, and 9.9
None
USEPA MHRW
Sodium Nitrite: Fisher, certified ACS, assay 99.7%, Lot #
080939
None
None
Mortality (LC50)
46
-------
Table 33. Test results for 96-hour toxicity test on Lampsilis siliquoidea with nitrite.
Results of a Lanwsilis siliauoidea 96-Hour Static Acute Toxicitv Test
Conducted 09/08/09 - 09/12/09 Using: Sodium Nitrite: Sigma Aldrich Cas. No. 7632-00-0
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
9.5 (9.9) mg/L
19.0 (19.6) mg/L
38.0 (39.1) mg/L
76.0 (82.0) mg/L
152.0 (158.1) mg/L
304.0 (3 12. 8) mg/L
Cumulative Percent Affected a
24-Hr
0
0
0
0
0
0
0
48-Hr
0
0
0
0
0
0
0
72-Hr
0
0
5
0
0
10
75
96-Hr
0
10
10
0
10
30
100
LC50 Values* (mg/L)
(EC50*)
24-Hr 48-Hr
72-Hr 96-Hr
>312.8 >312.8 240.6 177.0
96-Hour LC50* = 177.0 mg/L
LC50 95% Confidence Limits
24-Hr 48-Hr
LLNR NR
ULNR NR
72-Hr 96-Hr
209.9 145.0
275.7 215.0
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 and EC50
Confidence Limit Values: Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ and EC™ values are determined based on measured concentrations.
47
-------
Table 34. Analytical chemistry data for 96-hour toxicity test on Lanpsilis siliquoidea
with nitrite.
Nominal (Measured) Test
Concentration
Dilution water/Control
9.5(9.9)mg/L
19.0 (19.6) mg/L
38.0(39.1)mg/L
76.0 (82.0) mg/L
152.0 (158.1) mg/L
304.0 (3 12.8) mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
N-NO2" N-NO3ab Temperature pH D.O. Conductivity Alkalinity Hardness
(mg/L) (mg/L) (°C) (s.u.) (mg/L) (inmos) (mg/L) (mg/L)
-------
48-hr Toxicity of Nitrite on Chironomus dilutus
The 48-hr test to determine the toxicity of nitrite on C. dilutus was completed by GLEC.
Serial dilutions. Test organisms, 10-days old from the GLEC culture, were acclimated to
the dilution water (MHRW), test temperature and other test conditions prior to test
initiation. Once acclimated, test organisms were examined for any disease, stress,
parasites, etc. If free from ailments, test organisms were randomly assigned to the test
chambers (which were randomly assigned to testing locations); four replicates were used
per treatment with five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 2.4, 3.9, 6.6, 11,18 and 30
mg NO2-N/L.
Testing was conducted at 22 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Probit method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 35; test results are provided in Table 36. Analytical chemistry data are provided
in Table 37. Accompanying information, including raw laboratory data, analytical
chemistry data, reference toxicant data and statistical analyses, is provided in Appendix
10.
49
-------
Table 35. Test conditions for 48-hour toxicity test on Chironomus dilutus with
nitrite.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg N02-
N/L):
14. Measured Test Concentrations (average of samples
collected at test initiation and termination- mg N02-
N/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Chironomus dilutus, 10 days old, GLEC culture
Static, 48 hours
July 23-July 25, 2009
22 + 1
Ambient Laboratory, 10-20 uE/m2/s
16 h light, 8 h darkness
None
150 mL beaker
100 mL
5
4
20
30, 18, 11,6.6, 3.9, and 2.4
32.4,19,11,6.8,4.0, and 2.6
None
USEPA MHRW
Sodium Nitrite: Sigma Aldrich, 97 +%, ACS Reagent Cas.
No. 7632-00-0, Batch # 08316DJ
None
None
Mortality (LC50)
50
-------
Table 36. Test results for 48-hour toxicity test on Chironomus dilutus with nitrite.
Results of a Chironomus dilutus (tentans) 48-Hour Static Acute Toxicitv Test
Conducted 07/23/09 - 07/25/09 Using: Nitrite (Sodium Nitrite: Sigma Aldrich Cas. No. 7632-00-0)
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
2.4 (2.6) mg/L
3. 9 (4.0) mg/L
6.6 (6.8) mg/L
11 (11) mg/L
18 (19) mg/L
30 (32) mg/L
Cumulative Percent Affected a
24-Hr
0
(0)
0
(0)
0
(0)
0
(0)
0
(0)
20
(20)
50
(50)
48-Hr
0
(0)
0
(0)
0
(0)
10
(10)
20
(20)
50
(50)
100
(100)
72-Hr
96-Hr
LC50 Values* (mg/L)
24-Hr 48-Hr 72-
Hr 96-Hr
31.2 15.6 NA NA
48-Hour LC50* = 15.6 mg/L
LC50 95% Confidence Limits (mg/L)
24-Hr 48-Hr 72-
Hr 96-Hr
LL 25.4 13.2 NA NA
UL47.5 18.9 NA NA
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC5
Limit Values: Probit
o Confidence
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC,n values are determined based on measured concentrations.
51
-------
Table 37. Analytical chemistry data for 48-hour toxicity test on Chironomus dilutus
with nitrite.
Nominal (and Measured)
Test Concentrations
Dilution
water/Control
DayO
Day1
Day 2
Nitrite9 Nitrate" Temperature pH D.O. Conductivity Alkalinity Hardness Ammonia
(mg NO2-N/L) (mg NO3-N/L) (°C) (s.u.) (mg/L) (mmos) (mg/L) (mg/L) (mg/L)
<0.0011
0.008
0.008
0.000
0.000
O.0012
22.3
21.4
22.4
8.04
7.78
7.80
7.4
7.5
7.7
292
290
60
90
84
124
ND
ND
2.4 (2.6) mg/L
DayO
Day1
Day 2
2.7
2.6
2.5
2.6
0.400
0.115
22.7
21.4
22.4
8.01
7.80
7.81
7.5
7.5
7.7
291
290
3.9 (4.0) mg/L
DayO
Dav1
Day 2
3.9
4.1
4.0
0.097
3.885
22.4
21.4
22.4
8.02
7.84
7.85
7.6
7.5
7.5
330
334
6.6 (6.8) mg/L
DayO
Day1
Day 2
6.7
6.5
7.1
6.8
0.700
0.339
O.1129
22.5
21.4
22.5
8.. 05
7.89
7.88
7.6
7.5
7.5
381
394
11 (11) mg/L
DayO
Day1
Day 2
10.7
10.7
10.7
0.971
0.542
22.5
21.4
22.3
8.05
7.89
7.91
7.6
7.6
7.2
436
430
18(19) mg/L
DayO
Day1
Day 2
18.3
18.9
18.6
3.275
<0.2485
22.4
21.4
22.3
8.05
7.91
7.91
7.5
7.7
7.2
478
484
30 (32) mg/L
DavO
Day1
Day 2
32.9
32.0
32.4
< 1.13
0.949
22.5
21.4
22.3
8.03
7.95
7.99
7.7
7.7
7.3
525
530
60
80
64 116
ND
ND
ND
Nitrite Analysis Method SM 4500 NO2
Nitrate Analysis Method SM 4500 NO3
Not Detected; below detection limit
52
-------
96-hr Toxicity of Nitrite on Amphinemura delosa
The 96-hr test to determine the toxicity of nitrite on A delosa was completed by INKS.
Test organisms, field-collected nymphs, were acclimated to the dilution water (MHRW),
test temperature and other test conditions prior to test initiation. Once acclimated, test
organisms were examined for any disease, stress, parasites, etc. If free from ailments, test
organisms were randomly assigned to the test chambers (which were randomly assigned
to testing locations); four replicates were used per treatment with five organisms per
replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 12, 6.1, 3.0, 1.5, and 0.8 mg
N-NO2/L.
Testing was conducted at 12 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 38; test results are provided in Table 39. Analytical chemistry data are provided
in Table 40. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 11.
53
-------
Table 38. Test conditions for 96-hour toxicity test on Amphinemura delosa with
nitrite.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg N-NO2/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination- mg
N-N02/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Amphinemura delosa, nymphs, Field collected
Static, 96 hours
May 14- 18,2009
12 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
250 mL beaker
200 mL
4
4
16
12,6.1,3.0, 1.5, and 0.8
13,6.1,3.1, 1.5, and 0.7
None
USEPA MHRW
Sodium Nitrite: Fisher, certified ACS, assay 99.7%, Lot #
080939
None
None
Mortality (LC50)
54
-------
Table 39. Test results for 96-hour toxicity test on Amphinemura delosa with nitrite.
Results of a Amphinemura delosa 96-Hour Static Acute Toxicitv Test
Conducted 05/14/09 - 05/18/09 Using: Nitrite (Sodium Nitrite: Sigma Aldrich Cas. No. 7632-00-0)
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
0.8 (0.7) mg/L
1.5(1.5)mg/L
3.0 (3.1) mg/L
6. 1(6.1) mg/L
12.2 (12.7) mg/L
Cumulative Percent Affected a
24-Hr
0
12.5
12.5
50
68.75
75
48-Hr
6.25
12.5
31.25
100
100
100
72-Hr
6.25
18.75
81.25
100
100
100
96-Hr
6.25
25
81.25
100
100
100
LC50 Values* (mg/L)
(EC50*)
24-Hr
48-Hr 72-
3.6 1.7 1
96-Hour LC50* = 1.0 mg/L
Hr 96-Hr
0 1.0
LC50 95% Confidence Limits
24-Hr
LL2.2
UL5.8
48-Hr 72-
1.3 0
2.2 1
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not
Hr 96-Hr
9 0.8
2 1.2
reliable
Method(s) Used to Determine LC50 and EC50
Confidence Limit Values: Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ and EC™ values are determined based on measured concentrations.
55
-------
Table 40. Analytical chemistry data for 96-hour toxicity test on Amphinemura delosa
with nitrite.
Nominal (Measured) Test
Concentration
Dilution water/Control
0.8(0.7)mg/L
1.5(1.5)mg/L
3.0(3.1)mg/L
6.1(6.1)mg/L
12.2 (12.7) mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
N-NO2" N-NO3ab Temperature pH D.O. Conductivity Alkalinity Hardness
(mg/L) (mg/L) (°C) (s.u.) (mg/L) (umhos) (mg/L) (mg/L)
<0.08
<0.07
na
0.8
0.7
0.7
1.5
1.5
1.5
3.1
3.0
3.1
6.1
6.1
6.1
12.8
12.7
12.7
<0.08
<0.07
1.0
1.0
2.1
2.0
4.1
3.8
8.9
3.6
18.6
14.5
12.5
12.1
12.3
12.5
12.8
12.5
12.1
12.3
12.5
12.7
12.5
12.1
12.3
12.5
12.8
12.5
12.3
12.3
12.5
12.7
12.5
12.3
12.3
12.5
12.7
12.5
12.2
12.3
12.5
12.2
7.9
7.9
7.9
7.9
7.9
7.8
7.9
7.9
7.9
7.9
7.8
7.9
7.9
7.9
7.9
7.8
7.9
7.9
7.9
7.9
7.8
7.9
7.9
7.9
7.9
7.8
7.9
7.9
7.9
7.9
9.10
9.97
9.17
9.98
9.13
9.93
9.14
9.97
9.06
9.99
9.08
9.99
301
300
300
305
299
306
303
302
303
307
314
310
310
310
313
327
324
325
325
324
354
351
355
350
351
406
404
405
405
405
60
60
60
60
60
60
60
60
60
60
60
62
88
90
90
90
90
90
90
90
90
88
90
90
"Nitrite Analysis Method 354.1
b Nitrite + Nitrate Analysis Method 353.1
na = not applicable
56
-------
96-hr Toxicity of Nitrite on Sphaerium simile
The 96-hr test to determine the toxicity of nitrite on S. simile was completed by INHS.
Test organisms, juveniles released from field-collected adults, were acclimated to the
dilution water (MHRW), test temperature and other test conditions prior to test initiation.
Once acclimated, test organisms were examined for any disease, stress, parasites, etc. If
free from ailments, test organisms were randomly assigned to the test chambers (which
were randomly assigned to testing locations); four replicates were used per treatment with
five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 304, 152, 76, 38, 19, and 9.5
mg N-NO2/L.
Testing was conducted at 22 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 41; test results are provided in Table 42. Analytical chemistry data are provided
in Table 42. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 12.
57
-------
Table 41. Test conditions for 96-hour toxicity test on Sphaerium simile with nitrite.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg N-NO2/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination- mg
N-NO2/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Sphaerium simile, juveniles (released from field-collected
adults)
Static, 96 hours
August 17-21,2009
22 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
150 mL beaker
120 mL
5
4
20
304,152, 76,38,19, and 9.5
296,158, 82,39,19, and 9.4
None
USEPA MHRW
Sodium Nitrite: Fisher, certified ACS, assay 99.7%, Lot #
080939
None
None
Mortality (LC50)
58
-------
Table 42. Test results for 96-hour toxicity test on Sphaerium simile with nitrite.
Results of a Sphaerium simile 96-Hour Static Acute Toxicitv Test
Conducted 08/17/09 - 08/21/09 Using: Sodium Nitrite: Sigma Aldrich Cas. No. 7632-00-0
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
9.5 (9.4) mg/L
19.0 (19.2) mg/L
38.0 (38.8) mg/L
76. 1(82.2) mg/L
152.2 (157.7) mg/L
304.4 (296.1) mg/L
Cumulative Percent Affected a
24-Hr
0
0
0
0
0
0
0
48-Hr
0
0
0
0
0
30
60
72-Hr
0
0
0
0
0
50
60
96-Hr
0
0
15
40
50
95
100
LC50 Values* (mg/L)
24-Hr 48-Hr 72-
Hr 96-Hr
>296.1 240.0 178.7 55.7
96-Hour LC50* = 55.7 mg/L
LC50 95% Confidence Limits
24-Hr 48-Hr 72-
Hr 96-Hr
LLNR 171.1 65.1 43.0
ULNR 336.7 490.7 72.1
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not
reliable
Method(s) Used to Determine LC50 and EC50
Confidence Limit Values: Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ and EC™ values are determined based on measured concentrations.
59
-------
Table 43. Analytical chemistry data for 96-hour toxicity test on Sphaerium simile
with nitrite.
Nominal (Measured) Test Concentration
Dilution water/Control
9.5 (9.4)mg/L
19.0 (19.2) mg/L
38.0(38.8)mg/L
76. 1(82.2) mg/L
152.2 (157. 7) mg/L
304.4 (296.1) mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
N-NO2" N-NO3"'b Temperature pH D.O. Conductivity Alkalinity Hardness
(mg/L) (mg/L) (°C) (s.u.) (mg/L) (umhos) (mg/L) (mg/L)
<0.02
<0.02
na
9.6
9.2
9.4
19.3
19.8
19.1
19.7
19.2
38.9
38.7
38.8
79.2
85.3
82.2
153.5
162.1
157.7
293.1
299.2
296.1
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
22 3
22.9
22.7
22.7
22.7
22.4
22.8
22.7
22.6
22.7
22.5
22.8
22.6
22.7
22.7
22.4
22.8
22.7
22.7
22.7
22 3
22.8
22.6
22.7
22.7
22.4
22.8
22.7
22.7
22.8
22.5
22.8
22.7
22.6
22.8
8.0
7.7
7.7
7.7
7.7
8.1
7.7
7.3
7.6
7.6
8.1
7.7
7.3
7.6
7.6
8.2
7.7
7.4
7.6
7.6
8.2
7.6
7.5
7.6
7.6
8.2
7.7
7.5
7.6
7.5
8.2
7.7
7.5
7.6
7.7
7.74
7.14
7.17
7.31
7.51
7.81
6.49
6.30
6.67
6.78
7.79
6.59
7.09
7.10
6.89
7.80
6.25
6.86
6.78
6.75
7.82
5.82
6.81
6.26
6.04
7.82
7.03
6.02
6.09
6.09
7.82
7.03
5.46
5.76
5.70
302
304
313
316
317
383
384
389
393
395
466
465
470
474
481
626
623
629
633
639
941
941
948
958
971
1562
1558
1574
1577
1587
2750
2740
2750
2750
2740
62
62
62
64
62
64
64
66
72
76
96
110
120
126
88
90
90
90
90
90
88
90
88
90
88
90
88
90
a Nitrite Analysis Method 3
b Nitrite + Nitrate Analysis
54.1 NM =
Method 353.1
not measured
na = not applicable
60
-------
96-hr Toxicity of Nitrite on Hyalella azteca
The 96-hr test to determine the toxicity of nitrite on H. azteca was completed by GLEC.
H. azteca were collected from GLEC's laboratory culture. These organisms are
maintained in 10 gallon glass aquaria; plastic artificial turf and screen mesh serve as a
substrate for the culture. The tanks are filled with de-chlorinated Lake Michigan water
(City of Traverse City, Michigan water passed through an activated carbon filter).
Cultures are fed 50 mL of 4 g/L Tetrafin slurry daily. When visible algae are not
observed within the glass aquaria, algae (Selenastrum sp.) are used as a supplement to the
Tetrafin slurry. Additionally, on occasion, dried Aspen (Populus sp.) leaves are prepared
as a food supplement. The culture is maintained in a 16-hour light: 8-hour dark
photoperiod at a temperature between 23 and 26 °C.
Test organisms were acclimated to the dilution water (MHRW), test temperature and
other test conditions prior to test initiation. Once acclimated, test organisms were
examined for any disease, stress, parasites, etc. If free from ailments, test organisms were
randomly assigned to the test chambers (which were randomly assigned to testing
locations); four replicates were used per treatment with five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 0.40, 0.67, 1.1, 1.8 and 3.0
mg NO2-N/L.
Testing was conducted at 22 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso was determined using
the Probit method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 44; test results are provided in Table 45. Analytical chemistry data are provided
in Table 46. Accompanying information, including raw laboratory data, analytical
chemistry data, reference toxicant data and statistical analyses, is provided in Appendix
13.
61
-------
Table 44. Test conditions for 96-hour toxicity test on Hyalella azteca with nitrite.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg N02-
N/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination- mg N02-
N/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Hyalella azteca, 10 days old, GLEC culture
Static, 96 hours
August 6-August 10,2009
22 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
150 mL beaker
100 mL
5
4
20
3.0,1.8, 1.1, 0.67 and 0.40
4.1,2.4,1.4,0.81 and 0.54
None
USEPA MHRW
Sodium Nitrite: Sigma Aldrich, 97 +%, ACS Reagent Cas.
No. 7632-00-0, Batch # 08316DJ
None
None
Mortality (LC50)
62
-------
Table 45. Test results for 96-hour toxicity test on Hyalella azteca with nitrite.
Results of a Hvalella azteca 96-Hour Static Acute Toxicitv Test
Conducted 08/06/09 - 08/10/09 Using: Nitrite (Sodium Nitrite: Sigma Aldrich Cas. No. 7632-00-0)
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
0.40 (0.54) mg/L
0.67 (0.81) mg/L
1.1 (1.4) mg/L
1.8 (2. 4) mg/L
3. 0(4.1) mg/L
Cumulative Percent Affected a
24-Hr
0
(0)
0
(0)
10
(10)
10
(10)
30
(30)
60
(65)
48-Hr
0
(0)
0
(0)
10
(10)
15
(15)
40
(40)
60
(65)
72-Hr
0
(0)
0
(0)
10
(10)
30
(30)
60
(60)
80
(80)
96-Hr
0
(0)
0
(0)
35
(35)
50
(50)
70
(70)
90
(90)
LC50 Values* (mg/L)
24-Hr 48-Hr 72-Hr 96-Hr
3.6 3.2 2.1 1.5
96-Hour LC50* = 1.5 mg/L
LCSO 95% Confidence Limits
24-Hr 48-Hr 72-Hr 96-Hr
LL 2.7 2.4 1.7 1.2
UL6.2 5.2 2.8 1.9
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 Confidence
Limit Values: Probit
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ values are determined based on measured concentrations.
63
-------
Table 46. Analytical chemistry data for 96-hour toxicity test on Hyalella azteca with
nitrite.
