Toxicity 101 An Introduction to Whole Effluent Toxicity (WET) Testing

Transcription

1 Toxicity 101 An Introduction to Whole Effluent Toxicity (WET) Testing VA AWWA/VWEA 23 rd Annual Good Laboratory Practices Conference July 25, 2017 Charlottesville, VA Peter F. De Lisle, Ph.D. Coastal Bioanalysts, Inc. Gloucester, VA

2 Why WET? 1980s EPA finds effluents & streams toxic even though compliant with water quality standards Toxicity data are available for a relatively small percentage of all chemicals in use Sufficient toxicity data are available for limited number of chemical-specific surface water quality standards WET evaluates synergistic, additive and antagonistic effects of multiple toxicants WET evaluates matrix effects (e.g. DOC, ph, hardness) which may decrease or increase toxicity 2

3 WET TESTS IN YOUR PERMIT A. Testing Requirements B. Running the Tests C. Interpreting Test Results D. Dealing with Toxicity 3

4 1.0 TU c 0.3 TU a (Permit with WET compliance) (Permit with WET monitoring) From EPA-832-B

5 Acute WET Tests Short (48 h) exposure duration (e.g. swimming through discharge plume, stormwater) A single sample is used Test endpoint is survival only Intermittent and continuous (> 5 days) discharges WLA 1Q10 or end of pipe basis Permit reporting requirements: LC50 (concentration of effluent causing 50% mortality). TU A =100/LC50 NOAEC (highest concentration of effluent causing no statistically significant reduction in survival). Effluent dominated (>30%) stream. TU A =100/NOAEC 5

6 Chronic WET Tests: Long (6-7 days) exposure duration (e.g. partial life-cycle; population maintenance downstream) A minimum of 3 samples are used Multiple test endpoints: Survival (lethal endpoint) Growth or reproduction (sublethal endpoint) Continuous (> 5 days) discharges only WLA 7Q10 (steady-state) or dynamic basis Permit reporting requirements: NOEC (highest concentration of effluent causing no statistically significant reduction in lethal or sublethal endpoints). TU C =100/NOEC IC25 (concentration of effluent causing 25% reduction in sublethal endpoint). ). TU C =100/IC25 6

7 WET TESTS IN YOUR PERMIT A. Testing Requirements B. Running the Tests C. Interpreting Test Results D. Dealing with Toxicity 7

8 The Players Ceriodaphnia dubia Pimephales promelas (Fathead minnow) Oncorhynchus mykiss (Rainbow trout) Americamysis (Mysidopsis) bahia Cyprinodon variegatus 8

9 24-h old Fathead 7-day old Ceriodaphnia 7-day old Fathead 9

10 3-d old mysid Newly hatched Sheepshead larvae 12-d old Sheepshead 10

11 Fathead Minnow Cultures 11

12 Fathead Minnows Spawning 12

13 Fathead Minnows Eggs 13

14 Ceriodaphnia Cultures 14

15 Mysid cultures 15

16 Sample Collection Acute tests: 1 sample Chronic tests: 3 samples over 5 days 16

17 Sample hold time is 36 hours Samples must arrive at lab < 6 o C 17

18 Sample Prep Warm sample to 25 C (Add sea salts to 20 g/kg) Filter 60µm (if live indigenous organisms present) Check D.O. aerate if <4.0 mg/l or >saturation (8.3 mg/l freshwater, 7.4 mg/l saltwater) Conductivity ph TRC (if present at check-in) 18

19 Synthetic dilution waters used in VA tests Saltwater: Commercial artificial sea salts Salinity 20 g/kg (VA) Freshwater: Moderately-hard synthetic freshwater Hardness mg/l CaCO 3 ph Alkalinity mg/l CaCO 3 Conductivity µs/cm + 19

20 Toxicity Tests Test chambers are filled with dilutions of effluent Replicate chambers are tested for each concentration A lab control (blank) of standard dilution water is also tested Control 6.25% 12.5% 25% 50% 100% 20

21 Water quality is measured in test chambers Standardized conditions help make data comparable among labs Water quality (Temp, ph, D.O., salinity) must be within tolerance limits of test organisms and method specs Deviations from test conditions may confound interpretation of results or invalidate test 21

22 Plus: Hardness, alkalinity, ammonia, TRC on each new sample and dilution water. 22

23 Animals are randomly assigned to beakers. Species, replicates, duration, etc. depend on permit/protocol ACUTE 2 reps of 10 fish each CHRONIC 4 reps of 10 fish each 2 reps of 10 shrimp each 8 reps of 5 shrimp each 4 reps of 5 daphnids each 10 reps of 1 daphnid each 23

24 Each day tests are renewed with fresh solutions, water quality measured before & after renewal, animals fed Acute test may be static (VA) or renewal. Only mysids are fed in acute tests. 24

25 Over the time period of chronic tests the organisms grow and mature. Exposure to toxicants can result in decrease in survival and sublethal effects such as decreased growth (dry weight at test end) or reproduction (number of offspring or eggs). Control 6.25% 12.5% 25% 50% 100% 25

26 At the end of the test data are statistically analyzed relative to controls: NOEC or NOAEC: One-tailed hypothesis test at p=0.05. (H 0 : Control < Effluent treatment response) Point estimates LC50 (EC50), IC25 26

27 Mortality (%) Acute toxicity is usually reported as the LC 50. This is the calculated concentration predicted to kill 50% of the test organisms within test period LC50 = 34% Effluent Concentration (%) 27

