Presented to:

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

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 – LC50 with Mysids (WBM/SBM) • Tests the affect of the mud on the water column • LC50 = Lethal Concentration to 50% of the exposed organisms • Compliance target is an LC50 ≧ 30,000 ppm SPP. • SPP = Suspended Particulate Phase of 1 part mud to 9 parts seawater mixture

4. Drilling Fluid LC50 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 LC50Toxicity Test

6. Determining the LC50 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. SedimentToxicity Test Set-up • With organisms from the same batch and • randomly loaded: • A 96 Hour LC50 performed on the field sample • A 96 Hour LC50 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

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 LC50 Value for the Reference Drilling Fluid = STR LC50 Value for the Submitted SBM sample Ex: Ref MudLC50 = 42.9 ml/Kg Field Mud LC50 = 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. 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 Menidiaberyllina • The Invertebrate organism is Mysidopsisbahia

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. 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. 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. 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. DATA TABLE FOR Mysidopsis bahia SURVIVAL

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. On December 16th, 2013 “Partners in Compliance” >>>>>>>>>