Nominal (and Measured) Test
Concentrations
Dilution water/Control
Day 0
Day 1
Day 2
Day3
Day 4
Nitrite1
(mg NO2-N/L)
<0.0011
0.02
0.02
0.02
Nitrate"
mg NO3-N/L)
<0.0012
0.044
0.056
Temperature
(°C)
22.0
22.1
23.2
22.8
22.1
PH
(s.u.)
8.00
7.99
111
7.96
7.80
D.O.
(mg/L)
8.4
8.5
8.3
8.5
8.0
Conductivity
(mmhos)
300
290
Alkalinity
(mg/L)
60
60
Hardness
(mg/L)
80
80
Ammonia
(mg/L)
ND
0.40 (0.54) mg/L
DayO
Day 1
Day 2
Day3
Day 4
0.51
0.57
0.54
<0.0048
<0.0001
22.0
22.2
23.2
22.8
22.1
7.98
7.97
7.99
7.98
7.91
8.4
8.5
8.4
8.5
8.0
336
310
0.67(0.81) mg/L
DavO
Day 1
Day 2
DayS
Day 4
0.87
0.78
0.79
0.81
<0.0072
<0.0072
0.055
22.0
22.2
23.1
22.7
22.1
7.99
7.92
8.01
8.04
7.90
8.4
8.5
8.4
8.5
8.0
340
315
1.1 (1.4) mg/L
DavO
Day 1
Day 2
DayS
Day 4
1.44
1.43
1.44
<0.01
<0.01
22.0
22.2
23.1
22.8
22.2
8.00
7.95
8.06
8.06
7.90
8.4
8.5
8.5
8.6
8.0
340
325
1.8 (2. 4) mg/L
DayO
Day 1
Day 2
DayS
Day 4
2.40
2.47
2.44
<0.02
<0.02
22.0
22.2
23.1
22.8
22.2
8.04
7.90
8.06
8.04
7.96
8.4
8.5
8.5
8.6
8.0
341
325
3. 0(4.1) mg/L
DayO
Day 1
Day 2
DayS
Day 4
3.96
4.29
4.1
<0.05
<0.05
22.0
22.2
23.1
22.8
22.2
8.06
7.90
8.07
8.04
7.97
8.4
8.5
8.6
8.5
8.0
342
322
60
58
80
80
ND
" Nitrite Analysis Method SM 4500 NO2
b Nitrate Analysis Method SM 4500 NO3
ND Not Detect; below detection limit
64
-------
96-hr Toxicity of Nitrite on Pimephales promelas
The 96-hr test to determine the toxicity of nitrite on P. promelas was completed by
GLEC. Test organisms, collected from the GLEC laboratory culture, were acclimated to
the dilution water (de-chlorinated Lake Michigan water), test temperature and other test
conditions prior to test initiation. Once acclimated, test organisms were examined for any
disease, stress, parasites, etc. If free from ailments, test organisms were randomly
assigned to the test chambers (which were randomly assigned to testing locations); two
replicates were used per treatment with ten organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 3.9, 6.6, 11,18 and 30 mg
NO2-N/L.
Testing was conducted at 25 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Trimmed Spearman-Karber and Probit methods.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 47; test results are provided in Table 48. Analytical chemistry data are provided
in Table 49. Accompanying information, including raw laboratory data, analytical
chemistry data, reference toxicant data and statistical analyses, is provided in Appendix
14.
65
-------
Table 47. Test conditions for 96-hour toxicity test on Pimephales promelas with
nitrite.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg NC>2-
N/L):
14. Measured Test Concentrations (average of samples
collected at test initiation and termination- mg NC>2
N/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Pimephales promelas, (weight 0.12g and 18 mm length),
GLEC culture
Static, 96 hours
August 13-August 17,2009
25 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
4000 mL beaker
3500 mL
10
2
20
30,18, 11, 6.6, and 3.9
33,18. 11,5.8, and 4.1
None
De-chlorinated Lake Michigan Water
Sodium Nitrite: Sigma Aldrich, 97 +%, ACS Reagent Cas.
No. 7632-00-0, Batch # 08316DJ
None
None
Mortality (LC50)
66
-------
Table 48. Test results for 96-hour toxicity test on Pimephales promelas with nitrite.
Results of a Pimephales promelas 96-Hour Static Acute Toxicitv Test
Conducted 08/13/09 - 08/17/09 Using: Nitrite (Sodium Nitrite: Sigma Aldrich Cas. No. 7632-00-0)
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
3.9 (4.1) mg/L
6.6 (5.8) mg/L
11 (11) mg/L
18 (18) mg/L
30 (33) mg/L
Cumulative Percent Affected a
24-Hr
5
(5)
10
(10)
10
(10)
0
(0)
20
(20)
10
(25)
48-Hr
5
(5)
10
(10)
10
(10)
0
(0)
25
(25)
25
(25)
72-Hr
5
(5)
10
(10)
10
(10)
0
(0)
30
(30)
45
(65)
96-Hr
5
(5)
15
(15)
10
(10)
5
(15)
35
(45)
65
(65)
LCSO Values* (mg/L)
24-Hr 48-Hr 72-Hr 96-Hr
>33 >33 >33 27.0
96-Hour LC50* = 27.0 mg/L
LC50 95% Confidence Limits
24-Hr 48-Hr 72-Hr 96-Hr
LL NA NA NA 19.7
ULNA NA NA 41.4
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 Confidence
Limit: Trimmed Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ values are determined based on measured concentrations.
67
-------
Table 49. Analytical chemistry data for 96-hour toxicity test on Pimephales promelas
with nitrite.
Nominal (Measured) Test
Concentration
Dilution
water/Control
DayO
Day 1
Day 2
Dav3
Day 4
Nitrite3 Nitrateb Temperature pH D.O. Conductivity Alkalinity Hardness Ammonia
(mg NO2-N/L) (mg NO3-N/L) (°C) (s.u.) (mg/L) (mmos) (mg/L) (mg/L) (mg/L)
O.0011
0.01
0.01
0.23
0.23
0.26
1.15
25.0
24.6
24.2
24.5
24.7
7.99
7.66
7.55
7.59
7.67
7.69
8.1
6.7
6.1
6.0
6.0
8.2
6.7
5.8
6.1
290
304
102
100
144
136
ND
ND
3.9 (4.1) mg/L
DayO
Day 1
Day 2
Day3
Day 4
3.90
4.20
4.26
4.12
<0.11
<0.11
25.0
24.6
24.2
24.4
24.7
8.06
7.90
7.60
7.65
7.74
7.77
8.2
7.1
5.9
6.0
5.9
8.2
6.9
5.9
5.9
320
333
6.6 (5.8) mg/L
DayO
Day 1
Day 2
Day3
Day 4
5.18
6.39
5.78
0.52
<0.11
25.0
24.4
24.3
24.6
24.7
8.07
7.79
7.56
7.69
7.80
7.80
8.4
6.9
5.5
6.1
5.9
8.3
6.9
5.9
6.0
330
354
11 (11) mg/L
DayO
Day1
Day 2
Day3
Day 4
10.65
10.65
10.65
<0.25
<0.11
25.0
24.3
24.2
24.6
24.7
18(18) mg/L
DavO
Day1
Day 2
DayS
Day 4
17.96
17.35
17.66
0.34
4.88
25.0
24.3
24.3
24.3
24.6
8.08
7.79
7.63
7.74
7.80
7.81
8.08
7.79
7.63
7.69
7.75
7.68
8.5
7.3
6.5
6.3
5.7
8.3
7.2
6.1
5.9
360
386
8.5
7.3
6.0
6.0
5.9
8.4
7.3
6.1
6.1
424
437
30 (33) mg/L
DayO
Dav1
Day 2
DayS
Day 4
31.66
33.79
32.72
<0.61
<0.36
25.0
24.3
24.2
24.3
24.5
8.06
7.77
7.61
7.63
7.75
7.77
8.6
7.5
6.3
5.5
5.7
8.5
7.4
6.5
5.7
507
520
98
100
136
132
ND
ND
1 Nitrite Analysis Method SM 4500 NO2
b Nitrate Analysis Method SM4500 NO3
ND Not Detect; below detection limit
68
-------
32-day Toxicity of Nitrite on Pimephales promelas
The 32-day test to determine the toxicity of nitrite on P. promelas was completed by
GLEC. The fish were continuously exposed for 32 days to five concentrations of nitrite
(nominal concentrations of 1.9, 3.8, 7.6, 15 and 30 mg NO2-N/L - dilution factor of 0.5)
and to a dilution water control using a continuous flow-through system (Benoit et al.
1982). The temperature-controlled test concentration solutions were supplied to each test
chamber via the continuous flow-through system at a rate of approximately four
turnovers a day. There were four replicate test chambers for each treatment. The flow
through test was conducted at 25 ± 1 °C with a photoperiod of 16 hr light and 8 hr
darkness (ambient laboratory light).
After test concentrations had achieved steady state in the flow through system, the test
was initiated with < 24 hour old fertilized embryos. The embryos were randomly
assigned to incubation cups until each incubation cup contained 30 embryos. The
incubation cups were randomly assigned to the 2.5 L glass test chambers (1 cup per
chamber) and suspended in the test solutions from a rocker arm assembly. The rocker
arm assembly moves the incubation cups in a reciprocal motion within each test chamber.
Embryos were inspected on a daily basis and the number of live, hatched and dead
embryos was recorded. On Day 5 of the test (three days after first hatch), the surviving
fish were randomly thinned to achieve 20 fish in each test chamber. The remainder of the
surviving fish was discarded. The number of surviving fish was recorded at test
termination (32 days). In addition, the wet weights were recorded for each fish at test
termination. Because of the size range offish in each test chamber, all of the fish from
each test chamber were weighed together to determine average dry weight.
Instantaneous water temperature measurements made on August 11, 2009 (Day 4) ranged
from 25.5°C -26.3°C within individual replicates; this exceeded the allowed range of 25
± 1 °C in the toxicity testing method. However, the average water temperature (across the
duration of the test) in each replicate was always ± 0.3 °C of the target test temperature
(25 °C) in all treatments. In addition, the water temperature as recorded by the continuous
temperature logger was never outside the range outlined in the method. Therefore, the
water temperature exceedance likely had no effect on the results of this test.
In addition, on test day 24 there was an instance of low DO (5.8 mg/L) in replicate four
of the laboratory control. Low DO concentrations were never observed on any other day
of the test. Therefore, the one instance of low DO was an anomaly and likely had no
effect on the results of this test.
On August 27, 2009 (day 20) a diluter malfunction (stock solution metering pump)
occurred for 8 hours. Analytical chemistry samples were collected from replicate one of
each test concentration subsequent to this malfunction that analysis indicated that the test
solution concentrations were significantly less than the target concentrations. The
measured concentration of nitrite in the five test concentrations on day 20 was less than
50 percent of the time-weighted average measured concentration; the duration of the
malfunction was less than 10 percent (approximately 3.1 percent) of the total duration of
69
-------
the test. Therefore, the diluter malfunction likely had little, if any, affect on the results of
this test.
Once the test was complete, the LCso, NOEC and LOEC values were determined using
the average measured concentrations with the Spearman Karber and ANOVA methods
while LC25, LC20, LCio values were determined with the Probit method. ECso, £€25, £€20
and ECio values were estimated using EPA's TRAP.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 50; test results are provided in Table 51. Survival and growth data are provided
in Table 52 and analytical chemistry data are provided in Table 53. Accompanying
information, including raw laboratory data, analytical chemistry data and statistical
analyses, is provided in Appendix 15.
70
-------
Table 50. Test conditions for 32-day toxicity test on Pimephales promelas with
nitrite.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg NC>2-
N/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination- mg NC>2
N/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Pimephales promelas, (<24 hour fertilized embryos ),
GLEC culture
Continuous flow-through; 32 days
August 7-September 8, 2009
25 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
Live Brine Shrimp (Artemia nauplii) Twice daily
2.5 Liter glass Tank
2000 mL
30 eggs, thinned to 20 larvae after hatch
4
120 eggs, thinned to 80 larvae after hatch
30,15, 7.6, 3.8, and 1.9
30,15, 6.8, 3.4, and 1.7
Continuous flow through, 4 turnovers per day
De-chlorinated Lake Michigan Water
Sodium Nitrite: Sigma Aldrich, 97 +%, ACS Reagent Cas.
No. 7632-00-0, Batch # 08316DJ
None
None
Survival (LC50, LC25!LC20 and LC10,NOEC and LOEC) and
Growth (EC50, EC25> EC20 and EC10, NOEC and LOEC)
71
-------
Table 51. Test results for 32-day toxicity test on Pimephales promelas with nitrite.
Results of a Pimephales promelas
32-Dav Continuous Flow Chronic Toxicitv Test
Conducted 08/03/09 - 09/08/09 Using: Nitrite ( Sodium Nitrite: Sisma Aldrich Cas. No. 7632-00-0)
Test Solution Concentrations
Measured
Embryo Percent Hatch (%)
32-Day Mean Survival (%)
32-Day Average Biomass1 (mg )
Primary Control/
Dilution Water
Secondary
100
90.0
6.02
1.7 mg/L
97.5
88.8
6.11
3.4 mg/L
95.8
86.3
6.49
6.8 mg/L
97.5
78.8
6.17
15 mg/L
96.7
38.8a
2.95a
30 mg/L
99.2
Oa
Oa
Embryo Percent Hatch NOEC:
32-Day LC50*:
32-Day Survival NOEC:
32-Day Survival LOEC:
32-Day Growth NOEC:
32-Day Growth LOEC:
32-Day LC25:
30.2 mg/L 32-Day LC20:
11.3 mg/L (9. 7 mg/L- 13. 2 mg/L) 32-Day LC10:
6.8 mg/L 32-Day EC50:
15.0 mg/L 32-Day EC25:
6.8 mg/L 32-Day EC20:
15.0 mg/L 32-Day EC10:
7.0 mg/L (5.7 mg/L - 8.2 mg/L)
5.7 mg/L (4.3 mg/L - 7.0 mg/L)
2.3 mg/L (0.6 mg/L - 4.0 mg/L)
14.6 mg/L (12.7 mg/L - 16.8 mg/L)
1 1 .7 mg/L (6.7 mg/L - 20.4 mg/L)
11.0 mg/L (5.6 mg/L -21.8 mg/L)
9.3 mg/L (3.3 mg/L - 26.1 mg/L)
a: Significantly different from dilution water control (p< 0.05)
*: All LC, EC, NOEC and LOEC values are determined based on the average measured nitrite
concentration.
NOEC: No-Observed-Effect-Concentration
LOEC: Lowest-Observed-Effect-Concentration
'Biomass: Biomass is the average dry weight of the four replicates calculated by the total dry weight of
surviving organisms divided by the initial number of organisms (20).
72
-------
Table 52. Survival and growth data for 32-day toxicity test on Pimephales promelas
with nitrite.
Nominal (and Measured) Test
Concentration
Dilution
water/Control
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
Number of
Eggs at Test
Initiation
30
30
30
30
Number of
Hatched
Larvae
30
30
30
30
Number of
Dead Eggs
0
0
0
0
Percent
Hatched
Larvae
100.0
100.0
100.0
100.0
Number of
Larvae at Test
Termination
20
19
17
16
Percent Survival
at Test
Termination*
100.0
95.0
85.0
80.0
Biomass1
(mg)
5.72
6.28
5.73
6.33
Average 100.0 90.0 6.02
1.9(1.7)mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
29
30
30
28
1
0
0
2
96.7
100.0
100.0
93.3
16
17
18
20
80.0
85.0
90.0
100.0
6.86
5.69
5.77
6.12
Average 97.5 88.8 6.11
3.8(3.4)mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
29
26
30
30
1
4
0
0
96.7
86.7
100.0
100.0
19
12
19
19
95.0
60.0
95.0
95.0
6.37
7.04
6.26
6.28
Average 95.8 86.3 6.49
7.6(6.8)mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
28
29
30
30
2
1
0
0
93.3
96.7
100.0
100.0
16
16
19
12
80.0
80.0
95.0
60.0
6.55
6.28
5.51
6.34
Average 97.5 78.8 6.17
15(15)mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
30
28
29
29
0
2
1
1
100.0
93.3
96.7
96.7
9
2
9
11
45.0
10.0
45.0
55.0
3.27
0.88
3.43
4.22
Average 96.7 38.8 2.95
30 (30) mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
30
29
30
30
0
1
0
0
100.0
96.7
100.0
100.0
0
0
0
0
0.0
0.0
0.0
0.0
0.00
0.00
0.00
0.00
Average 99.2 0.0 0.00
' On Day 5 of the test, (three days after first hatch) the surviving fish were randomly thinned to 20 fish in each test chamber.
Percent surval at test termination is the number of surviving at test termination divided by 20.
Biomass: Biomass is the total dry weight of surviving organisms divided by the initial number of organisms (20)
73
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Table 53. Analytical chemistry data for 32-day toxicity test on Pimephales promelas
with nitrite.
Nominal (and Measured) Test
Conent rations
Dilution
Water/Control
Average
1.9 mg/L
(1.7 mg/L)
Average
3.8 mg/L
(3.4 mg/L)
Average
7.6 mg/L
(6.8 mg/L)
Average
15 mg/L
(15 mg/L)
Average
30 mg/L
(30 mg/L)
Average
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
NOi
(mg NO2-N/L)
0.00
(0.00-0.01)
0.01
(0.01-0.01)
0.00
(0.00-0.01)
0.01
(0.00-0.03)
0.01
1.44
(0.70*-2.04)
1.60
(0.88-1.95)
1.99
(1.52-2.41)
1.86
(1.46-2.47)
1.7
3.07
(1.40*-4.20)
3.5
(3.01-4.47)
3.65
(2.92-4.29)
3.56
(3.04-4.17)
3.4
6.36
(2.83*-7.34)
6.91
(5.78-7.85)
7.06
(6.09-8.22)
7.31
(6.09-8.13)
6.8
14.1
(4.63*-17.5)
15.0
(12.8-18.5)
15.9
(15.0-17.2)
15.3
(143-16.6)
15
30.0
(10.7*-51.8)
30.4
(25.0-34.4)
29.7
(21.0-42.3)
30.7
(28.6-32.6)
30
NO3
(mg NO3-N/L)
0.25
0.26
0.26
0.25
0.22
0.26
0.11
0.25
0.26
0.22
0.32
0.14
0.41
0.44
0.28
0.17
0.81
0.76
1.17
0.61
3.16
1.75
4.74
1.81
Temperature
(°Q
24.8
(24.0-25.4)
25.2
(24.3-26.3)
25.2
(24.4-25.7)
25.3
(24.2-26.2)
24.9
(24.1-25.5)
25.2
(24.0-26.0)
25.1
(24.5-25.8)
25.1
(24.1-26.0)
24.9
(24.0-25.6)
25.0
(24.1-26.1)
25.1
(24.5-25.9)
25.1
(24.2-25.9)
25.1
(24.1-25.8)
25.2
(24.2-25.9)
25.1
(24.3-25.7)
25.0
(24.3-26.1)
25.0
(24.0-26.0)
25.0
(24.2-25.8)
25.0
(24.3-25.7)
25.3
(24.5-26.1)
24.6
(24.0-25.4)
25.0
(24.3-26.3)
25.0
(24.3-25.8)
25.1
(24.5-26.2)
pH
(S.U.)
8.03
8.13
8.17
8.03
8.18
8.25
8.17
8.23
8.22
8.27
8.10
8.18
8.18
8.26
8.18
8.23
8.21
8.27
8.19
8.29
8.24
8.28
8.25
8.29
DO
(mg/L)
7.7
(6.9-8.5)
8.1
(7.7-9.1)
8.0
(7.4-9.4)
7.3
(5.8-8.2)
7.8
(6.7-9.1)
8.0
(7.7-8.3)
7.9
(7.7-8.0)
7.8
(6.7-9.1)
7.9
(7.0-9.2)
8.1
(7.7-9.2)
7.8
(7.5-8.1)
7.7
(6.9-8.2)
7.6
(7.0-8.0)
7.9
(7.3-9.1)
7.8
(7.4-8.0)
7.7
(6.4-9.1)
7.9
(7.2-9.1)
7.9
(7.6-8.3)
7.7
(7.6-8.0)
8.0
(7.0-9.2)
8.0
(7.4-9.4)
8.1
(7.5-9.0)
8.1
(8.0-8.2)
7.7
(7.1-8.3)
SC
(mmos)
298
304
301
315
324
318
328
292
326
359
368
357
417
431
426
528
545
454
Hardness
(mg/L)
118
132
146
132
140
132
136
132
144
124
136
132
Alkalinity
(mg/L)
105
102
103
106
105
104
105
106
104
104
103
104
Ammonia
(mg/L)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
DO: Dissolved Oxygen
SC: Specific Conductance
ND: Non Detect; below detection limit.
