TNI Logo The NELAC Institute

TNI Standards Guidance

Disclaimer: This material represents the opinion of its authors. It is intended solely as guidance and does not include any mandatory requirements except where such requirements are referenced. This guidance does not establish expectations of being implemented universally, exclusively, in whole, or in part.

This guidance does not establish or affect legal rights or obligations and is not finally determinative of the issues it addresses. It does not create any rights enforceable by any party in litigation with TNI, its accreditation bodies, or affiliated institutions. Any decisions made by TNI regarding requirements addressed in this guidance will be made by applying the applicable standards, policies or procedures to the relevant facts.


Module: Toxicity

Subject: Randomization of Testing Chambers

Question 1

Is randomization necessary or can the lab justify conducting the test without randomization?

While there is nothing in the TNI Volume 1 Module 7 (Quality Systems for Toxicity Testing) to assist us in addressing this question, there are several instances in EPA’s chronic WET guidance discussing the importance and requirement of randomizing both the addition of test organisms to test chambers and the placement of test chambers. The pertinent language describing this in the subsections are included below.

For example, per USEPA chronic WET guidance [EPA-821-R-02-013, EPA-821-R-02-014]:

" Statistical independence among observations is a critical assumption in all statistical analysis of toxicity data. One of the best ways to insure independence is to properly follow rigorous randomization procedures. Randomization techniques should be employed at the start of the test, including the randomization of the placement of tests organisms in the test chambers and randomization of the test chamber location within the array of chambers."

" All test chambers must be randomized using a template for randomization or by using a table of random numbers. Test chambers are randomized once at the beginning of the test (see Subsection When using templates, a number of different templates should be prepared, so that the same template is not used for every test. Randomization procedures must be documented with daily records."

" Randomize the position of test chambers at the beginning of the test (see Appendix A). Maintain the chambers in this configuration throughout the test."

" the test chambers must be randomly assigned to a board using a template (Figure 1) or by using random numbers (see Appendix A). Randomizing the position of test chambers as described in figure 1 (or equivalent) will assist in assigning test organisms using blocking by known parentage (Subsection 13.102.4). A number of different templates should be prepared, and the template used for each test should be identified on the data sheet. The same template must not be used for every test."

It is critical to pay attention to the specific wording of the method and/or permit regarding what is required or not. When method language for randomization includes ‘must’ phrases (and not 'should' phrases), a failure to use randomization procedures could cause tests to be considered invalid for reporting purposes. Tests should follow the methods specified in the National Pollutant Discharge Elimination System (NPDES) permit.

For example using the summary of test conditions for the C. dubia chronic study below (EPA-821-R-02-013, Method 1002.0) are the required conditions for this test (unless specified otherwise in the NPDES permit). Other items listed on the table are recommended. Tests should follow the method specified in the NPDES permit.

  • Static-renewal
  • Test temperature of 251C (recommended) with a maximum differential of 3C (required)
  • Daily renewal
  • Age: <24-h old within an 8-h period
  • 1 organism per test cup, placement assigned using blocking by known parentage
  • 10 replicates
  • 5 test concentrations & control (while this is required some states perform testing with only one effluent concentration and a control so this requirement is state specific)
  • Test duration: when 60% or more of the surviving control females have had three broods (maximum test duration of 8 days)
  • Endpoints: survival and reproduction
  • Test acceptability criteria (TAC): ≥80% survival of control organisms, ≥ 15 average neonates per surviving control females, ≥60% of surviving control females have had three broods
  • A minimum of 3 effluent samples per test with a maximum holding time of 36 h before first use, see Subsection 8.5.4 (EPA-821-R-02-013) for more info.



Short Term Methods For Estimating The Chronic Toxicity of Effluents and Receiving Water to Freshwater Organisms, Fourth Edition. October 2002. United States Environmental Protection Agency. Office of Water, Washington, D.C., EPA 821-R-02-013