28 frequency Acute and chronic toxicity may also be reported as the NOAEC or NOEC using hypothesis testing No significant difference. NOEC=12.5% 80 Mean=20 young in 12.5% effluent conc. Mean=23 young in control group No. Ceriodaphnia offspring 28

29 frequency At a confidence level of 95%, toxicity is predicted at 25% effluent concentration. There is 5% chance that there is no difference between the two groups (false positive) Significant (p=0.05) difference. LOEC=25% 80 Mean=11 young in 25% effluent conc. Mean=23 young in control group % Rejection Region No. Ceriodaphnia offspring 29

30 % Effect (Offspring reduction vs controls) Sublethal toxicity is also often expressed as the IC 25. This is the calculated concentration predicted to cause a 25% reduction in the endpoint (e.g. reproduction) compared to the control group IC 25 = 26% Effluent Concentration (%) 30

31 WET TESTS IN YOUR PERMIT A. Testing Requirements B. Running the Tests C. Interpreting Test Results D. Dealing with Toxicity 2012 Coastal Bioanalysts, Inc 31

32 The permit language (monitoring): The test dilutions should be able to determine compliance with the following endpoints: Acute NOAEC of 100% equivalent to a TU a of 1.00 Chronic NOEC of 69% equivalent to a TU c of 1.44 *Note: Chronic tests have two or more endpoints (survival and sublethal). Report the larger TU C value, which is always the sublethal endpoint. Example: NOEC survival = 50% (2.0 TU C ) and NOEC reproduction = 25% (4.0 TU C ); report 4.0 TU C 32

33 Portion of Toxcalc printout for acute Ceriodaphnia test for permit limit NOAEC = 100% (1.00 TU a ) 33

34 Portion of Toxcalc printout for chronic Ceriodaphnia test for permit limit NOEC = 69% (1.44 TU c ) 34

35 These tests met the permit endpoint of 1.00 TU a but did not meet permit endpoint of 1.44 TU c for the Ceriodaphnia 35

36 WET TESTS IN YOUR PERMIT A. Testing Requirements B. Running the Tests C. Interpreting Test Results D. Dealing with Toxicity 36

37 What s toxic, what s not. The dose makes the poison Paracelsus ( ) 37

38 Common Toxicants Identified in Wastewaters Polymers Metals (e.g. Zn, Cu, Ni) Organics (pesticides, surfactants) TDS acute >3000 µs/cm chronic >1500 µs/cm Chlorine NH 3 Biological factors (bacteria, fungi) 38

39 Ok, so what do we do? 39

40 1. False Positive WET Test Results Type I statistical error (5% NOEC/NOAEC Endpoints) Intra-laboratory variability Biological interference 40

41 Toxic Units (100/LC50) Large Intra-Laboratory Variability = Increased False Positive & Negative Rate Hypothetical Split Testing of Sam ple w ith Constant Toxicity Permit Limit (3.5 T.U.) Lab A (Mean = 2.34 T.U. CV = 0.32) Lab B (Mean = 2.33 T.U. CV = 0.08) Test Number 41

42 False Positives: Biological Interference Identification: Typically fish only, invertebrates usually not affected Replicate specific mortality (CV > 40%) No acute mortality; death begins day 3 or 4 Irregular concentration-response curve with good survival in synthetic dilution water controls Common in natural surface waters or effluents with surface water component (e.g. cooling waters, retention ponds) May be seasonal 42

43 2. Toxicity Identification Evaluation and Toxicity Reduction Evaluation Toxicity Reduction Evaluation A step-wise procedure to control toxicity (within permit limits) which may include identification, treatability, source evaluation and other measures to identify appropriate control methods. Toxicity Identification Evaluation A step-wise procedure involving characterization (class), identification, and confirmation of toxicant(s) Problems: Expensive and time consuming TIE Methods do not work well with samples of low toxicity Some toxicants expensive/difficult to remove Remedies that do require knowledge of causative toxicant(s) may be available 43

44 From EPA 600/6-91/003 44

45 From EPA 600/6-91/003 45

46 3. Chemical-specific Limits in Lieu of WET Limit Limits of whole effluent toxicity are not necessary where chemical-specific limits for the effluent are sufficient to attain and maintain applicable numeric and narrative water quality standards Problems: State may still require WET tests to confirm that chemical-specific limit is controlling toxicity In addition to identifying toxicant, safe concentrations will need to be established for inclusion in limit (toxicants with existing water quality standards already limited to concentrations controlling toxicity) 46

47 4. Dilution Solutions Solutions: Perform mixing study to determine if dilution criteria used by regulatory agency are appropriate Install diffuser at outfall Move outfall to larger receiving stream Decrease flow to outfall Problems: Costs of mixing studies, outfall relocation, diffuser installation Difficulties in obtaining easements for outfall relocation 47

48 Resources (Links and downloads to all relevant documents) Guidelines Establishing Test Procedures for the Analysis of Pollutants; Whole Effluent Toxicity Test Methods; Final Rule (2002) (Searchable toxicity database) National Whole Effluent Toxicity (WET) Implementation Guidance Under the NPDES Program (2004 Draft) EPA 832-B (Good explanation of program rationale, example calculation of RP) 48

49 Questions or further information: Pete De Lisle Coastal Bioanalysts, Inc Enterprise Court Gloucester, VA (Fax) 49