"August 20,2009 (day 20) a metering diluter malfunction occurred and analyitcal chemistry samples were collected.
74
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48-hr Toxicity of Nitrite on Ceriodaphnia dubia
The 48-hr test to determine the toxicity of nitrite on C. dubia was completed by GLEC.
Test organisms, < 24-hours old collected from the GLEC laboratory culture, were
acclimated to the dilution water (DMW), test temperature and other test conditions prior
to test initiation. Once acclimated, test organisms were examined for any disease, stress,
parasites, etc. If free from ailments, test organisms were randomly assigned to the test
chambers (which were randomly assigned to testing locations); four replicates were used
per treatment with five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 0.99, 1.6, 2.7, 4.6, and 7.6 mg
NO2-N/L.
Testing was conducted at 25 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LC50 value was determined
using the Probit and Spearman Karber methods.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 54; test results are provided in Table 55. Analytical chemistry data are provided
in Table 56. Accompanying information, including raw laboratory data, analytical
chemistry data, reference toxicant data and statistical analyses, is provided in Appendix
16.
75
-------
Table 54. Test conditions for 48-hour toxicity test on Ceriodaphnia dubia with
nitrite.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (N02-N/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination- N02-N/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Ceriodaphnia dubia, < 24 hours old, GLEC culture
Static, 48 hours
August 21-August 23,2009
25 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
30 mL beaker
15 mL
5
4
20
7.6,4.6,2.7,1.6, and 0.99
7.3, 4.7,2.8, 1.6, and 0.99
None
USEPA DMW
Sodium Nitrite: Sigma Aldrich, 97 +%, ACS Reagent Cas.
No. 7632-00-0, Batch # 08316DJ
None
None
Mortality (LC50)
76
-------
Table 55. Test results for 48-hour toxicity test on Ceriodaphnia dubia with nitrite.
Results of a Ceriodaphnia dubia 48-Hour Static Acute Toxicitv Test
Conducted 08/21/09 - 08/23/09 Using: Nitrite (Sodium Nitrite: Sigma Aldrich Cas. No. 7632-00-0)
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
1.0(1.0)mg/L
1.6(1.6)mg/L
2.7 (2.8) mg/L
4.6 (4.7) mg/L
7.6 (7.3) mg/L
Cumulative Percent Affected a
24-Hr
0
(0)
0
(0)
0
(0)
0
(0)
25
(25)
80
(80)
48-Hr
0
(0)
0
(0)
0
(0)
0
(0)
50
(50)
100
(100)
72-Hr
96-Hr
LC50 Values* (mg/L)
24-Hr 48-Hr 72-Hr 96-Hr
5.8 4.6 NA NA
48-Hour LC50* = 4.6 mg/L
LCSO* 95% Confidence Limits
24-Hr 48-Hr 72-Hr 96-Hr
LL 5.1 4.1 NA NA
UL6.6 5.1 NA NA
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 Confidence
Limit Values: Spearman-Karber and Probit
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC,n values are determined based on measured concentrations.
77
-------
Table 56. Analytical chemistry data for 48-hour toxicity test on Ceriodaphnia dubia
with nitrite.
Nominal (and Meas
Concentrati
Dilution
water/Control
Average
ured) Test
on
DayO
Day 1
Day 2
Nitrite1
(mg N02-N/L)
O.0011
0.0011
0.020
0.020
Nitrate"
(mg N03-N/L)
0.31
0.30
0.17
Temperature
(°C)
25.0
24.6
24.8
pH
(s.u.)
7.98
8.40
8.08
D.O.
(mg/L)
7.8
8.8
8.9
Conductivity
(mmos)
189
192
Alkalinity
(mg/L)
76
Hardness
(mg/L)
96
Ammonia
(mg/L)
ND
1.0 (1.0) mg/L
Average
DayO
Day 1
Day 2
0.99
0.98
0.99
0.12
0.33
25.0
24.7
24.8
8.10
8.32
8.12
7.8
8.8
9.0
206
210
1.6(1.6)mg/L
Average
DayO
Day1
Day 2
1.72
1.52
1.64
1.63
0.26
0.31
0.33
25.0
24.7
24.8
8.11
8.24
8.20
7.9
8.8
9.0
214
220
2.7 (2.8) mg/L
Average
DayO
Day 1
Day 2
2.98
2.62
2.80
0.05
0.37
25.0
24.6
24.8
8.12
8.24
8.22
8.0
8.8
9.0
223
229
4.6 (4.7) mg/L
Average
DayO
Day 1
Day 2
4.7
4.7
4.7
O.18
0.41
25.0
24.6
24.8
8.13
8.22
8.30
8.0
8.9
9.0
236
234
7.6 (7.3) mg/L
Average
DayO
Day 1
Day 2
7.6
7.0
7.3
O.11
0.11
25.0
24.6
24.8
8.14
8.21
8.36
8.0
9.0
9.0
257
246
76
108
ND
1 Nitrite Analysis Method SM 4500 NO2
b Nitrate Analysis Method SM4500 NO3
ND Not Detect; below detection limit
78
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7-day Toxicity of Nitrite on Ceriodaphnia dubia
The 7-day test to determine the toxicity of nitrite on C. dubia was completed by GLEC.
Test organisms, < 24-hours old collected from the GLEC laboratory culture, were
acclimated to the dilution water (DMW), test temperature and other test conditions prior
to test initiation. Once acclimated, test organisms were examined for any disease, stress,
parasites, etc. If free from ailments, test organisms were randomly assigned to the test
chambers (which were randomly assigned to testing locations); ten replicates were used
per treatment with one organism per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static-renewal conditions. Serial dilutions of the highest test
concentration (known weight of test chemical dissolved in a known volume of dilution
water) were made to prepare the following nominal test concentrations: 0.48, 0.95, 1.9,
3.8, and7.6mgNO2-N/L.
Testing was conducted at 25 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). On a daily basis, organisms were fed Selenastrum
capricornutum and YTC (at renewal) and examined for mortality. As outlined in ASTM
Method E1295 (ASTM 2006 b), the duration of this test is based primarily on brood
production. Therefore, because greater than 60% of surviving control females had three
broods at Day 6 of the test, the test was terminated early and no results were reported for
Day?.
Once the test was complete, the 48-hour and 6-day LCso values and 6-day NOEC and
LOEC values were determined using the average measured concentrations with the
Spearman Karber and ANOVA methods. LC25, LC20 and LCio values were determined
using the Probit method and ECso, £€25, EC20 and ECio values were estimated using EPA's
TRAP.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 57; test results are provided in Table 58. Analytical chemistry data are provided
in Table 59. Accompanying information, including raw laboratory data, analytical
chemistry data, reference toxicant data and statistical analyses, is provided in Appendix
17.
79
-------
Table 57. Test conditions for 7-day toxicity test on Ceriodaphnia dubia with nitrite.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg N02-
N/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination- mg N02-
N/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Ceriodaphnia dubia, < 24 hours old, GLEC culture
Three Brood Static-Renewal
August 21-August 27,2009
25 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
Daily: Selenastrum capricornutum and YTC at renewal
30 mL beaker
15 mL
1
10
10
7.6,3.8, 1.9, 0.95, and 0.48
7.3,3.6, 1.8,0.82, and 0.36
Daily
USEPA DMW
Sodium Nitrite: Sigma Aldrich, 97 +%, ACS Reagent Cas.
No. 7632-00-0, Batch # 08316DJ
Boardman River Culture Water
None
Survival (LC50, LC25!LC20 and LC10,NOEC and LOEC) and
Reproduction (EC50 EC25 EC20andEC10,NOEC and LOEC)
80
-------
Table 58. Test results for 7-day toxicity test on Ceriodaphnia dubia with nitrite.
Results of a Ceriodaphnia dubia 7-Day Static Renewal Chronic Toxicitv Test
Conducted 08/21/09 - 08/27/09 Using: Nitrite ( Sodium Nitrite: Sigma Aldrich Cas. No. 7632-00-0)
Nominal (Measured)
Concentrations
48-hour Survival (%)
6-day Mean Survival (%)
6-day Mean
Reproduction/Female
Primary Control/
Dilution Water
100
100
25.1
Secondary
Control
100
100
20.7
0.48 (0.36)
mg/L
100
100
25.0
0.95
(0.82)
mg/L
100
90
23.3
1.90(1.75)
mg/L
100
100
19.1
3.81 (3.56)
mg/L
100
90
3.6
7.61
(7.31)
mg/L
0
0
0
48-Hour LC50*
6-Day LC50*
6-Day Mean Survival (%) NOEC*
6-Day Mean Survival (%) LOEC*
6-Day Mean Reproduction per
Female NOEC*
6-Day Mean Reproduction per
Female LOEC*
6-Day LC25:
5.1 mg/L
4.7 mg/L (4.1 mg/L - 5.4 mg/L)
3.6 mg/L
7.3 mg/L
0.82 mg/L
1.8 mg/L
3.5 mg/L (2.5 mg/L - 4.6 mg/L)
6-Day LC20:
6-Day LC10:
6-Day EC50:
6-Day EC25:
6-Day EC20:
6-Day EC10:
3.3 mg/L (2.3 mg/L - 4.3 mg/L)
2.5 mg/L (1.4 mg/L - 3.6 mg/L)
2.4 mg/L (2.1 mg/L - 2.8 mg/L)
1.9 mg/L (1.5 mg/L -2.3 mg/L)
1 .7 mg/L (1 .4 mg/L - 2.2 mg/L)
1.4 mg/L (1.1 mg/L - 1.9 mg/L)
j>
AllLC, EC, NOEC and LOEC values are determined based on the analytical chemistry results.
NOEC: No Observed Effect Concentration
LOEC: Lowest Observed Effect Concentration
81
-------
Table 59. Analytical chemistry data for 7-day toxicity test on Ceriodaphnia dubia
with nitrite.
Nominal (and Measured)
Test Concentration
Dilution water/Control
Average
DayO™
Day1°
Day2N
Day 3°
Day4N
Day 5°
Day 6°
Nitrite1
(mg NO2-N/L)
<0.0011
<0.0011
0.006
0.005
0.002
0.02
0.01
0.02
0.01
Nitrate"
(mg NO3-N/L)
0.307
0.305
<0.0012
O.0012
0.258
0.061
0.079
0.074
0.0012
<0.0012
Temperature
(°C)
old
24.7
24.7
24.7
24.6
25.1
24.7
pH
(S.U.)
new" old0
8.00
8.16
8.20
8.21
8.10
8.12
8.35
8.47
8.88
8.56
8.47
8.31
D.O.
(mg/L)
new" old0
7.9
8.0
8.0
7.3
8.2
7.6
8.7
9.0
8.9
8.9
9.0
8.9
Conductivity
(mmhos)
183
195
180
197
187
181
Alkalinity
(mg/L)
76
74
58
Hardness
(mg/L)
96
100
88
Ammonia
(mg/L)
ND
ND
0.48 (0.366) mg/L
Average
DayON
Day1°
Day2N
Day 3°
Day4N
Day 5°
Day 6°
0.50
0.34
0.48
0.23
0.26
0.36
0.111
0.184
0.167
0.111
0.159
8.12
8.19
8.22
8.28
8.03
8.08
8.37
8.49
8.87
8.60
8.67
8.43
7.9
8.0
8.0
7.3
8.2
8.0
8.7
9.0
8.9
8.9
9.0
8.9
196
210
203
204
191
188
0.95(0.82) mg/L
Average
DayON
Day1°
Day2N
Day 3°
Day4N
Day 5°
Day 6°
1.00
0.80
0.85
0.74
0.72
0.82
0.239
0.255
0.357
0.172
0.251
8.16
8.20
8.23
8.29
8.05
8.10
8.37
8.50
8.87
8.64
8.64
8.44
8.0
8.0
8.0
7.3
8.2
7.8
8.7
9.0
8.9
8.9
9.3
8.9
206
217
218
208
199
194
1.90 (1.75) mg/L
Average
DayON
Day1°
Day2N
Day 3°
Day4N
Day 5°
Day 6°
1.94
1.61
1.98
1.70
1.61
1.67
1.75
0.151
0.233
0.323
0.145
0.215
0.124
24.7
24.8
24.4
24.6
25.2
24.7
8.15
8.19
8.23
8.29
8.07
8.09
8.39
8.54
8.87
8.61
8.66
8.39
8.0
8.0
8.0
7.4
8.2
7.9
8.7
9.0
8.9
8.9
9.0
8.9
213
225
231
215
206
201
3.81 (3.56) mg/L
Average
DayON
Day1°
Day2N
Day 3°
Day4N
Day 5°
Day 6°
3.77
3.81
3.62
3.29
3.38
3.57
0.291
0.041
0.558
0.818
0.332
8.16
8.15
8.23
8.27
8.08
8.09
8.40
8.54
8.84
8.67
8.67
8.43
8.1
8.0
8.0
7.2
8.2
7.9
8.7
9.0
8.9
8.9
9.1
8.9
227
238
244
229
222
215
76
76
74
76
76
104
108
104
108
108
ND
7.61 (7.31) mg/L
Average
DayON
Day1°
Day2N
Day 3°
Day4N
Day 5°
Day 6°
7.6
7.0
7.3
0.11
O.11
8.14
8.17
8.40
8.4
8.5
8.7
253
261
76
76
108
104
ND
1 Nitrite Analysis Method SM 4500 NO2
b Nitrate Analysis Method SM 4500 NO3
ND Not Detect; below detection limit
N "New" test solutions are fresh solutions prepared each day and are used to initiate the toxicity test and to renew the existing test solutions.
82
-------
Boron
Table 60 provides a summary of estimated LC50 values for the nine toxicity tests
performed using boron. LCso values ranged between 28.4 and >544 mg B/L.
Table 60. LCso estimates for toxicity tests performed using boron.
Test Species and Duration
Lampsilis siliquoidea 96 hr
Pimephales promelas - 96 hr
Pimephales promelas - 3 2 day
Ceriodaphnia dubia - 48 hr (pH 7.75)
Pimephales promelas - 96 hr (pH 6.75)
Pimephales promelas - 96 hr (pH 7.75)
Pimephales promelas - 96 hr (pH 8.75)
Ligumia recta - 96 hr
Megalonaias nervosa - 96 hr
LC50 (mg B/L)
137
101
28.4
76.9
70.6
137
133
147
>544
For each of the acute toxicity tests completed using boron, two tables were generated: the
first summarizes the test results for each toxicity test, including nominal and analytical
test concentration and LC50 estimates with confidence intervals; the second table
summarizes analytical chemistry data collected throughout the toxicity tests. The results
of chronic tests performed with boron were summarized in three tables: the first
summarizes nominal and analytical test concentrations, LCso estimates with confidence
intervals, NOEC and LOEC estimates, mean survival and mean biomass; the second table
summarizes replicate-specific survival and growth data and the third table summarizes
analytical chemistry data collected throughout the toxicity tests. Also discussed, if
applicable, are deviations from the guidance provided in the ASTM method used to
complete the toxicity testing.
83
-------
96-hr Toxicity of Boron on Lampsilis siliciuoidea
The 96-hr test to determine the toxicity of boron on L. siliquoidea was completed by
INHS. Test organisms, < 5-day old juveniles collected from the Missouri State University
laboratory culture, were acclimated to the dilution water (MHRW), test temperature and
other test conditions prior to test initiation. Once acclimated, test organisms were
examined for any disease, stress, parasites, etc. If free from ailments, test organisms were
randomly assigned to the test chambers (which were randomly assigned to testing
locations); four replicates were used per treatment with five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 500, 250, 125, 62.5, and 31.3
mg B/L.
Testing was conducted at 20 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LC50 value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 61; test results are provided in Table 62. Analytical chemistry data are provided
in Table 63. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 18.
84
-------
Table 61. Test conditions for 96-hour toxicity test on Lampsilis siliquoidea with
boron.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg B/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination-mg
B/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Lampsilis siliquoidea, juveniles <5 days old (Missouri State
University)
Static, 96 hours
June 03 - 07, 2009
20 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
50 mL beaker
40 mL
5
4
20
500, 250, 125, 62.5, and 31.3
524, 260, 140, 72, and 34
None
USEPA MHRW
Boric acid: Acros Organics, 99.6%, ACS Reagent (crystals)
Cas. No. 10043-35-3, Lot # B0124654 and Borax (sodium
tetraborate decahydrate), 99.5 +% (for analysis ACS), Cas.
No. 1303-96-5, Lot # A0256722
None
None
Mortality (LC50)
85
-------
Table 62. Test results for 96-hour toxicity test on Lampsilis siliquoidea with boron.
Results of a Lamysilis siliciuoidea 96-Hour Static Acute Toxicitv Test
Conducted 06/03/09 - 06/07/09 Using: Boric acid Cas. No. 10043-35-3 and Borax Cas. No.
1303-96-5
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
31.3(34)mg/L
62.5 (72) mg/L
125(140)mg/L
250 (260) mg/L
500 (524) mg/L
Cumulative Percent Affected a
24-Hr
5
0
0
10
15
15
48-Hr
5
10
25
15
25
20
72-Hr
5
25
30
35
60
90
96-Hr
5
35
35
45
90
95
LCSO Values* (mg/L)
24-Hr 48-Hr
>524 >524
72-Hr 96-Hr
181 137
96-Hour LCSO* = 137 mg/L
LC50 95% Confidence Limits
24-Hr 48-Hr
LL NR NR
ULNR NR
72-Hr 96-Hr
110 86
296 220
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 and EC50
Confidence Limit Values: Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC50 and EC50 values are determined based on measured concentrations.
86
-------
Table 63. Analytical chemistry data for 96-hour toxicity test on Lampsilis siliquoidea
with boron.
Nominal (Measured) Test
Concentration
Dilution water/Control
31.3(34)mg/L
62.5(72)mg/L
125 (140)mg/L
250(260)mg/L
500(524)mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
Boron" Temperature pH D.O. Conductivity Alkalinity Hardness
(mg/L) (°C) (s.u.) (mg/L) (umhos) (mg/L) (mg/L)
0.1
1.1
0.3
35
33
34
69
76
72
130
150
140
250
270
260
500
550
524
20.0
19.9
20.0
20.1
19.9
20.1
19.8
20.0
20.1
19.9
20.0
19.8
20.1
20.1
20.0
20.2
19.7
20.1
20.1
19.9
20.0
19.9
20.1
20.1
19.9
20.1
20.0
20.1
20.1
20.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.1
8.0
7.81
7.10
7.80
7.05
7.80
6.99
7.92
6.97
7.96
6.92
7.99
6.89
305
305
322
320
344
350
385
390
464
465
619
625
60
62
82
82
90
92
116
120
164
164
272
270
90
90
90
92
90
92
90
90
88
90
86
90
"Boron Analysis Method 200.7
87
-------
96-hr Toxicity of Boron on Megalonaias nervosa
The 96-hr test to determine the toxicity of boron onM nervosa was completed by INHS.
Test organisms, < 5-day old juveniles collected from the Genoa National Fish Hatchery,
were acclimated to the dilution water (MHRW), test temperature and other test conditions
prior to test initiation. Once acclimated, test organisms were examined for any disease,
stress, parasites, etc. If free from ailments, test organisms were randomly assigned to the
test chambers (which were randomly assigned to testing locations); four replicates were
used per treatment with five organisms per replicate. In one replicate of the 250 mg/L
treatment, a test organism was inadvertently crushed, but this was accounted for in the
LCso calculation.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 500, 250, 125, 62.5, and 31.3
mg B/L.
Testing was conducted at 20 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 64; test results are provided in Table 65. Analytical chemistry data are provided
in Table 66. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 19.
