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NPDES - ACUTE Bioassay

This toxicity test is conducted on municipal wastewaters that are directly discharged into receiving waters (streams, rivers, ocean, etc.) or on industrial dischargers who may or may not have their own outfall. There are several editions of the EPA's acute testing manual, each having minor differences which may significantly influence the outcome of the test. The most recent edition, the fifth (EPA-821-R-02-012) calls for using fish of one to 14 days in age and a minimum renewal at 48 hours. Both of these requirements tend to make this test more sensitive than previous editions of the test method. A detailed test protocol of this method is given below. Also the entire acute manual is available directly from EPA's homepage. You may access it by clicking here. Our laboratory is also certified to conduct toxicity tests following the older fourth edition protocol (EPA/600/4-90/027F). This manual may be still referenced in NPDES permits and differs little from the new manual. The choice of which protocol is often directed by regional water quality control boards and should be specified in the NPDES permit. Any protocol modifications are also usually listed in the NPDES permit.

The basic acute test uses a series of five test chambers each containing a specific concentration of the effluent diluted with standard laboratory water plus a control. A common dilution series such as: 100%, 50%, 25%, 12.5%, and 6.25% may be used. The dilution series may be specified in the NPDES permit and may differ between permits to reflect individual regulatory concerns. Ten test fish are placed into each chamber and their survival and water quality of the test chambers are monitored for 96 hours. All tests are conducted in replicate (2, 4, or 5 replicates, depending upon species). The number of fish surviving at each test concentration and in the control chamber at the end of the test are used to mathematically calculate the sample concentration toxic to 50% of the test fish. This known as the LC50. Toxicity units can then be calculated by dividing 100 by the LC50 value. For example, an LC50 of 75% would yield a TU of 1.3 TUa (100/75 Toxic Units acute). This test protocol can also be conducted as a percent survival bioassay where 100% sample is divided into two or more test tanks, fish are added, and water quality and survival are monitored for 96 hours. Results are then reported as percent survival in 100% effluent.

Other important considerations:

  1. Test organism - there are over 45 different species listed in the EPA manual, ranging from water fleas to rainbow trout, however fathead minnows are by far the most commonly used organism;
  2. Length of test - tests can range from 24 to 96 hours in length with 48 and 96 hour tests the most common;
  3. Static or static renewal - in static tests solutions are mixed at the beginning of the test with test organisms exposed to the same solution throughout the test, while in static renewal tests solutions are mixed and changed daily or at a minimum every 48 hours (these are much more labor intensive and therefore more expensive).

SPECIAL CONDITIONS: Sample hold time is 36 hours. Sample volumes are: one gallon for static and two and a half gallons for some static-renewal tests. Samples should be kept cold (4oC) prior to and during shipment to the laboratory. Sample containers, coolers and blue ice available upon request. We recommend shipping via Federal Express Priority Overnight Service. Please call our laboratory for a cost quote and/or additional information. Our number is (805) 650-0546.


  1. Test type: Static non-renewal, static-renewal, or flow-through
  2. Test duration: 24, 48, or 96 h
  3. Temperature: 20C 1C; or 25C 1C
  4. Light quality: Ambient laboratory illumination
  5. Light intensity: 10-20 E/m2/s (50-100 ft-c) (ambient laboratory levels)
  6. Photoperiod: 16 h light, 8 h darkness
  7. Test chamber size: 250 mL (minimum)
  8. Test solution volume: 200 mL (minimum)
  9. Renewal of test solutions: Minimum, after 48 h
  10. Age of test organisms: 1-14 days; 24-h range in age
  11. No. organisms per test chamber: Minimum, 10 for effluent and receiving water tests
  12. No. replicate chambers per concentration: Minimum, 2 for effluent tests Minimum, 4 for receiving water tests
  13. No. organisms per concentration: Minimum, 20 for effluent tests Minimum, 40 for receiving water tests
  14. Feeding regime: Artemia nauplii are made available while holding prior to the test; add 0.2 mL Artemia nauplii concentrate 2 h prior to test solution renewal at 48 h
  15. Test chamber cleaning: Cleaning not required
  16. Test solution aeration: None, unless DO concentration falls below 4.0 mg/L; rate should not exceed 100 bubbles/min
  17. Dilution water: Moderately hard synthetic water prepared using MILLIPORE MILLI-Q or equivalent deionized water and reagent grade chemicals or 20% DMW, receiving water, ground water, or synthetic water, modified to reflect receiving water hardness.
  18. Test concentrations: Effluents: Minimum of five effluent concentrations and a control Receiving Waters: 100% receiving water and a control
  19. Dilution series: Effluents: 0.5 dilution series Receiving Waters: None, or 0.5 dilution series
  20. Endpoint: Effluents: Mortality (LC50 or NOAEC) Receiving Waters: Mortality (Significant difference from control)
  21. Sampling and sample holding requirements: Effluents and Receiving Waters: Grab or composite samples are used within 36 h of completion of the sampling period.
  22. Sample volume required: 2 L for effluents and receiving waters
  23. Test acceptability criterion: 90% or greater survival in controls

Hazardous Waste » California: Title 22 CCR
Storm Water Runoff » Percent Survival
NPDES - Acute » EPA Protocol
NPDES - Chronic » Fathead Minnow / Water Flea / Algal / Inland Silverside / Abalone / Giant Kelp / Topsmelt