1. Presented to: Presented by: Kevin Dischler, Bioassay Lab Director Understanding Offshore Toxicity Testing – Beyond Pass or Fail

2. 2 Topics to be covered…. Drilling fluids – WBM and SBM Produced water Subsea-chemicals What are the limits? What do they mean? What makes a test valid? How are results derived?

3. Drilling Fluids – Toxicity Testing Aquatic Toxicity – LC 50 with Mysids (WBM/SBM) Tests the affect of the mud on the water column LC 50 = Lethal Concentration to 50% of the exposed organisms Compliance target is an LC 50 30,000 ppm SPP. SPP = S uspended P articulate P hase of 1 part mud to 9 parts seawater mixture

4. 4 Drilling Fluid LC 50 Test Set-up Control + 5 concentrations, 3 replicates, 20 shrimp per replicate, randomly loaded Temp: 20±2ºC Salinity: 20±2 ppt 14 hours of light, 10 hours of dark SPP created by mixing mud and saltwater pH is adjusted Test validity: Control must have at least 90% survival @ 96- hours

5. Aquatic LC 50 Toxicity Test

6. Determining the LC 50 % SPPSurvivors (of 60)Survival % Control (0%)5998.3 20%5896.7 40%5490 60%4270 80%2440 100%1118.3 LC 50 of this data set is 72.8% or 728,000 ppm SPP With the target being 30,000 ppm, this would be a Passing test

7. Why a second drilling fluid tox test?

8. Sediment Toxicity Test Measure the effect of the SBM coated cuttings on the sea floor Uses Leptocheirus plumulosus as the test organism Exposes the Leptos to a mixture of SBM and a formulated (man-made) sediment

9. Sediment Toxicity Test Set-up 96-Hour tests are set-up with 5 concentrations and a Control, 5 replicates, 20 Leptos per replicate Tests are continuously aerated Temp: 20±1ºC Salinity: 20±1 ppt Light cycle: 14 hours of light, 10 hours of dark Test validity: Check #1 - Controls must have at least 90% survival @ 96-hours Check #2 - Controls must have Coefficient of Variation of <40%

10. With organisms from the same batch and randomly loaded: A 96 Hour LC 50 performed on the field sample A 96 Hour LC 50 performed on the appropriate reference mud: - SBMs <11.0 ppg test with 9.0 ppg Ref Mud - SBMs 11.0-14.0 ppg test with 11.5 ppg Ref Mud - SBMs >14.0 ppg test vs. with 14.5 ppg Ref Mud Sediment Toxicity Test Set-up

11. A SedTox test with a Ref Mud - Randomly loaded organisms - Randomly placed on the test shelves

12. The Sediment Toxicity Ratio (STR) The compliance limit for an STR is 1.0 LC 50 Value for the Reference Drilling Fluid = STR LC 50 Value for the Submitted SBM sample Ex: Ref MudLC 50 = 42.9 ml/Kg Field Mud LC 50 = 197 ml/Kg 42.9 ÷ 197 = 0.2 Sediment Toxicity Ratio is 0.2 and is a Pass

13. But what if…..? SedTox can be a difficult test. So much so, that EPA allows it to be run three times. An STR of >1.0 on the first test is an initial failure. Not out of compliance yet. Test 2 must be run from same sample, same bottle. Test 3 must be run from a sample caught within 15 minutes of the first sample When averaging is needed, the LC50s are averaged, not the STRs.

14. 14 Produced Water Toxicity Test Chronic static renewal seven (7) day survival and growth test Measures the effect of Produced Water on the survival and growth of the test organisms. The Vertebrate organism is a Menidia beryllina The Invertebrate organism is Mysidopsis bahia

15. 15 Produced Water Toxicity Test Set-up Mysid portion: - 7 day old organisms - Control + 5 concentrations, 8 replicates of 5 shrimp - Temperature: 26±1ºC, Salinity: 25±2 ppt Menidia (minnow) portion: - 7 to 11 day old organisms - Control + 5 concentrations, 5 replicates of 8 fish - Temperature: 25±1ºC, Salinity: 25±2 ppt Both require: - 16 hours of light, 8 hours of dark - 0.5 Dilution series – concentrations halve and double

16. 16 Produced Water Toxicity Test Randomly loaded organisms Randomly placed on test shelves This is a renewal test. Test water carefully changed out daily, survivors counted and recorded daily.

17. 17 Looking for a Passing NOEC NOEC is No Observed Effect Concentration A NOEC for survival < Critical Dilution is considered a lethal effect and requires re-testing A NOEC for growth < Critical Dilution is considered a sub- lethal effect and requires re-testing NOEC > Critical Dilution required to pass

18. 18 What is a Critical Dilution? The Critical Dilution is determined by three factors used in Table 1 Appendix D from the GMG Permit - Highest dailly average flow rate for the previous 3 months prior to sampling - Diameter of the discharge pipe - Water depth from discharge pipe to seafloor What does the Critical Dilution represent? The Cormix model tells us that at a point 100 m from the outfall, a random sample pulled from the sea will contain that same % of effluent from the outfall.

19. Produced Water Toxicity Test At test completion, survival counts are recorded. The surviving organisms are dried overnight, cooled and then weighed

20. Produced Water Toxicity Test Validity Check #1: Controls must have a minimum of 80% survival @ 7 days Check #2: Mysids in Control must average 0.20 mg Check #3: Minnows in Control must average 0.50 mg Check #4: Coefficient of Variation for the Controls must be <40% Check #5: Unless significant effects are exhibited, the Coefficient of Variation for Critical Dilutions must be <40% Check #6: A Percent Minimum Significant Difference range of 11-37 for Mysid growth and 11-28 for Menidia growth shall be applied accordingly. A test failure can not be deemed invalid due to a CV exceedence

21. Time of ReadingControl (0%)0.04%0.08%0.16%0.32%0.64% 24 hours97.5 100 97.5100 48 hours9597.5100 95100 7 days87.595 82.595 CV%17.68.57 16.88.57 DATA TABLE FOR Mysidopsis bahia SURVIVAL Time of ReadingControl (0%)0.04%0.08%0.16%0.32%0.64% 24 hours97.5 100 97.5100 48 hours90 1009087.580 7 days77.58587.572.56555 CV%17.68.57 16.616.88.57

22. DATA TABLE FOR Mysidopsis bahia GROWTH

23. Sub-sea Fluid Toxicity testing Examples: Sub-sea wellhead preservation fluids, leak tracer fluids, umbilical storage fluids, riser tensioner fluids, etc. The permit sets the limit or Critical Dilution at 50 mg/l Same method as Produced Water Toxicity Test Same organisms and test criteria

24. 24 Partners in Compliance On December 16 th, 2013 >>>>>>>>>