-------
Table 64. Test conditions for 96-hour toxicity test on Megalonaias nervosa with
boron.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg B/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination-mg
B/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Megalonaias nervosa, juveniles <5 days old, Genoa
National Fish Hatchery
Static, 96 hours
October 16 - 20, 2009
20 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
50 mL beaker
40 mL
5
4
20
500, 250, 125, 62.5, and 31.3
544, 275, 140, 74, and 37
None
USEPA MHRW
Boric acid: Acros Organics, 99.6%, ACS Reagent (crystals)
Cas. No. 10043-35-3, Lot # B0124654 and Borax (sodium
tetraborate decahydrate), 99.5 +% (for analysis ACS), Cas.
No. 1303-96-5, Lot # A0256722
None
None
Mortality (LC50)
89
-------
Table 65. Test results for 96-hour toxicity test on Megalonaias nervosa with boron.
Results of a Mesalonaias nervosa 96-Hour Static Acute Toxicitv Test
Conducted 10/16/09 - 10/20/09 Using: Boric acid Cas. No. 10043-35-3 and Borax Cas. No. 1303-96-5
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
31.3(37)mg/L
62.5 (74) mg/L
125(140)mg/L
250 (275) mg/L
500 (544) mg/L
Cumulative Percent Affected a
24-Hr
0
0
0
0
0
0
48-Hr
0
0
0
0
5
0
72-Hr
0
0
5
5
10
0
96-Hr
0
0
10
15
20
5
LCSO Values* (mg/L)
24-Hr 48-Hr
>544 >544
72-Hr 96-Hr
>544 >544
96-Hour LCSO* = >544mg/L
LC50 95% Confidence Limits
24-Hr 48-Hr
LL NR NR
ULNR NR
72-Hr 96-Hr
NR NR
NR NR
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 and EC50
Confidence Limit Values: Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC50 and EC50 values are determined based on measured concentrations.
90
-------
Table 66. Analytical chemistry data for 96-hour toxicity test on Megalonaias nervosa
with boron.
Nominal (Measured) Test
Concentration
Dilution water/Control
31.3(37)mg/L
62.5(74)mg/L
125 (140) mg/L
250 (275) mg/L
500 (544) mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
Boron" Temperature pH D.O. Conductivity Alkalinity Hardness
(mg/L) (°C) (s.u.) (mg/L) (mmos) (mg/L) (mg/L)
<0.02
<0.02
na
36
38
37
72
76
74
140
140
140
270
280
275
520
570
544
21.0
20.8
20.5
20.3
20.6
21.0
20.5
20.5
20.4
20.6
21.0
20.8
20.5
20.3
20.5
21.0
20.9
20.6
20.5
20.7
21.0
20.8
20.6
20.5
20.8
21.0
20.9
20.6
20.4
20.8
7.8
7.7
7.8
8.0
8.0
8.0
7.9
7.9
8.0
8.0
8.0
7.9
7.9
8.0
8.0
7.9
8.0
8.0
8.1
8.1
7.9
8.0
8.0
8.1
8.1
7.9
8.0
8.0
8.1
8.2
7.95
8.27
8.21
8.21
7.62
8.21
8.32
8.22
8.28
8.15
8.20
8.34
8.22
8.27
8.25
8.25
8.38
8.23
8.29
8.27
8.23
8.40
8.21
8.25
8.28
8.24
8.32
8.20
8.25
8.23
300
303
305
290
337
320
320
320
330
351
340
343
345
347
364
381
389
390
401
417
460
461
461
488
504
613
616
618
638
654
60
68
70
72
86
90
110
115
160
178
266
276
88
90
00
00
00
00
00
00
00
00
00
00
00
00
00
00
88
86
88
* Boron Analysis Method 200.7
na=not applicable
91
-------
96-hr Toxicity of Boron on Ligumia recta
The 96-hr test to determine the toxicity of boron on L. recta was completed by INHS.
Test organisms, < 5-day old juveniles collected from the Missouri State University
laboratory culture, were acclimated to the dilution water (MHRW), test temperature and
other test conditions prior to test initiation. Once acclimated, test organisms were
examined for any disease, stress, parasites, etc. If free from ailments, test organisms were
randomly assigned to the test chambers (which were randomly assigned to testing
locations); four replicates were used per treatment with five organisms per replicate. One
replicate was mistakenly loaded with only four individuals, but this was accounted for in
the LCso calculation.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 500, 250, 125, 62.5, and 31.3
mg B/L.
Testing was conducted at 20 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 67; test results are provided in Table 68. Analytical chemistry data are provided
in Table 69. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 20.
92
-------
Table 67. Test conditions for 96-hour toxicity test on Ligumia recta with boron.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg B/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination-mg
B/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Ligumia recta, juveniles <5 days old, Missouri State
University
Static, 96 hours
September 10- 14,2009
20 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
50 mL beaker
40 mL
5
4
20
500, 250, 125, 62.5, and 31.3
510, 255, 130, 64, and 33
None
USEPA MHRW
Boric acid: Acros Organics, 99.6%, ACS Reagent (crystals)
Cas. No. 10043-35-3, Lot # B0124654 and Borax (sodium
tetraborate decahydrate), 99.5 +% (for analysis ACS), Cas.
No. 1303-96-5, Lot # A0256722
None
None
Mortality (LC50)
93
-------
Table 68. Test results for 96-hour toxicity test on Ligumia recta with boron.
Results of a Ligumia recta 96-Hour Static Acute Toxicity Test
Conducted 09/10/09 - 09/14/09 Using: Boric acid Cas. No. 10043-35-3 and Borax Cas. No. 1303-96-5
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
31.3(33)mg/L
62.5 (64) mg/L
125(130)mg/L
250 (255) mg/L
5 00 (5 10) mg/L
Cumulative Percent Affected a
24-Hr
0
0
0
0
0
0
48-Hr
0
0
0
0
0
0
72-Hr
0
0
0
0
0
0
96-Hr
0
30
30
50
58
100
LCSO Values* (mg/L)
24-Hr 48-Hr 72-
Hr 96-Hr
>510 >510 >510 147
96-Hour LCSO* = 147 mg/L
LC50 95% Confidence Limits
24-Hr 48-Hr 72-
Hr 96-Hr
LLNR NR NR 88
ULNR NR NR 246
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not
reliable
Method(s) Used to Determine LC50 and EC50
Confidence Limit Values: Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC50 and EC50 values are determined based on measured concentrations.
94
-------
Table 69. Analytical chemistry data for 96-hour toxicity test on Ligumia recta with
boron.
Nominal (Measured) Test
Concentration
Dilution water/Control
31.3(33)mg/L
62.5(64)mg/L
125(130)mg/L
250 (255) mg/L
500(510)mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
Boron" Temperature pH D.O. Conductivity Alkalinity Hardness
(mg/L) (°C) (s.u.) (mg/L) (umhos) (mg/L) (mg/L)
<0.02
<0.02
na
33
34
33
62
66
64
130
130
130
250
260
255
500
520
510
20.4
19.5
19.1
19.2
19.2
20.5
19.5
19.0
19.3
19.3
20.5
19.5
19.0
19.3
19.3
20.4
19.5
19.0
19.3
19.3
20.5
19.5
19.2
19.3
19.3
20.5
19.5
19.4
19.1
19.1
7.9
8.1
8.0
8.1
8.0
8.1
8.0
8.1
8.1
8.1
8.1
8.1
8.18
8.14
8.14
8.10
8.13
8.11
8.12
8.05
8.12
8.03
8.11
8.12
301
312
320
334
341
353
382
394
460
472
616
634
60
60
68
68
90
90
112
112
170
170
270
270
92
92
92
92
90
90
90
90
90
90
90
90
"Boron Analysis Method 200.7
na = not applicable
95
-------
96-hr Toxicity of Boron on Pimephales promelas
The 96-hr test to determine the toxicity of boron on P. promelas was completed by
GLEC. Test organisms, collected from the GLEC laboratory culture, were acclimated to
the dilution water (de-chlorinated Lake Michigan water), test temperature and other test
conditions prior to test initiation. Once acclimated, test organisms were examined for any
disease, stress, parasites, etc. If free from ailments, test organisms were randomly
assigned to the test chambers (which were randomly assigned to testing locations); two
replicates were used per treatment with ten organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 38.9, 64.8, 108, 180, 300 and
500mgB/L.
Testing was conducted at 25 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LC50 value was determined
using the Probit method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 70; test results are provided in Table 71. Analytical chemistry data are provided
in Table 72. Accompanying information, including raw laboratory data, analytical
chemistry data, reference toxicant data and statistical analyses, is provided in Appendix
21.
96
-------
Table 70. Test conditions for 96-hour toxicity test on Pimephales promelas with
boron.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg B/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination-mg B/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Pimephales promelas, (weight 0.12g and 19.8 mm length),
GLEC Culture
Static, 96 hours
September 2-September 6, 2009
25 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
4000 mL beaker
3500 mL
10
2
20
500, 300, 180, 108, 64.8, and 38.9
546, 352, 200, 123, 71.5, and 46.1
None
De-chlorinated Lake Michigan Water
Boric Acid: Sigma Aldrich, ACS Reagent >= 99.5% Cas.
No. 10043-35-3, Batch 118K0007 and Borax (sodium
tetraborate decahydrate) Sigma Aldrich, > 99.5%, ACS
reagent, Cas. No. 1303-96-4, Lot # 118K0172
None
None
Mortality (LC50)
97
-------
Table 71. Test results for 96-hour toxicity test on Pimepehales promelas with boron.
Results of a Pimephales promelas _ 96-Hour Static Acute Toxicitv Test
Conducted 09/02/09 - 09/06/09 Using: Boron (Boric Acid: Sigma Aldrich Cas No. 10043 35-3)
(Borax: Sigma Aldrich Cas No. 1303-96-4)
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
38.9(46.1)mg/L
64.8(71.5)mg/L
108 (123) mg/L
180(200)mg/L
300 (352) mg/L
500 (546) mg/L
Cumulative Percent Affected a
24-Hr
0
(0)
0
(0)
0
(0)
0
(0)
0
(0)
0
(0)
45
(45)
48-Hr
0
(0)
0
(0)
0
(0)
0
(0)
5
(5)
65
(65)
100
(100)
72-Hr
0
(0)
0
(0)
0
(5)
20
(20)
60
(60)
100
(100)
100
(100)
96-Hr
0
(0)
0
(0)
15
(15)
70
(70)
100
(100)
100
(100)
100
(100)
LCSO* Values (mg/L)
24-Hr 48-Hr
>546 312
72-Hr 96-Hr
173 101
96-Hour LC50 *= 101 mg/L
LCSO *95% Confidence Limits
24-Hr 48-Hr
LL NA 271
UL NA 353
72-Hr 96-Hr
150 88.3
200 116
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 Confidence
Limit Values: Probit
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ values are determined based on measured concentrations.
98
-------
Table 72. Analytical chemistry data for 96-hour toxicity test on Pimephales promelas
with boron.
Nominal (and M
Test Concent
Dilution
water/Control
easured)
•ations
DayO
Day 1
Day 2
Day3
Day 4
Boron
(mg/L)
0.03
0.04
0.035
Temperature
(°C)
25.0
24.7
24.8
24.8
24.7
PH
(S.U.)
8.01
7.73
7.54
7.65
7.95
D.O.
(mg/L)
8.0
6.3
5.9
5.7
6.0
6.0
6.1
5.5
6.1
Conductivity
(mmos)
290
278
Alkalinity
(mg/L)
104
100
Hardness
(mg/L)
144
132
Ammonia
(mg/L)
ND
38.9 ( 46.1 )mg/L
DayO
Day 1
Day 2
Day3
Day 4
54.7
37.4
46.1
25.0
24.6
24.8
24.9
24.7
8.16
8.12
8.02
8.05
8.07
8.0
6.7
6.4
6.0
6.4
6.8
6.5
5.9
6.5
318
323
64.8 (71 .5) mg/L
DayO
Day 1
Day 2
Day3
Day 4
82
61
71.5
25.0
24.7
24.8
24.8
24.7
8.14
8.17
8.05
8.11
8.02
7.9
7.7
4.6
6.3
5.4
6.7
6.4
4.5
6.4
335
346
108(123)mg/L
DayO
Day 1
Day 2
Day3
Day 4
146
99.1
123
25.0
24.8
25.0
24.9
24.8
8.18
8.18
8.15
8.13
8.15
8.0
7.0
6.5
5.9
6.7
6.4
6.2
6.4
6.0
362
376
180(200) mg/L
DayO
Day 1
Day 2
Day3
Day 4
223
176
200
25.0
25.1
24.9
24.9
24.8
8.18
8.17
8.14
8.11
8.13
8.0
6.2
6.1
5.9
5.3
6.3
5.8
4.7
5.3
406
422
146
136
300 (352) mg/L
DayO
Day 1
Day 2
Day3
Day 4
388
316
352
25.0
24.7
24.8
24.8
8.17
8.17
8.17
8.15
8.0
7.0
6.8
5.8
6.3
5.4
5.0
476
500 (546) mg/L
DavO
Day 1
Day 2
Day3
Day 4
585
507
546
25.0
24.7
24.8
8.14
8.15
8.13
8.0
7.0
7.4
7.1
7.5
597
316
168
ND
a Boron Analysis EPA 200.8
ND Not Detect; below detection limit
99
-------
32-day Toxicity of Boron on Pimephales promelas
The 32-day test to determine the toxicity of boron on P. promelas was completed by
GLEC. The fish were continuously exposed for 32 days to five concentrations of boron
(nominal concentrations of 6.3, 12.5, 25, 50 and 100 mg B/L) and to a dilution water
control using a continuous flow-through system (Benoit et al. 1982). The temperature-
controlled test concentration solutions were supplied to each test chamber via the
continuous flow-through system at a rate of approximately four turnovers a day. There
were four replicate test chambers for each treatment. The flow through test was
conducted at 25 ± 1 °C with a photoperiod of 16 hr light and 8 hr darkness (ambient
laboratory light).
After test concentrations had achieved steady state in the flow through system, the test
was initiated with < 24 hour old fertilized embryos. The embryos were randomly
assigned to incubation cups until each incubation cup contained 30 embryos. The
incubation cups were randomly assigned to the 2.5 L glass test chambers (1 cup per
chamber) and suspended in the test solutions from a rocker arm assembly. The rocker
arm assembly moves the incubation cups in a reciprocal motion within each test chamber.
Embryos were inspected on a daily basis and the number of live, hatched and dead
embryos was recorded. On Day 8 of the test (four days after first hatch), the surviving
fish were randomly thinned to achieve 20 fish in each test chamber. The remainder of the
surviving fish was discarded. The number of surviving fish was recorded at test
termination (32 days). In addition, the wet weights were recorded for each fish at test
termination. Because of the size range offish in each test chamber, all of the fish from
each test chamber were weighed together to determine average dry weight.
Instantaneous water temperature measurements made on September 12 (Day 2: 23.5°C-
24.5°C), October 1 (Day 21: 23.7°C-24.3°C), October 9 (Day 29: 25.9°C-26.5°C),
October 11 (Day 31: 23.7°C-24.1°C), and October 12 (Day 32: 23.8°C-24.5°C) exceeded
the allowable range of 25 ± 1 °C in the toxicity testing method. However, the overall
average water temperatures (across the duration of the test) in each replicate were within
± 0.5 °C of the target test temperature (25 °C) in all treatments. Therefore, the water
temperature exceedances noted above likely had no effect on the results of this study.
On September 19 and 20, 2009 (test days 9 and 10) 60-90 percent mortality occurred in
replicates one and two of the laboratory control. It is of GLEC's opinion that the
equipment used during the thinning procedure on test day 8 contributed toxicity to these
two control replicates, resulting in the high fish mortality. This high mortality was
communicated to the EPA Work Assignment Manager and GLEC was advised to
continue the test with the assumption that further control mortality would result in test
failure. No further toxicity was observed in the remaining control fish throughout the test.
However, because of the mortality observed in the control treatments, only replicates
three and four of the laboratory control were used in the survival and growth statistical
comparisons.
100
-------
Once the test was complete, the LCso, NOEC and LOEC values were determined using
the average measured concentrations with the Spearman Karber and ANOVA methods.
LC25; LC20 and LCio values were determined using the Probit method and EC50, EC25,
EC2o and ECio values were estimated using EPA's TRAP.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 73; test results are provided in Table 74. Survival and growth data are provided
in Table 75 and analytical chemistry data are provided in Table 76. Accompanying
information, including raw laboratory data, analytical chemistry data and statistical
analyses, is provided in Appendix 22.
101
-------
Table 73. Test conditions for 32-day toxicity test on Pimephales promelas with
boron.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination-mg/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Pimephales promelas, (<24 hour fertilized embryos ),
GLEC Culture
Continuous flow-through; 32 days
September 10-October 12, 2009
25 + 1
Ambient Laboratory, 10-20 uE/m2/s
16 h light, 8 h darkness
Live Brine Shrimp (Artemia nauplii) Twice daily
2.5 Liter glass Tank
2000 mL
30 eggs, thinned to 20 larvae after hatch
4
120 eggs, thinned to 80 larvae after hatch
100, 50.0, 25.0, 12.5 and 6.25 mg/L-boron
112, 56.5, 27.4, 12.9, and 5.90 mg/L-nitrate
Continuous flow through, 4 turnovers per day
De-Chlorinated Lake Michigan Water
Boric Acid: Sigma Aldrich, ACS Reagent >= 99.5% Cas.
No. 10043-35-3, Batch 118K0007 and Borax (sodium
tetraborate decahydrate) Sigma Aldrich, > 99.5%, ACS
reagent, Cas. No. 1303-96-4, Lot # 118K0172
None
None
Survival (LC50, LC25)LC20 and LC10,NOEC and LOEC) and
Growth (EC50 EC25 EC20 and EC10, NOEC and LOEC)
102
-------
Table 74. Test results for 32-day toxicity test on Pimepehales promelas with boron.
Results of a Pimephales promelas
Conducted 09/10/09 - 10/12/09
Test Solution Concentrations
Measured
Primary
Control/
Dilution Water
32-Day Continuous Flow Chronic Toxicitv Test
Using: Boron ( Boric Acid: Sigma Aldrich Cas. No. 10043-35-3
Borax: Sigma Aldrich Gas. No. 1303-96-4)
5.90 mg/L 12.9 mg/L 27.4 mg/L 56.5 mg/L
112mg/L
Embryo Percent Hatch (%)
100
100
100
100
100
100
32-Day Mean Survival (%)
97.5
92.5
58.S
1.3a
32-Day Average Biomass1 (mg )
10.1
7.27 ;
7.37;
4.29a
Embryo Percent Hatch NOEC:
32-Day LC50*:
32-Day Survival NOEC:
32-Day Survival LOEC:
32-Day Growth NOEC:
32-Day Growth LOEC:
32-Day LC25:
100 mg/L 32-Day LC20:
28.4 mg/L (25.5 mg/L-31.7 mg/L) 32-Day LC10:
12.9 mg/L 32-Day EC50:
27.4 mg/L 32-Day EC25:
<5.9mg/L 32-Day EC20:
5.9 mg/L 32-Day EC10:
20.8 mg/L (18.2 mg/L - 23.4 mg/L)
18.6 mg/L (15.9 mg/L -21.3 mg/L)
12.8 mg/L (9.6 mg/L - 16.0 mg/L)
28.7 mg/L (23.7 mg/L - 34.6 mg/L)
25.1 mg/L (17.1 mg/L - 36.7 mg/L)
24.2 mg/L (14.3 mg/L -41.0 mg/L)
21.9 mg/L (8.6 mg/L - 56.0 mg/L)
*: All LC, EC, NOEC and LOEC values are determined based on the average measured boron concentration.
NOEC: No-Observed-Effect-Concentration
LOEC: Lowest-Observed-Effect-Concentration
'Biomass: Biomass is the average dry weight of the four replicates calculated by the total dry weight of surviving
organisms divided by the initial number of organisms (20).
103
-------
Table 75. Survival and growth data for 32-day toxicity test on Pimephales promelas
with boron.
