5.1 Ceriodaphnia  was first used as a toxicity test organism by Mount and Norberg (4). Introduced for use in effluent and ambient water evaluations, Ceriodaphnia have also been a valuable addition to single chemical test procedures.

5.2 Protection of a population requires prevention of unacceptable effects on the number, weight, health, and uses of the individuals of that species, or species for which the test species serves as a surrogate. A three-brood toxicity test is conducted to help determine changes in survival and the number of neonates produced that result from exposure to the test material.

5.3 Results of three-brood toxicity tests with C. dubia might be used to predict chronic or partial chronic effects on species in field situations as a result of exposure under comparable conditions.

5.4 Results of three-brood toxicity tests with C. dubia might be compared with the chronic sensitivities of different species and the chronic toxicities of different materials, and to study the effects of various environmental factors on results of such tests.

5.5 Results of three-brood toxicity tests with C. dubia might be useful for predicting the results of chronic tests on the same test material with the same species in another water or with another species in the same or a different water. Most such predictions are based on the results of acute toxicity tests, and so the usefulness of the results of a three-brood toxicity test with C. dubia might be greatly increased by also reporting the results of an acute toxicity test (see Guides E729 and E1192) conducted under the same conditions. In addition to conducting an acute test with unfed C. dubia, it might also be desirable to conduct an acute test in which the organisms are fed the same as in the three-brood test, to see if the presence of that concentration of that food affects the results of the acute test and the acute chronic ratio (see 10.4.1).

5.5.1 A 48 or 96-h EC50 or LC50 can sometimes be obtained from a three-brood toxicity test with a known test material, but often all the concentrations in the test will be below the EC50 or LC50. In addition, it is usually desirable to know the EC50 or LC50 before beginning the three-brood test, as a means to determine the concentrations for use in the chronic test (see 10.4.1). It should be noted that results from an acute test may not necessarily correspond to those of a chronic test, due to the addition of food to the chronic test.

5.6 Three-brood toxicity tests with C. dubia might be useful for studying biological availability of, and structure activity relationships between, test materials.

5.7 Results of three-brood toxicity tests with C. dubia can vary with temperature, quality and quantity of food, quality of the dilution water, condition of the test organisms, and other factors.

5.8 Results of three-brood toxicity tests with C. dubia might be an important consideration when assessing the hazards of materials to aquatic organisms (see Guide E1023), or when deriving water quality criteria for aquatic organisms.

1.1 This guide describes procedures for obtaining data concerning the adverse effects of an effluent or a test material (added to dilution water, but not to food) on Ceriodaphnia dubia Richard 1894, during continuous exposure throughout a portion of the organism's life. These procedures should also be useful for conducting life cycle toxicity tests with other Cladocera (Guide E1193), although modifications will be necessary.

1.2 These procedures are applicable to most chemicals, either individually or in formulations, commercial products, or known mixtures, that can be measured accurately at the necessary concentrations in water. With appropriate modifications these procedures can be used to conduct tests on temperature, dissolved oxygen, pH, and on such materials as aqueous effluents (see also Guide E1192), leachates, oils, particulate matter, sediments (see also Guide E1383), and surface waters. Renewal tests might not be applicable to materials that have high oxygen demand, are highly volatile, are rapidly biologically or chemically transformed, or sorb to test chambers. If the concentration of dissolved oxygen falls below 4 mg/L or the concentration of test material decreases by more than 20 % in test solution(s) between renewals, more frequent renewals might be necessary.

1.3 Other modifications of these procedures might be justified by special needs or circumstances. Results of tests conducted using unusual procedures are not likely to be comparable to results of many other tests. Comparisons of results obtained using modified and unmodified versions of these procedures might provide useful information on new concepts and procedures for conducting three-brood toxicity tests with C. dubia.

1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Specific hazard statements are given in Section 8.

1.5 This guide is arranged as follows:

Section

Referenced Documents

2

Terminology

3

Summary of Guide

4

Significance and Use

5

Apparatus

6

 Facilities

6.1

 Construction Materials

6.2

 Test Chambers

6.3

 Cleaning

6.4

Reagents and Materials

7

Hazards

8

Dilution Water

9

 Requirements

9.1

 Source

9.2

 Treatment

9.3

 Characterization

9.4

Test Material

10

 General

10.1

 Stock Solution

10.2

 Effluent

10.3

 Test Concentration(s)

10.4

 Collection

10.5

 Sample Containers

10.6

 Preservation

10.7

 Treatment

10.8

Test Organisms

11

 Species

11.1

 Age

11.2

 Source

11.3

 Brood Stock

11.4

 Food

11.5

 Handling

11.6

 Quality

11.7

Procedure

12

 Demonstration of Feasibility

12.1

 Experimental Design

12.2

 Dissolved Oxygen

12.3

 Temperature

12.4

 Preparing Test Solutions

12.5

 Conditioning Test Chambers

12.6

 Beginning a Test

12.7

 Renewing Test Solutions

12.8

 Duration of Test

12.9

 Biological Data

12.10

 Other Measurements

12.11

Analytical Methodology

13

Acceptability of Test

14

Calculation

15

Report

16

Appendixes

 Food

Appendix X1

 Culture Techniques

Appendix X2

 Test Chambers

Appendix X3

 Statistical Guidance

Appendix X4