BASICS OF TOXICITY TESTING WITH CERIODAPHNIA DUBIA

1. BASICS OF TOXICITY TESTING WITH CERIODAPHNIA DUBIA LAURA SHEALY SHEALY CONSULTING, LLC

2. TOPICS FOR DISCUSSION • PURPOSE OF TOXICITY TESTING • WET TEST PROCEDURES • DATA INTERPRETATION

3. PURPOSE OF WHOLE EFFLUENT TOXICITY • DEFINITION Whole effluent toxicity (WET) is the aggregate toxic effect of an effluent sample measured directly by an aquatic toxicity test.

4. PURPOSE OF WHOLE EFFLUENT TOXICITY • Expanded Definition WET tests utilize live organisms to measure actual biological responses to an effluent and, therefore, integrates the effects of all chemicals present in the effluent.

5. PURPOSE OF WHOLE EFFLUENT TOXICITY Why Perform Toxicity Testing? Measuring all potentially toxic chemicals in a sample would not be feasible (>50 million in CAS registry) WET tests are performed in order to indirectly measure all potentially toxic chemicals present WET predicts the potential for an effluent to have an adverse effect on the in-stream aquatic population

6. TOXICITY TESTING IN PERMITS • Early 1980’s toxicity testing was adapted by newly created EPA from testing chemicals to testing Whole Effluent Discharges • Mid 1980’s - Acute monitoring and NPDES • limits used on a routine basis • Late 1980’s/early 1990’s - Began use of chronic monitoring and chronic NPDES limits

7. WET TEST PROCEDURES Acute Test • Duration of 24, 48, or 96 hours • Five effluent concentrations and a laboratory control • Compare survival of the organisms exposed to the effluent treatments to the control

8. WET TEST PROCEDURES Chronic Test • Duration of 7 or more Days, or other endpoint • Five effluent concentrations and a laboratory control • Effluent concentrations bracket the CTC • Compare survival and reproduction (or growth) of the organisms exposed to the effluent treatments to the control

9. WET TEST PROCEDURES TEST SPECIES • FRESHWATER DISCHARGES Ceriodaphniadubia(water flea) Pimephalespromelas (fathead minnows)

10. Ceriodaphniadubia • Why we use Ceriodaphnia – 2 Main Reasons 1) Sensitivity Very Sensitive Toxicity of copper to C. dubia is 6 ppb in pure water 2) Practicality Easy to Culture in laboratory setting Very prolific (20 - 40 young/female in 7 days) Parthenogenetic - asexual reproduction Almost exclusively female in lab so do not have to worry about fertilization

11. PARTHENOGENESIS LIFE CYCLE OF C. DUBIA STR

12. PARTHENOGENESIS LIFE CYCLE OF C. DUBIA STR STRESS

13. LIFE CYCLE OF C. DUBIA SEXUAL REPRODUCTION STRESS MALE PROGENY FEMALE PROGENY EPHIPPIA

14. CLIENT PERMIT • Acute or Chronic Test • Test Species • Test Frequency • Sampling Requirements • Targeted effluent concentration (Concentration to Pass) ‘Target’ Effluent Concentration may be calculated as the Instream Waste Concentration or the Chronic Test Concentration. Test design should bracket this concentration.

15. STEP BY STEP PROCEDURE STEP 1: Samples logged into laboratory Receipt temperature and TRC taken on each sample

16. STEP BY STEP PROCEDURE STEP 2:Obtain C. dubia for testing 60 neonates/test • Ceriodaphnia neonates must meet the following criteria: • Must be <24 hours old • Must be released with the same 8 hour time period • Must be able to track to brood female (parent) – blocking design • Brood parent must be less than 14 days old • Brood parent must have average of 20 offspring/female during 1st 3 broods

17. STEP BY STEP PROCEDURE Step 3 Start Ceriodaphnia Test 1 newborn Ceriodaphnia “neonate” per test cup After 3 or 4 days she starts releasing offspring Prepare Test Dilutions Control + 5 effluent concentrations

18. STEP BY STEP PROCEDURE Step 4:Transfer ‘Mom’ to fresh test solution and record any mortality and/or reproduction. Typical Effluent Usage Mon. Sample – Tues. and Wed. Wed. Sample – Thurs. and Fri. Fri. Sample – Sat., Sun. and Mon.

19. TRANSFERING FEMALES OLD CUP NEW CUP NEONATES

20. BENCHSHEET

21. STEP BY STEP PROCEDURE Step 5:Feeding Each cup is fed 0.1ml YCT and 0.1ml algae each day (200,000 algae cells per test chamber)

22. STEP BY STEP PROCEDURE Step 6:Perform Water Chemistry Daily Daily Temperature Dissolved Oxygen pH

23. STEP BY STEP PROCEDURE Step 7:At end of test analyze data and determine if pass or fail and perform quality control checks

24. STEP BY STEP PROCEDURE Step 8:Call with any failures

25. DATA INTERPRETATION Hypothetical Chronic Test Typical dose response where percent effect increases as the concentration of effluent in the mixture increases.

26. DATA INTERPRETATION Hypothetical Chronic Test #1 – “TYPICAL RESPONSE” Typical dose response where percent effect increases as the concentration of effluent in the mixture increases. IC25 would be somewhere between 12.5% effluent and 25.0% effluent. Control 6.25% 12.5% 25% 50% 100% 25.0 22.0 (12%) 19.0 (24%) 10.1 (60%) 5.5 (78%) 0 (100%) Percent = % reduction in the #young/female COMPARED TO CONTROL

27. DATA INTERPRETATION Hypothetical Chronic Test #2 Hormesis Observed Hormesis is the term for generally favorable biological responses to low exposures to toxins IC25 would be somewhere between 6.25% effluent and 12.5% effluent. Control 6.25% 12.5% 25% 50% 100% 25.0 31.0 (0%) 19.0 (29%) 10.1 (64%) 5.5 (80%) 0 (100%) Percent = % reduction in the #young/female COMPARED TO CONTROL

29. QUESTIONS?