Nominal (and Measured) Test
Concentration
Dilution
water/Control
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
Number of
Eggs at Test
Initiation
30
30
30
30
Number of
Hatched
Larvae
30
30
30
30
Number of
Dead Eggs
0
0
0
0
Percent
Hatched
Larvae
100.0
100.0
100.0
100.0
Number of
Larvae at Test
Termination
1
4
19
20
Percent Survival
at Test
Termination*
5.0
20.0
95.0
100.0
Biomass (mg)
1.27
4.83
9.62
10.52
Average 100.0 97.5 2 10.07 2
6.25 (5. 9) mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
30
30
30
30
0
0
0
0
100.0
100.0
100.0
100.0
17
18
20
20
85.0
90.0
100.0
100.0
6.69
6.84
7.34
8.21
Average 100.0 93.8 7.27
12.5 (12.9) mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
30
30
30
30
0
0
0
0
100.0
100.0
100.0
100.0
17
19
19
19
85.0
95.0
95.0
95.0
6.99
8.53
6.77
7.17
Average 100.0 92.5 7.37
25. 0(27.4) mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
30
30
30
30
0
0
0
0
100.0
100.0
100.0
100.0
10
10
13
14
50.0
50.0
65.0
70.0
3.76
3.62
5.05
4.73
Average 100.0 58.8 4.29
50.0 (56.5) mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
30
30
30
30
0
0
0
0
100.0
100.0
100.0
100.0
0
0
1
0
0.0
0.0
5.0
0.0
0.00
0.00
0.25
0.00
Average 100.0 1.3 0.06
100 (11 2) mg/L
Replicate # 1
Replicate # 2
Replicate # 3
Replicate # 4
30
30
30
30
30
30
30
30
0
0
0
0
100.0
100.0
100.0
100.0
0
0
0
0
0.0
0.0
0.0
0.0
0.00
0.00
0.00
0.00
Average 100.0 0.0 0.00
* On Day 8 of the test, (four days after first hatch) the surviving fish were randomly thinned to 20 fish in each test chamber. Percent
surval at test termination is the number of surviving at test termination divided by 20.
Biomass: Biomass is the total dry weight of surviving organisms divided by the initial number of organisms (20)
Due to a technician error on day 8, only replicates 3 and 4 were used in the growth and survival analysis for the laboratory control
104
-------
Table 76. Analytical chemistry data for 32-day toxicity test on Pimephales promelas
with boron.
Nominal (and Measured) Test
Conent rations
Dilution
Water/Control
Replicate #
1
Replicate #
2
Replicate #
3
Replicate #
4
Boron
(mg/L)
0.0
(0.0-0.1)
0.0
(0.0-0.1)
0.1
(0.0-0.4)
0.0
(0.0-0.1)
Temperature
(°C)
25.1
(23.7-26.0)
24.9
(23.8-26.3)
24.9
(24.2-26.0)
24.9
(24.1-25.5)
PH
(s.u.)
8.08
8.11
8.16
8.17
DO
(mg/L)
7.5
(7.0-8.1)
7.3
(6.8-7.8)
7.2
(6.9-7.4)
7.3
(7.1-7.5)
SC
(mmos)
290
296
300
304
Hardness
(mg/L)
112
132
NM
NM
Alkalinity
(mg/L)
100
99
NM
NM
Ammonia
(mg/L)
ND
ND
ND
ND
Average Boron
ni2/L 0
6.25 mg/L
(5.9 mg/L)
Replicate #
1
Replicate #
2
Replicate #
3
Replicate #
4
5.8
(4.9-6.6)
6.0
(4.9-7.0)
6.0
(5.0-7.1)
5.9
(5.1-6.6)
24.6
(23.9-25.6)
24.9
(24.0-26.1)
25.0
(24.1-26.2)
24.8
(23.9-25.4)
8.16
8.17
8.21
8.18
7.5
(7.0-7.9)
7.3 (6.7
8.0)
7.1
(6.6-7.5)
7.1
(6.7-7.6)
301
305
305
309
128
132
NM
NM
106
104
NM
NM
ND
ND
ND
ND
Average Boron
mg/L 5.9
12.5 mg/L
(12.9 mg/L)
Replicate #
1
Replicate #
2
Replicate #
3
Replicate #
4
13.0
(11.6-13.9)
13.1
(11.5-14.2)
13.1
(11.6-14.7)
12.3
(11.0-13.8)
24.8
(23.7-25.9)
24.9
(24.1-26.1)
25.1
(24.2-26.5)
25.0
(24.2-25.6)
8.17
8.20
8.25
8.21
7.4
(6.8-8.1)
7.5
(6.9-8.0)
7.3
(7.0-7.6)
7.4
(7.1-7.8)
307
308
310
313
132
130
NM
NM
110
107
NM
NM
ND
ND
ND
ND
Average Boron
mg/L 12.9
25.0 mg/L
(27.4 mg/L)
Replicate #
1
Replicate #
2
Replicate #
3
Replicate #
4
28.6
(23.8-34.3)
28.0
(25.1-32.5)
26.2
(22.7-31.1)
26.3
(22.9-31.2)
24.6
(23.7-25.7)
24.7
(23.8-26.2)
24.8
(23.8-26.1)
24.5
(23.5-25.2)
8.18
8.21
8.12
8.22
7.5
(7.1-8.0)
7.5
(7.2-8.0)
7.4
(7.1-7.8)
7.4
(7.1-7.7)
314
316
317
320
128
128
NM
NM
116
113
NM
NM
ND
ND
ND
ND
Average Boron
mg/L 27.4
50.0 mg/L
(56.5 mg/L)
Replicate #
1
Replicate #
2
Replicate #
3
Replicate #
4
56.1
(48.2-65.4)
56.4
(46.9-63.3)
57.4
(45.8-67.9)
56.2
(45.3-63.4)
24.8
(23.9-25.7)
24.7
(24.1-26.0)
24.8
(23.9-26.0)
24.8
(23.9-25.4)
8.21
8.23
8.27
8.24
7.6
(7.1-8.5)
7.5
(7.2-8.1)
7.5
(7.1-8.3)
7.5
(7.3-8.0)
331
333
351
337
132
130
NM
NM
128
127
NM
NM
ND
ND
ND
ND
Average Boron
mg/L 56.5
100 mg/L (112
mg/L)
Replicate #
1
Replicate #
2
Replicate #
3
Replicate #
4
111
(98.9-117)
111
(93.7-117)
111
(97.2-122)
113
(103-123)
24.8
(23.6-26.0)
25.0
(23.9-26.2)
25.0
(24.4-26.2)
24.9
(24.0-25.6)
8.22
8.23
8.27
8.24
7.6
(7.0-8.4)
7.6
(7.2-8.4)
7.4
(7.0-8.0)
7.6
(7.3-8.0)
363
367
367
370
128
130
NM
NM
152
150
NM
NM
ND
ND
ND
ND
Average Boron
mp/T. 112
DO: Dissolved Oxygen
SC: Specific Conductance
ND: Non Detect; below detection limit.
NM: Not Measured
105
-------
48-hr Toxicity of Boron (pH 7.75) on Ceriodaphnia dubia
The 48-hr test to determine the toxicity of boron (pH 7.75) on C. dubia was completed by
GLEC. The C. dubia were continuously exposed for 48-hours to five concentrations of
boron and to a dilution water control with a target test pH of 7.75 (range of 7.65 to 7.85)
using a continuous flow-through system (modified Benoit mini-dilutor) and an in-line pH
adjustment/metering unit. The pH of both the dilution water and stock solution were
adjusted by using a 1:1 ratio of sulfuric acid and hydrochloric acid (acid solution). The
pH adjusted stock solution was delivered to mixing cells and diluted with pH adjusted,
de-chlorinated Lake Michigan water to achieve target nominal concentrations of boron
and a target test pH of 7.75.
Due to the buffering capacity of the borax and boric acid solution used to prepare the
boron concentrations, EPA agreed that GLEC should target the dilution water control pH
at 7.75 (range of 7.65 to 7.85). The five test concentrations were targeted to a pH of ± 0.1
pH unit from the pH value defined at test initiation in each test concentration (i.e.
regardless of whether or not the pH in the test concentrations were ±0.1 pH units from
that observed in the control water). The temperature-controlled test concentration
solutions were supplied to each test chamber via the continuous flow-through system at a
rate of approximately four turnovers a day. There were four replicate test chambers for
each treatment. The flow through test was conducted at 25 ± 1 °C with a photoperiod of
16 hr light and 8 hr darkness (ambient laboratory light).
After test concentrations had achieved steady state in the flow through system, the test
was initiated with < 24 hour old C. dubia collected from the GLEC laboratory culture.
Although these organisms were cultured in, and acclimated to, the dilution water's
"natural" pH (typically between a pH of 7.9 and 8.2), they were not acclimated to the pH-
adjusted dilution water prior to test initiation for two reasons: 1) GLEC does not maintain
a laboratory culture of C. dubia in de-chlorinated Lake Michigan water maintained at a
pH of 7.75 and 2) adequate acclimation of the organisms to the pH-adjusted dilution
water would have jeopardized the age requirement (< 24 hour old at test initiation) for
test organisms required under the toxicity testing method (ASTM 2007).
The C. dubia were randomly assigned to test cups until each test cup contained five C.
dubia. The test cups were randomly assigned to the 2.5 L glass test chambers (1 cup per
chamber) and suspended in the test solutions from a rod. C. dubia were counted on a
daily basis and the number of live C. dubia was recorded. pH was recorded twice a day,
at a minimum of eight hours apart (i.e. morning and evening). The number of surviving
C. dubia was recorded at test termination (48-hours).
The test was completed at the following nominal boron concentrations: 25.0, 50.0, 100,
200, and 400 mg/L. The average pH for the dilution water control measured in the C.
dubia toxicity test for the 48-hour test period was 7.77. The average pH over the 48-hour
test duration for the five test concentrations of 27.6, 49.8, 118, 223, and 391 mg/L was
7.92, 8.03, 8.03, 8.07, and 8.06, respectively.
106
-------
Once the test was complete, the LCso was determined using the average measured test
concentrations with the Probit method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 77; test results are provided in Table 78. Analytical chemistry data are provided
in Table 79. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 23.
Table 77. Test conditions for 48-hour toxicity test on Ceriodaphnia dubia with boron
(pH 7.75).
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination-mg/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Ceriodaphnia dubia, < 24 hours old, GLEC Culture
Continuous flow-through, 48 hours
October 23-October 25, 2009
25 + 1
Ambient Laboratory, 10-20 uE/m /s
16 h light, 8 h darkness
None
2.5 Liter glass Tank
2000 mL
10
2
20
400,200, 100, 50.0, and 25.0
391,223, 118,49.8, and 27.6
Continuous flow through, 4 turnovers per day
De-Chlorinated Lake Michigan Water
Boric Acid: Sigma Aldrich, ACS Reagent >= 99.5% Cas.
No. 10043-35-3, Batch 118K0007 and Borax (sodium
tetraborate decahydrate) Sigma Aldrich, > 99.5%, ACS
reagent, Cas. No. 1303-96-4, Lot # 118K0172
None
None
Mortality (LC50)
107
-------
Table 78. Test results for 48-hour toxicity test on Ceriodaphnia dubia with boron at
pH 7.75.
Results of a Ceriodaphnia dubia 48-Hour Static Acute Toxicitv Test
Conducted 10/23/09 - 10/25/09 Using: Boron fBoric Acid: Sigma Aldrich Cas No
(Borax: Sigma Aldrich Cas No.
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
25.0 (27.6) mg/L
50.0 (49.8) mg/L
100 (11 8) mg/L
200 (223) mg/L
400 (391) mg/L
Cumulative Percent Affected a
24-Hr
0
(0)
0
(0)
0
(0)
15
(15)
75
(75)
100
(100)
48-Hr
0
(0)
0
(0)
25
(25)
75
(75)
100
(100)
100
(100)
72-Hr
96-Hr
24-Hr
172
1303-96-4)
. 10043-35-3)
LC50 Values* (mg/L)
48-Hr 72-
Hr 96-Hr
76.9 NA NA
48-Hour LC50* = 76.9 mg/L
LC50* 95% Confidence
24-Hr
LL 144
UL 202
48-Hr 72-
Limits
Hr 96-Hr
62.3 NA NA
94.9 NA NA
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not
reliable
Method(s) Used to Determine LC50 Confidence
Limit Values: Probit
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ values are determined based on measured concentrations.
108
-------
Table 79. Analytical chemistry data for 48-hour toxicity test on Ceriodaphnia dubia
with boron at pH 7.75.
Nominal (and Measured) Test
Conent rations
Dilution
Water/Control
Average
25mg/L
(27.6 mg/L)
Average
50 mg/L
(49.8 mg/L)
Average
100 mg/L
(118 mg/L)
Average
Replicate #
1
Replicate #
2
Replicate #
Replicate #
4
Replicate #
1
Replicate #
2
Replicate #
Replicate #
4
Replicate #
1
Replicate #
2
Replicate #
3
Replicate #
4
Replicate #
1
Replicate #
2
Replicate #
3
Replicate #
4
Boron
(mg/L)
0.05
(0.04-0.05)
0.06
(0.04-0.07)
0.05
(0.03-0.07)
0.06
(0.05-0.07)
0.05
27.9
(27.2-28.5)
27.3
(25.9-28.0)
27.4
(27.1-27.8)
28.0
(27.2-28.9)
27.6
49.8
(47.4-52.9)
50.1
(47.8-53.7)
49.3
(47.5-52.7)
49.8
(47.8-53.5)
49.8
120
(111-127)
120
(112-124)
118
(110-130)
115
(105-125)
118
Temperature
(°C)
24.6
(24.5-24.6)
25.0
(24.9-25.1)
25.2
(25.1-25.2)
24.9
(24.9-24.9)
24.8
24.7
(24.5-24.8)
25.1
(25.0-25.2)
24.8
(24.7-24.9)
24.8
(24.8-24.9)
24.7
24.6
(24.5-24.7)
24.8
(24.7-24.8)
24.8
(24.7-24.9)
24.8
(24.7-24.9)
24.6
24.8
(24.6-24.9)
24.9
(24.8-25.0)
24.8
(24.7-24.9)
24.9
(24.6-25.1)
24.8
pH
(s.u.)
7.65
(7.53-7.75)
7.75
(7.70-7.82)
7.84
(7.77-7.94)
7.71
(7.56-7.79)
7.77
7.89
(7.80-7.95)
7.96
(7.89-8.06)
7.92
(7.87-8.00)
7.97
(7.89-8.06)
7.92
8.00
(7.96-8.07)
8.04
(7.97-8.11)
8.00
(7.96-8.07)
8.04
(7.97-8.11)
8.03
8.07
(8.03-8.13)
8.08
(8.02-8.14)
8.07
(8.02-8.13)
8.07
(8.00-8.14)
8.03
DO
(mg/L)
7.7
(6.5-8.7)
7.9
(6.9-8.6)
7.7
(7.1-8.1)
7.5
(6.4-8.1)
7.7
7.3
(5.9-8.1)
7.7
(7.2-8.0)
7.6
(6.7-8.1)
7.7
(7.1-8.1)
7.6
7.4
(6.4-7.9)
7.7
(7.1-8.0)
7.8
(7.3-8.1)
7.5
(6.5-8.1)
7.6
7.5
(6.7-8.0)
7.6
(6.8-8.0)
7.5
(6.4-8.1)
7.6
(6.9-8.0)
7.5
SC
(mmos)
320
328
322
323
346
350
348
364
365
364
411
412
411
Hardness
(mg/L)
146
146
138
138
144
144
128
128
Alkalinity
(mg/L)
47
47
50
50
57
57
77
77
Ammonia
(mg/L)
ND
ND
ND
ND
ND
ND
ND
ND
200 mg/L
(223 mg/L)
Average
400 mg/L
(391 mg/L)
Average
Replicate #
1
Replicate #
2
Replicate #
Replicate #
4
Replicate #
1
Replicate #
2
Replicate #
Replicate #
4
227
(221-232)
222
(216-228)
218
(211-224)
225
(222-228)
223
392
(381-399)
391
(386-396)
391
(388-394)
391
(384-401)
391
24.7
(24.6-24.8)
24.5
(24.4-24.7)
24.7
(24.6-24.8)
25.1
(24.8-25.3)
24.8
24.5
(24.4-24.6)
24.6
(24.4-24.8)
24.5
(24.3-24.8)
24.6
(24.4-24.7)
24.6
8.08
(8.02-8.15)
8.08
(8.03-8.15)
8.09
(8.02-8.15)
8.07
(8.01-8.14)
8.07
8.06
(8.00-8.13)
8.07
(8.01-8.14)
8.07
(8.01-8.14)
8.06
(8.01-8.13)
8.06
7.7
(7.2-8.0)
7.5
(6.6-8.0)
7.7
(7.1-8.0)
7.6
(6.7-8.1)
7.6
7.6
(6.7-8.0)
7.7
(7.1-8.0)
7.7
(7.2-8.0)
7.5
(6.4-8.0)
7.6
475
477
476
587
589
588
132
132
132
132
105
105
167
167
ND
ND
ND
ND
DO: Dissolved Oxygen
SC: Specific Conductance
ND: Non Detect; below detection limit.
NM: Not Measured
109
-------
96-hour Toxicity of Boron (pH 6.75) on Pimephales promelas
The 96-hr test to determine the toxicity of boron (pH 6.75) on P. promelas was
completed by GLEC. The P. promelas (collected from the GLEC laboratory culture)
were continuously exposed for 96-hours to five concentrations of boron and to a dilution
water control with a target test pH of 6.75 (range of 6.65 to 6.85) using a continuous
flow-through system (modified Benoit mini-dilutor) and an in-line pH adjustment/
metering unit. The pH of both the dilution water and stock solution were adjusted by
using a 1:1 ratio sulfuric acid and hydrochloric acid (acid solution). The pH adjusted
stock solution was delivered to mixing cells and diluted with pH adjusted Lake Michigan
water to achieve target nominal concentrations of boron and a target test pH of 6.75. Due
to the buffering capacity of the borax and boric acid solution used to prepare the boron
concentrations, EPA agreed that GLEC should target to a pH of ± 0.1 pH unit from the
pH value defined at test initiation in each test concentration (i.e. regardless of whether or
not the pH in the test concentrations were ±0.1 pH unit from that observed in the control
water). The temperature-controlled test concentration solutions were supplied to each test
chamber via the continuous flow-through system at a rate of approximately four
turnovers a day. There were two replicate test chambers for each treatment. The flow
through test was conducted at 25 ± 1 °C with a photoperiod of 16 hr light and 8 hr
darkness (ambient laboratory light).
After test concentrations had achieved steady state in the flow through system, the test
was initiated with the P. promelas. Although these organisms were cultured in, and
acclimated to, the dilution water's "natural" pH (typically between a pH of 7.9 and 8.2),
they were not acclimated to the pH-adjusted dilution water prior to test initiation for two
reasons: 1) GLEC does not maintain a laboratory culture of P. promelas in de-
chlorinated Lake Michigan water maintained at a pH of 6.75 and 2) adequate acclimation
of the organisms to the pH-adjusted dilution water may have jeopardized the weight
requirement (0.1-5 g at test initiation) for test organisms required under the toxicity
testing method (ASTM 2007).
The P. promelas were randomly assigned to the 2.5 L glass test chambers until each test
chamber contained ten P. promelas. P. promelas were counted on a daily basis and the
number of live P. promelas was recorded. pH was recorded twice a day, at a minimum of
eight hours apart (i.e., morning and evening). The number of surviving P. promelas was
recorded at test termination (96-hours).
The test was completed at the following nominal boron concentrations: 25.0, 50.0, 100,
200, and 400 mg/L (dilution factor of 0.5). The average pH for the dilution water control
measured in the P. promelas toxicity test for the 96-hour test period was 6.67. The
average pH over the 96-hour test duration for the five test concentrations of 32.9, 55.2,
122, 224, and 394 mg/L was 6.85, 6.93, 7.18, 7.28, and 7.33, respectively.
