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TNI Standards Guidance

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Module: Toxicity

Subject: Test Organism Criteria

Question 1

One of the stipulations for neonates to be selected for initiating a Ceriodaphnia chronic bioassay is that the parent organism must have a mean of 20 neonates by the time _ 60% of surviving females have a third brood. It appears that this as a “should” and not a “must” – is that accurate?  (NOTE: these paragraphs are cited from EPA-821-R-02-013) Cultures which are properly maintained should produce at least 20 young per adult in three broods (seven days or less). Typically, 60 adult females (one board) will produce more than the minimum number of neonates (120) required for two tests. Records should be maintained on the survival of brood organisms and number of offspring at each renewal. Greater than 20% mortality of adults, or less than an average of 20 young per female would indicate problems, such as poor quality of culture media or food.  Cultures that do not meet these criteria should not be used as a source of test organisms.

There are differences between culture and testing guidelines, and this question seems to overlap the two. The statement about the neonates used in a test must come from parent organisms that have a mean of 20 neonates (in seven days or less; see Chronic WET guidance and of EPA-821-R-02-013), is a “should” and not a “must”. This is a recommendation to insure minimum control criteria are met at the end of the test. Each lab may develop their own way of choosing test organisms, but as long as the test method age and parentage requirements are met, and the lab-defined protocols in an SOP or other quality system document are followed, not meeting this condition on occasion would be acceptable, unless there are records of inconsistent results or repeated control failures.  However, individual states could have rules in place to make this a requirement and not a recommendation.

For testing, the test acceptability criteria (TAC) for the C. dubia short-term chronic WET test per the guidance is a MUST for survival (≥80%) and reproduction (≥15 average young per surviving adult female).  See section 13.12.1 and Table 3 in the EPA chronic WET guidance, EPA-R-02-013


EPA WET Methods as codified at 40 CFR 136.3, Table 1A

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

Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition.  October 2002. United States Environmental Protection Agency. Office of Water, Washington, D.C., EPA 821-R-02-014

2009 TNI V1M2§5.9.3c The quality control protocols specified by the laboratory’s SOP shall be followed (see § of V1M2.)  The laboratory shall ensure that the essential standards outlines in Technical Modules or mandated methods or regulations (whichever are more stringent) are incorporated into their method manuals.  When it is not apparent which is more stringent, the QC in the mandated method or regulations is to be followed.