The hourly water temperatures as recorded by the continuous temperature logger did
show that test temperatures fell outside the temperature allowance in the early morning
on October 29, 2009 (readings of 23.8 and 23.9 °C). However, instantaneous water
110
-------
temperatures measured by GLEC technicians on October 28 and later in the morning on
October 29 never fell outside the acceptable range as outlined in the method. Therefore,
these water temperature exceedances were very brief and likely had no effect on the
results of this study.
Once the test was complete, the LCso was determined using the average measured test
concentrations with the Probit and Spearman method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 80; test results are provided in Table 81. Analytical chemistry data are provided
in Table 82. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 24.
Table 80. Test conditions for 96-hour toxicity test on Pimephales promelas with
111
-------
boron (pH 6.75).
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination-mg/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Pimephalespromelas, (weight 0.1 Ig andl8.4 mm length),
GLEC Culture
Continuous flow-through, 96 hours
October 29-November 02, 2009
25 + 1
Ambient Laboratory, 10-20 uE/m /s
16 h light, 8 h darkness
None
2.5 Liter glass Tank
2000 mL
10
2
20
400,200, 100, 50.0, and 25.0
394,224, 122, 55.2, and 32.9
Continuous flow through, 4 turnovers per day
De-chlorinated Lake Michigan Water
Boric Acid: Sigma Aldrich, ACS Reagent >= 99.5% Cas.
No. 10043-35-3, Batch 118K0007 and Borax (sodium
tetraborate decahydrate) Sigma Aldrich, > 99.5%, ACS
reagent, Cas. No. 1303-96-4, Lot # 118K0172
None
None
Mortality (LC50)
112
-------
Table 81. Test results for 96-hour toxicity test on Pimephales promelas with boron at
pH 6.75.
Results of a Pimephc
Conducted 10/29/09 -
lies promelas _
11/02/09 Usi
96-Hour Static Acute Toxicitv Test
ne: Boron (Boric Acid: Sigma Aldrich Cas No. 10043-35-3)
fBorax: Siema Aldrich Cas No. 1303-96-4)
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
25.0 (32.9) mg/L
50.0 (55.2) mg/L
100 (122) mg/L
200 (224) mg/L
400 (394) mg/L
Cumulative Percent Affected a
24-Hr
0
(0)
5
(5)
5
(5)
0
(0)
5
(5)
10
(10)
48-Hr
0
(0)
5
(5)
5
(5)
0
(0)
5
(5)
95
(95)
72-Hr
0
(0)
10
(10)
5
(5)
30
(30)
65
(65)
100
(100)
96-Hr
0
(0)
10
(10)
20
(20)
90
(90)
100
(100)
100
(100)
24-Hr
>394
LC50* Values (mg/L)
48-Hr 72-
Hr 96-Hr
297 163 70.6
96-Hour LC50 *= 70.6 mg/L
LC50 *95% Confidence
24-Hr
LL NA
UL NA
48-Hr 72-
Limits
Hr 96-Hr
285 130 58.3
310 205 86.3
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not
reliable
Method(s) Used to Determine LC50 Confidence
Limit Values: Probit and Spearman
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All EC™ values are determined based on measured concentrations.
113
-------
Table 82. Analytical chemistry data for 96-hour toxicity test on Pimephales promelas
with boron at pH 6.75.
Nominal (and Measured) Test
Conent rations
Dilution
Water/Control
Average
25 mg/L
(32.9 mg/L)
Average
50 mg/L
(55.2 mg/L)
Average
lOOmg/L
(122 mg/L)
Average
Replicate #
1
Replicate #
4
Replicate #
1
Replicate #
3
Replicate #
1
Replicate #
3
Replicate #
1
Replicate #
3
Boron
(mg/L)
0.04
(0.04-0.04)
0.06
(0.05-0.06)
0.05
32.8
(30.3-38.6)
33.1
(30.4-39.0)
32.9
55.2
(51.8-62.5)
55.2
(51.1-61.1)
55.2
120
(112-126)
123
(119-129)
122
Temperature
(°C)
24.4
(24.0-24.8)
24.5
(24.0-24.8)
24.5
24.5
(24.1-24.9)
24.6
(24.1-25.0)
24.5
24.4
(24.0-24.6)
24.6
(24.3-24.8)
24.5
24.5
(24.0-25.1)
24.5
(24.1-24.9)
24.5
pH
(s.u.)
6.62
(6.48-6.69)
6.73
(6.63-6.88)
6.67
6.82
(6.73-6.93)
6.88
(6.80-6.98)
6.85
6.94
(6.86-7.04)
6.93
(6.83-7.04)
6.93
7.17
(7.11-7.25)
7.19
(7.13-7.26)
7.18
DO
(mg/L)
7.3
(6.7-8.0)
7.2
(6.7-8.3)
7.3
7.2
(6.6-8.0)
7.6 (7.2
8.2)
7.4
7.4
(6.8-8.0)
7.3
(6.5-8.1)
7.4
7.4
(6.8-8.1)
7.6
(7.1-8.3)
7.5
SC
(mmos)
327
343
332
362
365
363
382
351
371
440
447
442
Hardness
(mg/L)
130
130
130
130
126
126
130
130
Alkalinity
(mg/L)
70
7
10
10
11
11
18
18
Ammonia
(mg/L)
ND
ND
ND
ND
ND
ND
ND
ND
200 mg/L
(224 mg/L)
Average
400 mg/L
(394 mg/L)
Average
Replicate #
9
Replicate #
4
Replicate #
1
Replicate #
4
223
(219-234)
224
(217-232)
224
391
(372-407)
397
(368-426)
394
24.3
(24.0-24.9)
25.3
(25.1-25.5)
24.8
24.3
(24.0-24.6)
24.6
(24.3-25.1)
24.4
7.28
(7.22-7.35)
7.28
(7.20-7.35)
7.28
7.33
(7.26-7.38)
7.32
(7.24-7.37)
7.33
7.2
(6.5-8.2)
7.3
(6.8-8.2)
7.2
7.4
(6.9-8.1)
7.5
(7.1-8.2)
7.4
518
527
521
670
696
678
128
128
126
126
25
25
41
41
ND
ND
ND
ND
DO: Dissolved Oxygen
SC: Specific Conductance
ND: Non Detect; below detection limit.
NM: Not Measured
114
-------
96-hr Toxicity of Boron (pH 7.75) on Pimephales promelas
The 96-hr test to determine the toxicity of boron (pH 7.75) on P. promelas was
completed by GLEC. The P. promelas (collected from the GLEC laboratory culture)
were continuously exposed for 96-hours to five concentrations of boron and to a dilution
water control with a target test pH of 7.75 (range of 7.65 to 7.85) using a continuous
flow-through system (modified Benoit mini-dilutor) and an in-line pH
adjustment/metering unit. The pH of both the dilution water and stock solution were
adjusted by using a 1:1 ratio of sulfuric acid and hydrochloric acid (acid solution). The
pH adjusted stock solution was delivered to mixing cells and diluted with pH adjusted
Lake Michigan water to achieve target nominal concentrations of boron and a target test
pH of 7.75. Due to the buffering capacity of the borax and boric acid solution used to
prepare the boron concentrations, EPA agreed that GLEC should target the dilution water
control pH at 7.75 (range of 7.65 to 7.85). The five test concentrations were targeted to a
pH of ± 0.1 pH unit from the pH value defined at test initiation in each test concentration
(i.e. regardless of whether or not the pH in the test concentrations were ± 0.1 pH unit
from that observed in the control water). The temperature-controlled test concentration
solutions were supplied to each test chamber via the continuous flow-through system at a
rate of approximately four turnovers a day. There were two replicate test chambers for
each treatment. The flow through test was conducted at 25 ± 1 °C with a photoperiod of
16 hr light and 8 hr darkness (ambient laboratory light).
After test concentrations had achieved steady state in the flow through system, the test
was initiated with the P. promelas. Although these organisms were cultured in, and
acclimated to, the dilution water's "natural" pH (typically between a pH of 7.9 and 8.2),
they were not acclimated to the pH-adjusted dilution water prior to test initiation for two
reasons: 1) GLEC does not maintain a laboratory culture of P. promelas in de-
chlorinated Lake Michigan water maintained at a pH of 7.75 and 2) adequate acclimation
of the organisms to the pH-adjusted dilution water may have jeopardized the weight
requirement (0.1-5 g at test initiation) for test organisms required under the toxicity
testing method (ASTM 2007).
The P. promelas were randomly assigned to the 2.5 L glass test chambers until each test
chamber contained ten P. promelas. P. promelas were counted on a daily basis and the
number of live P. promelas was recorded. pH was recorded twice a day, at a minimum of
eight hours apart (i.e. morning and evening). The number of surviving P. promelas was
recorded at test termination (96-hours).
The test was completed at the following nominal boron concentrations: 25.0, 50.0, 100,
200, and 400 mg/L (dilution factor of 0.5). The average pH for the dilution water control
measured in the P. promelas toxicity test for the 96-hour test period was 7.68. The
average pH over the 96-hour test duration for the five test concentrations of 28.6, 50.9,
121, 223, and 392 mg/L was 7.88, 7.98, 8.05, 8.07, and 8.06, respectively.
Once the test was complete, the LCso was determined using the average measured test
concentrations with the Spearman method.
115
-------
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 83; test results are provided in Table 84. Analytical chemistry data are provided
in Table 85. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 25.
Table 83. Test conditions for 96-hour toxicity test on Pimephales promelas with
boron (pH 7.75).
116
-------
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination-mg/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Pimephalespromelas, (weight 0.12g and 22.0 mm length),
GLEC Culture
Continuous flow-through, 96 hours
October 23-October 27, 2009
25 + 1
Ambient Laboratory, 10-20 uE/m2/s
16 h light, 8 h darkness
None
2.5 Liter glass Tank
2000 mL
10
2
20
400,200, 100, 50.0, and 25.0
392,223, 121, 50.9, and 28.6
Continuous flow through, 4 turnovers per day
De-chlorinated Lake Michigan Water
Boric Acid: Sigma Aldrich, ACS Reagent >= 99.5% Cas.
No. 10043-35-3, Batch 118K0007 and Borax (sodium
tetraborate decahydrate) Sigma Aldrich, > 99.5%, ACS
reagent, Cas. No. 1303-96-4, Lot # 118K0172
None
None
Mortality (LC50)
Table 84. Test results for 96-hour toxicity test on Pimephales promelas with boron at
pH 7.75.
117
-------
Results of a Pimephc
Conducted 10/23/09 -
lies promelas_
10/27/09 Usi
96-Hour Static Acute Toxicitv Test
ng: Boron (Boric Acid: Sigma Aldrich Cas No. 10043-35-3)
( Borax: Sigma Aldrich Cas No. 1303-96-4)
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
25.0 (28.6) mg/L
50.0 (50.9) mg/L
100 (121) mg/L
200 (223) mg/L
400 (392) mg/L
Cumulative Percent Affected a
24-Hr
0
(0)
0
(0)
0
(0)
0
(0)
5
(5)
10
(10)
48-Hr
0
(0)
0
(0)
0
(0)
0
(0)
10
(10)
95
(95)
72-Hr
0
(0)
0
(0)
0
(0)
0
(0)
65
(65)
100
(100)
96-Hr
0
(0)
0
(0)
0
(5)
21
(21)
100
(100)
100
(100)
LC50* Values (mg/L)
24-Hr
48-Hr 72-
Hr 96-Hr
>392 289 202 137
96-Hour LC50 *= 137 mg/L
LC50 *95% Confidence
24-Hr
LL NA
UL NA
48-Hr 72-
Limits
Hr 96-Hr
260 178 118
320 229 158
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not
Method(s) Used to Determine LC5
Limit Values: Spearman
reliable
o Confidence
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC50 values are determined based on measured concentrations.
Table 85. Analytical chemistry data for 96-hour toxicity test on Pimephales promelas
with boron at pH 7.75.
118
-------
Nominal (and Measured) Test
Conent rations
Dilution
Water/Control
Average
25 mg/L
(28.4 mg/L)
Average
50 mg/L
(50.9 mg/L)
Average
100 mg/L
(121 mg/L)
Average
Replicate #
1
Replicate #
4
Replicate #
1
Replicate #
3
Replicate #
1
Replicate #
3
Replicate #
1
Replicate #
3
Boron
(mg/L)
0.05
(0.04-0.05)
0.06
(0.05-0.07)
0.05
28.7
(27.2-31.2)
28.6
(27.1-32.1)
28.6
51.0
(47.4-54.7)
50.9
(47.5-55.5)
50.9
121
(111-127)
121
(110-130)
121
Temperature
(°C)
24.8
(24.5-25.3)
25.1
(24.9-25.4)
24.9
24.9
(24.5-25.4)
25.0
(24.7-25.4)
24.9
24.7
(24.5-25.1)
24.9
(24.7-25.4)
24.8
24.9
(24.6-25.3)
25.0
(24.7-25.4)
24.9
pH
(s.u.)
7.63
(7.49-7.75)
7.72
(7.56-7.85)
7.68
7.86
(7.70-7.95)
7.91
(7.80-8.00)
7.88
7.97
(7.80-8.07)
7.98
(7.90-8.07)
7.98
8.06
(7.97-8.13)
8.05
(7.96-8.13)
8.05
DO
(mg/L)
7.3
(6.5-8.0)
7.3
(6.4-8.1)
7.3
7.2
(5.9-8.1)
7.4
(6.7-8.1)
7.3
7.2
(6.4-8.0)
7.4
(6.7-8.1)
7.3
7.4
(6.7-8.1)
7.1
(6.2-8.1)
7.3
SC
(mmos)
351
321
336
375
345
360
393
361
377
436
410
423
Hardness
(mg/L)
146
146
138
138
146
146
128
128
Alkalinity
(mg/L)
41
41
49
49
57
57
77
77
Ammonia
(mg/L)
ND
ND
ND
ND
ND
ND
ND
ND
200 mg/L
(223 mg/L)
Average
Replicate #
2
Replicate #
4
222
(216-228)
225
(222-228)
223
24.6
(24.4-25.1)
25.2
(24.8-25.6)
24.9
8.07
(7.99-8.15)
8.07
(8.00-8.14)
8.07
7.3
(6.6-8.0)
7.3
(6.7-8.1)
7.3
500
476
488
132
132
107
107
ND
ND
400 mg/L
(392 mg/L)
Average
Replicate #
1
Replicate #
4
392
(381-399)
392
(384-401)
392
24.7
(24.4-25.1)
24.7
(24.4-25.2)
24.7
8.07
(8.00-8.13)
8.06
(8.01-8.13)
8.06
7.3
(6.7-8.0)
7.3
(6.4-8.0)
7.3
616
590
603
128
128
168
168
ND
ND
DO: Dissolved Oxygen
SC: Specific Conductance
ND: Non Detect; below detection limit.
NM: Not Measured
119
-------
96-hr Toxicity of Boron (pH 8.75) on Pimephales promelas
The 96-hr test to determine the toxicity of boron (pH 8.75) on P. promelas was
completed by GLEC. The P. promelas (collected from the GLEC laboratory culture)
were continuously exposed for 96-hours to five concentrations of boron and to a dilution
water control with a target test pH of 8.75 (range of 8.65 to 8.85) using a continuous
flow-through system (modified Benoit mini-dilutor) and an in-line pH
adjustment/metering unit. The stock solutions used for the test were prepared by diluting
a known weight (grams) of borax and boric acid to a known volume of dilution water (de-
chlorinated Lake Michigan water). The pH of both the dilution water and stock solution
were adjusted by using sodium hydroxide. The pH adjusted stock solution was delivered
to mixing cells and diluted with pH adjusted Lake Michigan water to achieve target
nominal concentrations of boron and a target test pH of 8.75. The temperature-controlled
test concentration solutions were supplied to each test chamber via the continuous flow-
through system at a rate of approximately four turnovers a day. There were two replicate
test chambers for each treatment. The flow through test was conducted at 25 ± 1 °C with
a photoperiod of 16 hr light and 8 hr darkness (ambient laboratory light).
After test concentrations had achieved steady state in the flow through system, the test
was initiated with the P. promelas. Although these organisms were cultured in, and
acclimated to, the dilution water's "natural" pH (typically between a pH of 7.9 and 8.2),
they were not acclimated to the pH-adjusted dilution water prior to test initiation for two
reasons: 1) GLEC does not maintain a laboratory culture of P. promelas in de-
chlorinated Lake Michigan water maintained at a pH of 8.75 and 2) adequate acclimation
of the organisms to the pH-adjusted dilution water may have jeopardized the weight
requirement (0.1-5 g at test initiation) for test organisms required under the toxicity
testing method (ASTM 2007).
The P. promelas were randomly assigned to the 2.5 L glass test chambers until each test
chamber contained ten P. promelas. P. promelas were counted on a daily basis and the
number of live P. promelas was recorded. pH was recorded twice a day, at a minimum of
eight hours apart (i.e., morning and evening). The number of surviving P. promelas was
recorded at test termination (96-hours).
The test was completed at the following nominal boron concentrations: 25.0, 50.0, 100,
200, and 400 mg/L (dilution factor of 0.5). The average pH for the dilution water control
measured in the P. promelas toxicity test for the 96-hour test period was 8.75. The
average pH over the 96-hour test duration for the five test concentrations of 21.1, 42.4,
112, 219, and 376 mg/L was 8.72, 8.70, 8.70, 8.70, and 8.67, respectively.
Instantaneous water temperature measurements made on February 13, 14, 15 and 16
(Days 1-4: 23.6°C-24.7°C) exceeded the allowable range of 25 ± 1 °C outlined in the
toxicity testing method. However, the average water temperatures across the duration of
the test in each replicate were always ± 0.9 °C of the target test temperature (25 °C) in all
treatments. In addition, the water temperature as recorded by the continuous temperature
logger did show that the test temperature fell outside the temperature allowance during
120
-------
the times of 0430 to 0830 on those days. However, due to the relatively small number of
temperature readings measured outside the range, these water temperature exceedances
likely had little effect on the results of this study.
Once the test was complete, the LCso was determined using the average measured test
concentrations with the Probit method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 86; test results are provided in Table 87. Analytical chemistry data are provided
in Table 88. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 26.
Table 86. Test conditions for 96-hour toxicity test on Pimephales promelas with
121
-------
boron (pH 8.75).
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination-mg/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Pimephalespromelas, (weight 0.13g and 22.8 mm length),
GLEC Culture
Continuous flow-through, 96 hours
February 12-February 16,2010
25 + 1
Ambient Laboratory, 10-20 uE/m /s
16 h light, 8 h darkness
None
2.5 Liter glass Tank
2000 mL
10
2
20
400,200, 100, 50.0, and 25.0
376,219, 112,42.4, and 21.1
Continuous flow through, 4 turnovers per day
De-chlorinated Lake Michigan Water
Boric Acid: Sigma Aldrich, ACS Reagent >= 99.5% Cas.
No. 10043-35-3, Batch 118K0007 and Borax (sodium
tetraborate decahydrate) Sigma Aldrich, > 99.5%, ACS
reagent, Cas. No. 1303-96-4, Lot # 118K0172
None
None
Mortality (LC50)
122
-------
Table 87. Test results for 96-hour toxicity test on Pimephales promelas with boron at
pH 8.75.
Results of a Pimephc
Conducted 02/12/10 -
lies promelas _ 96-Hour Static Acute Toxicitv Test
02/16/10 Usins: Boron (Boric Acid: Sisma Aldrich Cas No. 10043-35-3)
(Borax: Sigma Aldrich Cas No. 1303-96-4)
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
25.0(21.1)mg/L
50.0 (42.4) mg/L
100(1 12) mg/L
200 (2 19) mg/L
400 (376) mg/L
Cumulative Percent Affected a
24-Hr
0
(0)
0
(0)
0
(0)
0
(0)
5
(5)
10
(10)
48-Hr
0
(0)
0
(0)
0
(0)
0
(0)
15
(15)
65
(65)
72-Hr
0
(0)
0
(0)
0
(0)
5
(5)
60
(60)
100
(100)
96-Hr
5
(5)
0
(0)
0
(0)
30
(30)
95
(95)
100
(100)
24-Hr
>373
LCSO* Values (mg/L)
48-Hr 72-
Hr 96-Hr
325 195 133
96-Hour LCSO *= 133 mg/L
LC50 *95% Confidence
24-Hr
48-Hr 72-
Limits
Hr 96-Hr
LL NA 276 165 110
UL NA 412 227 157
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 Confidence
Limit Values: Probit
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC50 values are determined based on measured concentrations.
123
-------
Table 88. Analytical chemistry data for 96-hour toxicity test on Pimephales promelas
with boron at pH 8.75.
Nominal (and Measured) Test
Conent rations
Dilution
Wat er/Cont rol
Average
25mg/L
(21.1 mg/L)
Average
50mg/L
(42.4 mg/L)
Average
Replicate #
1
Replicate #
2
Replicate #
1
Replicate #
3
Replicate #
9
Replicate #
4
Boron
(mg/L)
0.04
(0.03-0.04)
0.03
(0.03-0.03)
0.03
21.9
(21.7-22.2)
20.2
(18.0-21.7)
21.1
42.6
(37.0-46.0)
42.7
(36.3-45.4)
42.4
Temperature
(°C)
24.2
(23.8-24.6)
24.4
(23.8-25.2)
24.3
24.3
(23.8-24.8)
24.2
(23.8-25.2)
24.2
24.3
(23.8-25.2)
24.3
(23.8-24.8)
24.3
PH
(s.u.)
8.74
(8.68-8.83)
8.75
(8.71-8.81)
8.75
8.72
(8.67-8.77)
8.71
(8.63-8.77)
8.72
8.69
(8.62-8.72)
8.72
(8.67-8.77)
8.70
DO
(mg/L)
7.6
(7.4-7.7)
7.4
(7.1-7.6)
7.5
7.5
(7.4-7.7)
7.3
(7.1-7.4)
7.4
7.1
(6.8-7.6)
7.2
(7.1-7.4)
7.2
SC
(mmos)
290
299
295
331
336
333
368
370
369
Hardness
(mg/L)
126
126
110
110
116
116
Alkalinity
(mg/L)
111
111
133
133
156
156
Ammonia
(mg/L)
ND
ND
ND
ND
ND
ND
100 mg/L
(112 mg/L)
Average
Replicate #
1
Replicate #
4
111
(93.5-125)
113
(96.3-128)
112
24.3
(23.9-25.2)
24.3
(23.6-25.0)
24.3
8.71
(8.65-8.76)
8.70
(8.63-8.76)
8.70
7.3
(7.1-7.5)
7.4
(7.2-7.6)
7.3
472
464
471
116
116
235
235
ND
ND
200 mg/L
(219 mg/L)
Average
Replicate #
1
Replicate #
4
207
(185-254)
231
(202-264)
219
24.3
(23.6-24.8)
24.5
(23.9-25.2)
24.4
8.70
(8.64-8.73)
8.70
(8.64-8.74)
8.70
7.3
(7.0-7.6)
7.2
(7.1-7.4)
7.3
628
638
633
120
120
340
340
ND
ND
400 mg/L
(376 mg/L)
Average
Replicate #
1
Replicate #
4
382
(359-412)
370
(353-399)
376
24.1
(24.0-24.5)
24.1
(23.8-24.8)
24.1
8.67
(8.62-8.73)
8.67
(8.62-8.71)
8.67
7.2
(6.9-7.3)
7.2
(7.1-7.3)
7.2
844
845
845
108
108
510
510
ND
ND
DO: Dissolved Oxygen
SC: Specific Conductance
ND: Non Detect; below detection limit.
NM: Not Measured
124
-------
Manganese
Table 89 provides a summary of estimated LC50 values for the two toxicity tests
performed using manganese. LCso values ranged between 31.5 and 43.3 mg Mn/L.
Table 89. LCso estimates for toxicity tests performed using manganese.
Test Species and Duration
Lampsilis siliquoidea 96 hr
Megalonaias nervosa - 96 hr
LC50 (mg Mn/L)
43.3
31.5
For each of the acute toxicity tests completed using manganese, two tables were
generated: the first summarizes the test results for each toxicity test, including nominal
and analytical test concentration and LCso estimates with confidence intervals; the second
table summarizes analytical chemistry data collected throughout the toxicity tests. Also
discussed, if applicable, are deviations from the guidance provided in the ASTM method
used to complete the toxicity testing.
125
-------
96-hr Toxicity of Manganese on Lampsttis siliquoidea
The 96-hr test to determine the toxicity of manganese on L. siliquoidea was completed by
INHS. Test organisms, < 5-day old juveniles collected from the Missouri State University
laboratory culture, were acclimated to the dilution water (MHRW), test temperature and
other test conditions prior to test initiation. Once acclimated, test organisms were
examined for any disease, stress, parasites, etc. If free from ailments, test organisms were
randomly assigned to the test chambers (which were randomly assigned to testing
locations); four replicates were used per treatment with five organisms per replicate. One
replicate was mistakenly loaded with only 4 individuals, but this was accounted for in the
LCso calculation.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 150, 75, 37, 18.8, 9.4, and 4.7
mg Mn/L.
Testing was conducted at 20 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 90; test results are provided in Table 91. Analytical chemistry data are provided
in Table 92. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 27.
126
-------
Table 90. Test conditions for 96-hour toxicity test on Lampsilis siliquoidea with
manganese.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg Mn/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination- Mn
mg/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Lampsilis siliquoidea, juveniles <5 days old, Missouri State
Univbersity
Static, 96 hours
September 8 - 12,2009
20 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
50 mL beaker
40 mL
5
4
20
150, 75,37, 18.8, 9.4, and 4.7
154.9, 72.5, 34.5,18.5, 10.1 and 4.5
None
USEPA MHRW
Manganese sulfate monohydrate: Fisher Scientific, ACS
grade assay, 98.7%, Cas. No. 7785-87-7, Lot # 086316 and
manganese chloride tetrahydrate, Fisher Scientific, certified
ACS Assay 99.8% Cas. No. 7773-01-5, Lot # 081484
None
None
Mortality (LC50)
127
-------
Table 91. Test results for 96-hour toxicity test on Lampsilis siliquoidea with
manganese.
Results of a Lamysilis siliauoidea)
Conducted 09/08/09 - 09/12/09
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
4.7 (4.5) mg/L
9.4(10.1)mg/L
18. 8 (18. 5) mg/L
37 (34.5) mg/L
75 (72.5) mg/L
150 (154.9) mg/L
96-Hour Static Acute Toxicitv Test
Usine: Maneanese sulfate Cas. No. 7785-87-7 &manganese
chloride Cas. No. 7773-01-5
Cumulative Percent Affected a
24-Hr
0
0
0
0
0
70
95
48-Hr
0
0
0
0
0
100
100
72-Hr
0
0
0
0
5.2
100
100
96-Hr
0
0
0
0
21.0
100
100
LC50 Values* (mg/L)
24-Hr 48-Hr
62.8 50.0
72-Hr 96-Hr
48.2 43.3
96-Hour LC50* = 43.3 mg/L
LC50 95% Confidence Limits
24-Hr 48-Hr
72-Hr 96-Hr
LL52.7 NR 45.0 38.1
UL74.9 NR 51.7 49.2
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 and EC50
Confidence Limit Values: Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC50 and EC50 values are determined based on measured concentrations.
128
-------
Table 92. Analytical chemistry data for 96-hour toxicity test on Lampsilis siliquoidea
with manganese.
Nominal (Measured) Test
Concentration
Manganese" Temperature pH D.O. Conductivity Alkalinity Hardness
(mg/L) (°C) (s.u.) (mg/L) (inmos) (mg/L) (mg/L)
Dilution water/Control
4.7 (4. 5) mg/L
9.4 (10.1) mg/L
18.8 (18.5) mg/L
37 (34.5) mg/L
75 (72.5) mg/L
150 (154.9) mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
<0.01
<0.01
na
4.5
4.6
4.5
9.2
11.0
10.1
19.0
18.0
18.5
34.0
35.0
34.5
73.0
72.0
72.5
150.0
160.0
154.9
20.4
20.3
20.4
19.6
19.1
20.5
20.2
20.3
19.5
19.1
20.4
20.3
20.4
19.6
19.0
20.4
20.2
20.3
19.6
19.0
20.5
20.2
20.3
19.6
19.0
20.5
20.2
20.3
19.7
19.1
20.5
20.3
20.4
19.7
19.2
7.9
7.9
7.9
7.9
7.9
7.9
7.7
7.9
7.7
7.9
7.7
7.9
7.7
7.9
7.08
7.50
7.83
7.69
7.87
7.65
7.83
7.72
7.94
7.76
7.88
7.70
7.88
7.80
316
322
324
328
341
345
376
380
447
450
582
590
840
850
62
62
62
62
62
62
62
62
62
62
62
62
62
62
92
90
98
100
110
110
120
124
152
152
220
224
*
*
"Manganese Analysis Method 200.7
* interference in hardness measurement
na = not applicable
129
-------
96-hr Toxicity of Manganese on Megalonaias nervosa
The 96-hr test to determine the toxicity of manganese onM nervosa was completed by
INHS. Test organisms, < 5-day old juveniles collected from the Genoa National Fish
Hatchery, were acclimated to the dilution water (MHRW), test temperature and other test
conditions prior to test initiation. Once acclimated, test organisms were examined for any
disease, stress, parasites, etc. If free from ailments, test organisms were randomly
assigned to the test chambers (which were randomly assigned to testing locations); four
replicates were used per treatment with five organisms per replicate. In one replicate of
the control a test organism was inadvertently crushed, but this was accounted for in the
LCso calculation.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 300, 150, 75, 37.5, and 18.8
mg Mn/L.
Testing was conducted at 20 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 93; test results are provided in Table 94. Analytical chemistry data are provided
in Table 95. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 28.
130
-------
Table 93. Test conditions for 96-hour toxicity test on Megalonaias nervosa with
manganese.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg Mn/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination-mg
Mn/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Megalonaias nervosa, juveniles <5 days old (Genoa
National Fish Hatchery)
Static, 96 hours
October 23 - 27, 2009
20 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
50 mL beaker
40 mL
5
4
20
300, 150, 75, 37.5, and 18.8
290, 140, 72, 34, and 18
None
USEPA MHRW
Manganese sulfate monohydrate: Fisher Scientific, ACS
grade assay, 98.7%, Cas. No. 7785-87-7, Lot # 086316 and
manganese chloride tetrahydrate, Fisher Scientific, certified
ACS Assay 99.8% Cas. No. 7773-01-5, Lot # 081484
None
None
Mortality (LC50)
131
-------
Table 94. Test results for 96-hour toxicity test on Megalonaias nervosa with
manganese.
Results of a Mesalonaias nervosa
Conducted 10/23/09 - 10/27/09
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
18.8(1 8) mg/L
37.5 (34) mg/L
75 (72) mg/L
150 (140) mg/L
300 (290) mg/L
96-Hour Static Acute Toxicitv Test
Usine: Maneanese sulfate Cas. No. 7785-87-7 & manganese
chloride Cas. No. 7773-01-5
Cumulative Percent Affected a
24-Hr
0
0
30
95
100
100
48-Hr
0
0
40
100
100
100
72-Hr
0
0
65
100
100
100
96-Hr
0
0
65
100
100
100
LC50 Values* (mg/L)
24-Hr 48-Hr
41.6 37.5
72-Hr 96-Hr
31.5 31.5
96-Hour LC50* = 31.5 mg/L
LC50 95% Confidence Limits
24-Hr 48-Hr
72-Hr 96-Hr
LL 35.6 32.2 27.2 27.2
UL48.8 43.6 36.6 36.6
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 and EC50
Confidence Limit Values: Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC50 and EC50 values are determined based on measured concentrations.
132
-------
Table 95. Analytical chemistry data for 96-hour toxicity test on Megalonaias nervosa
with manganese.
Nominal (Measured) Test Concentration Manganese" Temp. pH D.O. Cond. Alkalinity Hardness Ammonia
(mg/L) (°C) (s.u.) (mg/L) (mmos) (mg/L) (mg/L) (mg/L)
Dilution
water/Control
18.8 (18.0) mg/L
37.5 (34.0) mg/L
75 (72.0) mg/L
150 (140.0) mg/L
300 (290) mg/L
DayO
Day 1
Day 2
Day 3
Day 4 total
Day 4 dissolved
DayO
Day 1
Day 2
Day 3
Day 4 total
Day 4 dissolved
DayO
Day 1
Day 2
Day 3
Day 4 total
Day 4 dissolved
DayO
Day 1
Day 2
Day 3
Day 4 total
Day 4 dissolved
DayO
Day 1
Day 2
Day 3
Day 4 total
Day 4 dissolved
DayO
Day 1
Day 2
Day 3
Day 4 total
Day 4 dissolved
<0.01
0.1
0.7
0.1
18.0
18.0
16.0
18.0
33.0
35.0
34.0
34.0
70.0
74.0
68.0
72.0
140.0
140.0
150.0
140.0
290.0
290.0
290.0
290.0
20.8
20.9
20.9
20.9
20.8
20.8
20.9
20.9
20.9
20.8
20.8
20.9
20.9
20.9
20.8
20.8
20.9
20.8
20.9
20.9
20.8
20.9
20.8
20.9
20.9
20.8
20.9
20.8
20.9
20.9
8.0
8.1
8.1
8.0
8.0
7.9
7.9
7.7
7.6
7.8
7.9
7.7
7.6
7.6
7.7
7.9
7.7
7.6
7.5
7.7
7.8
7.7
7.6
7.5
7.6
7.7
7.7
7.6
7.5
7.6
8.34
8.04
8.13
8.31
8.00
8.33
8.10
8.19
8.31
7.92
8.26
8.11
8.25
8.42
7.94
8.29
8.07
8.26
8.32
7.91
8.33
8.03
8.22
8.40
7.88
8.27
7.98
8.20
8.22
7.94
303
297
303
305
315
375
362
371
379
383
445
442
458
478
495
582
569
567
581
589
841
826
817
851
848
1333
1301
1300
1325
1340
60
60
60
60
60
60
60
62
60
62
60
62
90
92
112
112
*
*
*
*
*
*
*
*
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
"Manganese Analysis Method 200.7
* interference in hardness measurement
Temp. = temperature; Cond. = conductivity
133
-------
Fluoride
Table 96 provides a summary of estimated LC50 values for the two toxicity tests
performed using fluoride. LCso values ranged between 13.4 and 62.0 mg F/L.
Table 96. LCso estimates for toxicity tests performed using fluoride.
Test Species and Duration
Sphaerium simile - 96 hr
Hyalella azteca - 96 hr
LC50(mgF/L)
62.0
13.4
For each of the acute toxicity tests completed using fluoride, two tables were generated:
the first summarizes the test results for each toxicity test, including nominal and
analytical test concentration and LCso estimates with confidence intervals; the second
table summarizes analytical chemistry data collected throughout the toxicity tests.
134
-------
96-hr Toxicity of Fluoride on Sphaerium simile
The 96-hr test to determine the toxicity of fluoride on S. simile was completed by INHS.
Test organisms, juveniles released from field collected adults, were acclimated to the
dilution water (MHRW), test temperature and other test conditions prior to test initiation.
Once acclimated, test organisms were examined for any disease, stress, parasites, etc. If
free from ailments, test organisms were randomly assigned to the test chambers (which
were randomly assigned to testing locations); four replicates were used per treatment with
five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 800, 400, 200, 100, and 50
mg F/L.
Testing was conducted at 22 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LC50 value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 97; test results are provided in Table 98. Analytical chemistry data are provided
in Table 99. Accompanying information, including raw laboratory data, analytical
chemistry data and statistical analyses, is provided in Appendix 29.
135
-------
Table 97. Test conditions for 96-hour toxicity test on Sphaerium simile with fluoride.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg F/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination-mg
F/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Sphaerium simile, juveniles (released from field-collected
adults)
Static, 96 hours
July 13-17,2009
22 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
150 mL beaker
120 mL
5
4
20
800, 400, 200, 100, and 50
800, 390, 185, 88 and 44
None
USEPA MHRW
Sodium fluoride: Acros Organics, 99+% for analysis ACS,
Cas. No. 7681-49-5, Lot # A0243428
None
None
Mortality (LC50)
136
-------
Table 98. Test results for 96-hour toxicity test on Sphaerium simile with fluoride.
Results of a Sphaerium simile 96-Hour Static Acute Toxicitv Test
Conducted 07/13/09 - 07/17/09 Using: Sodium fluoride: Sigma Aldrich Cas. No. 7681-49-5
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
50 (44) mg/L
100(88)mg/L
200 (185) mg/L
400 (390) mg/L
800 (800) mg/L
Cumulative Percent Affected a
24-Hr
0
0
0
0
0
0
48-Hr
0
0
0
0
5
5
72-Hr
0
0
0
35
50
25
96-Hr
5
25
75
100
100
100
LC50 Values* (mg/L)
24-Hr 48-Hr 72-
Hr 96-Hr
>800 >800 >800 62
96-Hour LCSO* = 62 mg/L
LC50 95% Confidence Limits
24-Hr 48-Hr 72-
Hr 96-Hr
LLNR NR NR 51
ULNR NR NR 75
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not
reliable
Method(s) Used to Determine LC50 and EC50
Confidence Limit Values: Spearman-Karber
Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
• All LC50 and EC50 values are determined based on measured concentrations.
137
-------
Table 99. Analytical chemistry data for 96-hour toxicity test Sphaerium simile with
fluoride.
Nominal (Measured) Test
Concentration
Dilution water/Control
50 (44) mg/L
100(88)mg/L
200 (185) mg/L
400 (390) mg/L
800 (800) mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
Fluoride" Temperature pH D.O. Conductivity Alkalinity Hardness
(mg/L) (°C) (s.u.) (mg/L) (umhos) (mg/L) (mg/L)
<0.5
<0.5
na
45
44
44
86
91
88
190
180
185
400
380
390
800
800
800
22.9
22.9
22.8
22.7
22.9
22.8
22.8
22.8
22.7
22.9
22.8
22.9
22.8
22.7
22.9
22.9
22.8
22.9
22.8
22.9
22.9
22.9
22.8
22.8
23.0
23.0
22.9
22.9
22.8
22.9
8.0
8.0
8.1
8.0
8.1
8.1
8.0
8.1
8.2
7.9
8.2
8.0
7.76
7.97
8.00
7.63
8.05
7.82
7.92
7.83
7.70
8.04
7.80
8.11
7.95
7.67
7.53
7.82
8.05
7.82
6.63
6.03
7.78
7.99
7.79
4.88
5.49
7.82
8.05
8.02
6.51
6.89
307
310
555
558
785
784
1264
1268
2210
2210
4050
4090
62
62
64
68
66
68
78
82
120
120
160
160
96
96
94
80
80
62
76
50
64
30
14
2
* Fluoride Analysis Method 300.0
na = not applicable
138
-------
96-hr Toxicity of Fluoride on Hyalella azteca
The 96-hr test to determine the toxicity of fluoride on H. azteca was completed by
GLEC. H. azteca were collected from GLEC's laboratory culture. These organisms are
maintained in 10 gallon glass aquaria; plastic artificial turf and screen mesh serve as a
substrate for the culture. The tanks are filled with de-chlorinated Lake Michigan water
(City of Traverse City, Michigan water passed through an activated carbon filter).
Cultures are fed 50 mL of 4 g/L Tetrafin slurry daily. When visible algae are not
observed within the glass aquaria, algae (Selenastrum sp.) are used as a supplement to the
Tetrafin slurry. Additionally, on occasion, dried Aspen (Populus sp.) leaves are prepared
as a food supplement. The culture is maintained in a 16-hour light: 8-hour dark
photoperiod at a temperature between 23 and 26 °C.
Test organisms were acclimated to the dilution water (MHRW), test temperature and
other test conditions prior to test initiation. Once acclimated, test organisms were
examined for any disease, stress, parasites, etc. If free from ailments, test organisms were
randomly assigned to the test chambers (which were randomly assigned to testing
locations); four replicates were used per treatment with five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 7.8, 12.9, 21.6, 36.0, 60.0,
and lOOmgF/L.
Testing was conducted at 22 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LCso value was determined
using the Probit and Spearman Karber methods.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 100; test results are provided in Table 101. Analytical chemistry data are
provided in Table 102. Accompanying information, including raw laboratory data,
analytical chemistry data, reference toxicant data and statistical analyses, is provided in
Appendix 30.
139
-------
Table 100. Test conditions for 96-hour toxicity test on Hyalella azteca with fluoride.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Target or Nominal Test Concentrations (mg F/L):
14. Analytical Test Concentrations (average of samples
collected at test initiation and termination-mg F/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Hyalella azteca, 14 days old, GLEC culture
Static, 96 hours
September 17- September 21, 2009
22 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
150 mL beaker
100 mL
5
4
20
100, 60,36,21.6,12.9, and 7.8
89.4, 58.9, 32.7, 22.8, 14.3, and 8.5
None
USEPA MHRW
Sodium Fluoride: Sigma Aldrich, 99+% ACS Reagent Cas.
No. 7681-49-4, Batch # 06810JJ
None
None
Mortality (LC50)
140
-------
Table 101. Test results for 96-hour toxicity test on Hyalella azteca with fluoride.
Results of a Hvalella azteca 96-Hour Static Acute Toxicitv Test
Conducted 09/17/09 - 09/21/09 Using: Fluoride (Sodium Fluoride Sigma Aldrich Cas. No. 7681-49-4)
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
7.8 (8.5) mg/L
12.9 (14.3) mg/L
2 1.6 (22. 8) mg/L
36 (32.7) mg/L
60 (58.9) mg/L
100 (89. 4) mg/L
Cumulative Percent Affected a
24-Hr
0
(0)
0
(0)
0
(0)
20
(20)
100
(100)
100
(100)
100
(100)
48-Hr
0
(0)
0
(0)
45
(45)
80
(80)
100
(100)
100
(100)
100
(100)
72-Hr
0
(0)
0
(0)
70
(70)
90
(90)
100
(100)
100
(100)
100
(100)
96-Hr
0
(0)
0
(0)
70
(70)
95
(95)
100
(100)
100
(100)
100
(100)
LC50 Values* (mg/L)
24-Hr 48-Hr 72-Hr 96-Hr
25.1 16.0 13.8 13.4
96-Hour LC50* = 13.4 mg/L
LCSO 95% Confidence Limits
24-Hr 48-Hr 72-Hr 96-Hr
LL 23.3 13.9 12.0 11.8
UL27.1 18.3 15.6 15.2
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 Confidence
Limit Values: Probit and Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ values are determined based on measured concentrations.
141
-------
Table 102. Analytical chemistry data for 96-hour toxicity test on Hyalella azteca
with fluoride.
Nominal (and M
Test Concent
Dilution
water/Control
easured)
ations
DayO
Day 1
Day 2
Day3
Day 4
Fluoride3
(mg/L)
ND
ND
ND
ND
Temperature
(°C)
22.0
21.9
22.1
22.0
22.2
PH
(S.U.)
7.84
7.79
7.79
7.84
7.81
D.O.
(mg/L)
8.0
7.5
7.1
7.9
8.0
Conductivity
(mmhos)
300
325
Alkalinity
(mg/L)
54
68
Hardness
(mg/L)
SO
88
Ammonia
(mg/L)
ND
7.8 (8.5) mg/L
DayO
Day 1
Day 2
Day3
Day 4
8.4
8.6
8.5
22.0
22.0
22.1
22.0
22.2
7.84
7.78
7.83
7.91
7.87
8.0
7.5
7.3
7.7
8.0
368
363
12.9(14.3) mg/L
DayO
Day 1
Day 2
Day3
Day 4
14.1
14.5
14.3
22.0
22.0
22.1
21.9
22.2
7.85
7.85
7.88
7.97
7.89
8.0
7.5
7.4
8.1
8.0
393
377
21.6(22.8) mg/L
DayO
Day 1
Day 2
Day3
Day 4
22.0
23.5
22.8
22.0
21.9
22.0
21.8
21.9
7.85
7.90
7.89
8.02
7.97
8.0
7.5
7.3
8.0
8.0
432
422
66
100
36 (32.7) mg/L
DayO
Day 1
Day 2
Day3
Day 4
33.4
32.0
32.7
22.0
21.9
7.88
7.91
8.0
7.5
502
60(58.9) mg/L
DayO
Day 1
Day 2
Day3
Day 4
58.1
59.6
58.9
22.0
21.9
7.88
7.92
8.0
7.5
622
100(89.4) mg/L
DayO
Day 1
Day 2
Day3
Day 4
91.0
87.7
89.4
22.0
21.9
7.89
7.99
8.0
7.5
781
72
70
68
68
ND
a Fluoride Analysis EPA 300.0
ND Not Detect; below detection limit
142
-------
Chloride
Table 103 provides a summary of the estimated LC50 value for the toxicity test performed
using chloride.
Table 103. LCso estimate for toxicity test performed using chloride.
Test Species and Duration
Musculium transversum - 96 hr
LC50(mgCl/L)
1,930
For the acute toxicity test completed using chloride, two tables were generated: the first
summarizes the test results for the toxicity test, including nominal and analytical test
concentration and LCso estimates with confidence intervals; the second table summarizes
analytical chemistry data collected throughout the toxicity test.
143
-------
96-hr Toxicity of Chloride on Musculium transversum
The 96-hr test to determine the toxicity of chloride on M. transversum was completed by
INHS. Test organisms, juveniles released from field collected adults, were acclimated to
the dilution water (MHRW), test temperature and other test conditions prior to test
initiation. Once acclimated, test organisms were examined for any disease, stress,
parasites, etc. If free from ailments, test organisms were randomly assigned to the test
chambers (which were randomly assigned to testing locations); four replicates were used
per treatment with five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 8,000, 4,018, 2,028, 1,032,
and535mgCl/L.
Testing was conducted at 22 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LC50 value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 104; test results are provided in Table 105. Analytical chemistry data are
provided in Table 106. Accompanying information, including raw laboratory data,
analytical chemistry data and statistical analyses, is provided in Appendix 31.
144
-------
Table 104. Test conditions for 96-hour toxicity test on Musculium transversum with
chloride.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg Cl/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination-
mg/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Musculium transversum, juveniles (released from field-
collected adults)
Static, 96 hours
September 04 - 08,2009
22 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
150 mL beaker
120 mL
5
4
20
8000,4018, 2028, 1032, and 535
7750, 3899, 1900, 985 and 505
None
EPA MHRW
Sodium chloride: Sigma Aldrich, 99% pure ACS Reagent
Cas. No. 7647-14-5, Lot # B0110043
None
None
Mortality (LC50)
145
-------
Table 105. Test results for 96-hour toxicity test on Musculium transversum with
chloride.
Results of a Musculium transversum
Conducted 09/04/09 - 09/08/09
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
535 (505) mg/L
1032 (985) mg/L
2028 (1900) mg/L
4018 (3899) mg/L
8000 (7750) mg/L
96-Hour Static Acute Toxicitv Test
Using: Sodium chloride: Sigma Aldrich Cas. No. 7647-14-5
Cumulative Percent Affected a
24-Hr
0
0
0
0
100
100
48-Hr
5
0
0
15
100
100
72-Hr
5
0
0
15
100
100
96-Hr
5
0
0
50
100
100
LC50 Values* (mg/L)
24-Hr 48-Hr 72-
Hr 96-Hr
2722 2455 2455 1930
96-Hour LC50* = 1930 mg/L
LC50 95% Confidence Limits
24-Hr 48-Hr 72-
Hr 96-Hr
LLNR 2200 2200 1655
ULNR 2740 2740 2251
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not
reliable
Method(s) Used to Determine LC50 and EC50
Confidence Limit Values: Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ and EC™ values are determined based on measured concentrations.
146
-------
Table 106. Analytical chemistry data for 96-hour toxicity test on Musculium
transversum with chloride.
Nominal (Measured) Test
Concentration
Chloride" Sulfateb Temperature pH D.O. Conductivity Alkalinity Hardness
(mg/L) (mg/L) (°C) (s.u.) (mg/L) (umhos) (mg/L) (mg/L)
Dilution water/Control
535 (505) mg/L
1032 (985) mg/L
2028 (1900) mg/L
4018 (3899) mg/L
8000 (7750) mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
33
39
36
490
520
505
970
1000
985
1900
1900
1900
3800
58
59
58
59
59
59
58
60
58
4000
3899
7700
7800
7750
59
60
60
23.0
22.9
23.0
23.0
23.0
22.9
23.0
22.9
22.9
23.0
22.8
22.9
22.8
22.9
23.0
22.9
23.0
22.9
23.0
23.0
22.9
23.0
22.7
22.9
22.9
22.8
22.9
22.9
22.8
22.9
8.1
8.0
8.1
8.0
8.1
8.0
8.0
8.0
8.0
8.1
8.0
7.9
7.93
8.13
7.99
8.14
8.00
8.22
8.05
8.14
8.05
8.06
8.07
8.14
385
378
2010
2000
3620
3640
6700
6730
12520
12470
24200
24000
62
62
62
62
62
62
62
62
62
62
62
62
48
48
48
48
48
48
48
48
48
48
48
48
" Chloride Analysis Method 300.0
b Sulfate Analysis Method 300.0
147
-------
Sulfate
Table 107 provides a summary of estimated LC50 values for the two toxicity tests
performed using sulfate. LCso values ranged between 1,483 and 3,378 mg
Table 107. LCso estimates for toxicity tests performed using sulfate.
Test Species and Duration
Ligumia recta - 96 hr
Megalonaias nervosa - 96 hr
LC50(mgS04/L)
1,483
3,378
For each of the acute toxicity tests completed using sulfate, two tables were generated:
the first summarizes the test results for each toxicity test, including nominal and
analytical test concentration and LCso estimates with confidence intervals; the second
table summarizes analytical chemistry data collected throughout the toxicity tests.
148
-------
96-hr Toxicity ofSulfate on Ligumia recta
The 96-hr test to determine the toxicity of sulfate on L. recta was completed by INHS.
Test organisms, < 5-day old juveniles collected from the Missouri State University
laboratory culture, were acclimated to the dilution water (MHRW), test temperature and
other test conditions prior to test initiation. Once acclimated, test organisms were
examined for any disease, stress, parasites, etc. If free from ailments, test organisms were
randomly assigned to the test chambers (which were randomly assigned to testing
locations); four replicates were used per treatment with five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 5,000, 2,545, 1,317, 703, and
397 mg SO4/L.
Testing was conducted at 20 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LC50 value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 108; test results are provided in Table 109. Analytical chemistry data are
provided in Table 110. Accompanying information, including raw laboratory data,
analytical chemistry data and statistical analyses, is provided in Appendix 32.
149
-------
Table 108. Test conditions for 96-hour toxicity test on Ligumia recta with sulfate.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg SO4/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination-mg
S04/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Ligumia recta, juveniles <5 days old, Missouri State
University
Static, 96 hours
September 10- 14,2009
20 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
50 mL beaker
40 mL
5
4
20
5,000, 2,545, 1,317, 703, and 397
5,038, 2,349, 1,200, 665, and 370
None
USEPA MHRW
Sodium sulfate anhydrous: Sigma Aldrich, 99% pure Cas.
No. 7757-82-6, Lot # A0231747
None
None
Mortality (LC50)
150
-------
Table 109. Test results for 96-hour toxicity test on Ligumia recta with sulfate.
Results of a Lisumia recta 96-Hour Static Acute Toxicitv Test
Conducted 09/10/09 - 09/14/09 Using: Sodium sulfate: Sigma Aldrich Cas. No. 7757-82-6
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
397 (370) mg/L
703 (665) mg/L
13 17 (1200) mg/L
2545 (2349) mg/L
5000 (5038) mg/L
Cumulative Percent Affected a
24-Hr
0
0
0
0
0
100
48-Hr
0
0
0
0
0
100
72-Hr
0
0
0
0
80
100
96-Hr
5
0
5
15
100
100
LC50 Values* (mg/L)
24-Hr 48-Hr
72-Hr 96-Hr
3440 3440 1938 1483
96-Hour LC50* = 1483 mg/L
LC50 95% Confidence Limits
24-Hr 48-Hr
LLNR NR
ULNR NR
72-Hr 96-Hr
1705 1321
2203 1665
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not reliable
Method(s) Used to Determine LC50 and EC50
Confidence Limit Values: Spearman-Karber
a Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
* All LC™ and EC™ values are determined based on measured concentrations.
151
-------
Table 110. Analytical chemistry data for 96-hour toxicity test on Ligumia recta with
sulfate.
Nominal (Measured) Test
Concentration
Sulfate11 Chloride"
(mg/L) (mg/L)
Temperature pH D.O. Conductivity Alkalinity Hardness
(°C) (s.u.) (mg/L) (inmos) (mg/L) (mg/L)
Dilution water/Control
397 (370) mg/L
703 (665) mg/L
13 17 (1200) mg/L
2545 (2349) mg/L
5000 (5038) mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
83
85
84
380
360
370
660
670
665
1200
1200
1200
2300
3.0
2.5
2.6
2.7
2.8
2.9
3.9
3.3
4.3
2400
2349
4700
5400
5038
4.8
5.9
6.1
19.8
19.7
19.8
19.2
19.2
19.8
19.7
19.8
19.2
19.2
19.8
19.8
19.9
19.2
19.2
19.8
19.9
19.9
19.2
19.2
19.8
19.9
20.0
19.2
19.2
19.9
19.9
20.0
19.2
19.2
7.9
8.0
8.0
8.2
8.1
8.3
8.0
8.3
8.1
8.4
8.1
8.4
7.93
7.85
7.95
7.87
7.95
7.92
7.94
7.96
7.99
7.95
8.08
8.00
309
308
1029
1032
1691
1716
2840
2990
5280
5380
9570
9680
60
62
60
62
60
60
60
60
60
60
62
64
92
92
92
92
92
92
92
92
92
92
94
94
" Sulfate Analysis Method 300.0
b Chloride Analysis Method 300.0
152
-------
96-hr Toxicity ofSulfate on Megalonaias nervosa
The 96-hr test to determine the toxicity of sulfate onM nervosa was completed by INHS.
Test organisms, < 5-day old juveniles collected from the Genoa National Fish Hatchery,
were acclimated to the dilution water (MHRW), test temperature and other test conditions
prior to test initiation. Once acclimated, test organisms were examined for any disease,
stress, parasites, etc. If free from ailments, test organisms were randomly assigned to the
test chambers (which were randomly assigned to testing locations); four replicates were
used per treatment with five organisms per replicate.
Organisms were exposed to a dilution water control and the test chemical at varying
concentrations under static conditions. Serial dilutions of the highest test concentration
(known weight of test chemical dissolved in a known volume of dilution water) were
made to prepare the following nominal test concentrations: 5,000, 2,545, 1,317, 703, and
397 mg SO4/L.
Testing was conducted at 20 ± 1 °C with a photoperiod of 16 hr light and 8 hr dark
(ambient laboratory light). Organisms were not fed for the duration of the test and were
examined daily for mortality. Once the test was complete, the LC50 value was determined
using the Spearman-Karber method.
A summary of the toxicity test conditions present throughout the assessment are provided
in Table 111; test results are provided in Table 112. Analytical chemistry data are
provided in Table 113. Accompanying information, including raw laboratory data,
analytical chemistry data and statistical analyses, is provided in Appendix 33.
153
-------
Table 111. Test conditions for 96-hour toxicity test on Ligumia recta with sulfate.
Summary of Toxicity Test Conditions
1. Test Species and Age:
2. Test Type and Duration:
3. Test Dates:
4. Test Temperature (°C):
5. Light Quality:
6. Photoperiod:
7. Feeding Regime:
8. Size of Test Vessel:
9. Volume of Test Solutions:
10. No. of Test Organisms per Test Vessel:
11. No. of Test Vessels per Treatment:
12. Total No. of Test Organisms per Treatment:
13. Test Concentrations (mg SO4/L):
14. Analytical Test Concentrations (geometric mean of
samples collected at test initiation and termination- mg
S04/L):
15. Renewal of Test Solutions:
16. Dilution and Primary Control Water:
17. Test Material:
18. Secondary Control Water:
19. Aeration:
20. Endpoints Measured:
Megalonaias nervosa, juveniles <5 days old, Genoa
National Fish Hatchery
Static, 96 hours
October 16 - 20, 2009
20 + 1
Ambient Laboratory, 10-20 uE/mVs
16 h light, 8 h darkness
None
50 mL beaker
40 mL
5
4
20
5,000, 2,545, 1,317, 703, and 397
5,130, 2,498, 1,249, 690, and 380
None
EPA MHRW
Sodium sulfate anhydrous: Sigma Aldrich, 99% pure Cas.
No. 7757-82-6, Lot # A0231747
None
None
Mortality (LC50)
154
-------
Table 112. Test results for 96-hour toxicity test on Megalonaias nervosa with sulfate.
Results of a Mesalonaias nervosa
Conducted 10/16/09 - 10/20/09
Nominal (Measured)
Concentrations
Primary Control/
Dilution Water
397 (380) mg/L
703 (690) mg/L
13 17 (1249) mg/L
2545 (2498) mg/L
5000 (5 130) mg/L
96-Hour Static Acute Toxicitv Test
Using: Sodium sulfate: Sigma Aldrich Cas. No. 7757-82-6
Cumulative Percent Affected a
24-Hr
0
5
0
0
0
100
48-Hr
0
5
0
0
0
100
72-Hr
0
10
5
0
10
100
96-Hr
0
10
5
0
10
100
LC50 Values* (mg/L)
24-Hr 48-Hr 72-
Hr 96-Hr
3,564 3,564 3,378 3,378
96-Hour LC50* = 3,378 mg/L
LC50 95% Confidence Limits
24-Hr 48-Hr 72-
Hr 96-Hr
LL NR NR 3,021 3,021
ULNR NR 3,777 3,777
LL = Lower Limit
UL = Upper Limit
NR = Confidence Intervals are not
reliable
Method(s) Used to Determine LC50 and EC50
Confidence Limit Values: Spearman-Karber
Cumulative percent affected is the total percentage of test organisms observed dead, immobile, exhibiting
loss of equilibrium or other defined endpoints.
• All LC50 and EC50 values are determined based on measured concentrations.
155
-------
Table 113. Analytical chemistry data for 96-hour toxicity test on Megalonaias
nervosa with sulfate.
Nominal (Measured) Test
Concentration
Sulfate11 Chloride Temperature pH D.O. Conductivity Alkalinity Hardness
(mg/L) (mg/L) (°C) (s.u.) (mg/L) (mmos) (mg/L) (mg/L)
Dilution
water/Control
397 (380) mg/L
703 (690) mg/L
13 17 (1249) mg/L
2545 (2498) mg/L
5000 (5130) mg/L
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
DayO
Day 1
Day 2
Day 3
Day 4
83
90
86
380
380
380
680
700
690
1200
1300
1249
2400
2.4
2.4
2.4
2.4
2.6
2.5
2.7
2.8
2.7
3.1
3.3
3.2
4.0
2600
2498
4700
5600
5130
4.3
4.1
5.7
6.3
6.0
21.0
20.9
20.5
20.9
20.6
21.0
20.8
20.4
20.9
20.7
21.0
20.9
20.4
20.9
20.7
21.0
21.0
20.5
20.9
20.8
21.0
20.9
20.5
20.8
20.8
21.0
21.0
20.5
20.8
20.9
7.8
7.8
7.8
7.9
7.9
8.0
7.9
7.9
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.1
8.0
8.0
8.0
8.0
8.2
8.1
8.1
8.2
8.1
8.3
8.2
8.2
8.3
8.2
7.95
8.30
8.21
8.28
8.18
8.24
8.38
8.22
8.40
8.20
8.21
8.35
8.23
8.29
8.25
8.28
8.32
8.28
8.40
8.24
8.22
8.30
8.20
8.38
8.20
8.26
8.28
8.00
8.32
8.37
300
300
308
324
364
1021
1016
1020
1021
1104
1702
1695
1698
1696
1853
2940
2920
2930
2940
3230
5320
5280
5295
5290
5600
9550
9540
9540
9540
10320
60
68
60
68
62
68
62
68
62
68
62
68
88
94
00
00
88
90
88
90
90
90
92
94
92
* Sulfate Analysis Method 300.0
b Chloride Analysis Method 300.0
156
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