Janet Gardner and Fernando Garcia (Amyris)Karen Eisenreich (US EPA)Sean Gallagher (Wildlife International)Hank Krueger (Wildlife International)Mention of trade names, products, or services does not convey official EPA approval,endorsement, or recommendation.Case Study 1: Chronic aquatic toxicity testing ofFarnesane, a hydrocarbon with low watersolubility1Problem statementIs Farnesane, a hydrocarbon with low water solubility, chronically toxic tofreshwater organisms?This talk presents:• The rationale for EPA recommended testing.• The technical challenges Amyris and Wildlife International encountered& lessons learned.• How EPA used the data to arrive atthe final decision to drop the PMN.2OverviewFarnesaneanditsphysical‐chemicalproperties(Amyris)EPAPMNassessmentresults&testingrecommendations(EPA)OverallPMNtimelineandlessonslearned(Amyris)Resultsandimplications forfinalPMNdecision(Amyris/EPA)Keyquestions/challengesassociatedwithtesting(WildlifeInternational)3What is Farnesane?• Amyris has introduced a renewable diesel fuel and jet fuel blend thatmeets the rigorous performance requirements set for these fuels.• Farnesane is sold as renewable diesel in metropolitan areas in Brazilfor use in buses.• Complies with ASTM D975 Diesel Fuel Specifications and it mayqualify under EPA RFS II.• Farnesane is in final stages of validation by the US military for use indiesel and jet fuel blends.“Farnesane”dodecane,2,6,10‐trimethylCAS3891‐98‐34Physical chemical properties of farnesaneWaterSolubilityModeled(μg/L)WaterSolubilityExperimental(μg/L)LogKowLogKocModeledHenryLawConstantModeled(atm‐m3/mole)Half‐lifeinwatermodeledHalf‐life inwaterexperimental5.3 0.25>7.20(Experimental)7.49(EPIWINmodel)4.3‐6.52.19×101(Bondmethod)6.58×101(GroupMethod)Modelriver:1.5hoursModellake:5.8days3.5days(seawater)Graphiccourtesyofhttp://www.silicones.eu/sustainability‐environment/silicones‐in‐the‐environmentBasedonexperimentalandmodeledresults:•Farnesanehaslowwatersolubilityandhasapredictedhalf‐lifeinwaterof<21days•Volatilizationfromwatertoairislikely•MayadsorbtosoilsandsedimentsAmyrisdidnothaveexperimentalsolubilityorpartitiondataattimeofPMNsubmission;thiswouldhavebeenhelpfulinformationtohaveupfront.ModeledresultswereobtainedfromEPISuiteunlessotherwisenoted(runon7/3/14)Predicted ecotoxicityTestOrganism TestType TestEndPointPredicted(mg/L)Fish 96‐hr LC50 *Daphnid 48‐hr LC50 *GreenAlgae 96‐hr EC50 *Fish‐ChronicValue 0.001Daphnid‐ChronicValue 0.001GreenAlgae‐ChronicValue 0.010QSARPredictionsforFarnesane*NoEffectsatSaturationWatersolubilityestimatedtobe0.0053mg/LLogKowestimatedtobe7.49FarnesanewasclassifiedasaNeutralOrganic.In the absence of experimental data,ecotoxicity concentrations arepredicted using SAR methods and (Q)SAR models.Concentrations of concern•Acutehazardendpointswerepredictedtobenoeffectsatsaturation.•ChronicConcentrationofConcern=ChronicValue/10=1μg/L•EPAdoesnottypicallysetCOCs<1μg/Lduetocostsorlimitationsinreliableanalyticalmethodstotestbelow1μg/L.Therefore,novalues<1μg/L(traditionallowerdetectionlimitforstandardanalyticalequipment)shouldbereportedunlessexperimentaldataonthechemicalorappropriateanalogstronglysupportaCOC<1μg/L.Surface water predictions•Inordertopredictrisk,potentialenvironmental(surfacewater)exposureconcentrations(PEC)arepredicted.•KeyexposureendpointsneededfromE‐FASTtocompleteaquaticriskassessmentare:–“PredictedEnvironmentalConcentration”(PEC)a.k.a“SurfaceWaterConcentration”(SWC).•ThePECistheconcentrationofthechemicalcalculatedtobeinreceivingwaters.TheSWCwerepredictedtobe24.5μg/Land122μg/Lforprocessingoperations.Risk characterization•“NumberofDaysExceeded”or“%yearexceeded”determinesthepotentialforChronicRisk.•SWCof24.5μg/Land122μg/LexceededthechronicCOCof1μg/Lby47and50daysperyear,respectively.•TherewasconcernforchronicriskbecausetheSWCexceedtheCOC>20days.•Basedontheestimatedchronicvalues,farnesane’sp‐chemproperties,andthepotentialforchronicrisk,thedecisionwastorecommendchronicecotoxicitytestingtoconfirmhazardconcerns.–Thechronicbaseset:FishEarly‐LifeStage,ChronicDaphnia,andAlgaetestswererecommended.Tests conducted:• Faced with options of SNUR orConsent Order, Amyris put PMN insuspension and contracted Wildlife International to conduct testing:1. A 96-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA(Pseudokirchneriella subcapitata) (U.S. EPA OCSPP Number850.4500–Statictest2. EARLY LIFE-STAGE (ELS) TOXICITY TEST WITH THE FATHEADMINNOW (Pimephales promelas) (OPPTS Number 850.1400)–Flow‐throughtest3. A FLOW-THROUGH LIFE-CYCLE TOXICITY TEST WITH THECLADOCERAN (Daphnia magna) (OPPTS Number 850.1300)–Flow‐throughtestAlltestsconductedusingmeasuredconcentrationsandinaccordancewith:SPECIALCONSIDERATIONSFORCONDUCTINGAQUATICLABORATORYSTUDIES(OPPTS850.1000)101) Would the testing be technically feasible given the low watersolubility of farnesane? Would it be possible to detect farnesaneat μg/L levels of toxicity?2) Should solvent be used to create stock solution for testing?3) What was source of inconsistent recoveries?4) How would EPA use the test data in decision making?11Testing questions/challengesStabilityAdsorbtionDegradation(HydrolysisandPhytolysis)VolatilityFunctionalSolubilityIs testing feasible?Challenge #1: Determining water solubility for testing• Physical and chemical data indicated that the materialwould be difficult to mix in water.• Modeled water solubility is 5.3μg/L, high Kow, volatility,adsorption.• In literature, farnesane has low water solubility of 0.25μg/L(Parkerton et al., 2007).13Resolution of Challenge #1• Wildlife International conducted “Functional Water Solubility” test todetermine feasible aquatic toxicity testing concentrations.• Farnesane has low water solubility.– Material would not go into solution in initial trial, therefore solventwas used at same rate as testing.– Saturated aqueous solutions of the test substance were preparedusing 0.1 ml acetone; Nominal concentration of 100μg a.i./L in 14-Lclear Water Accommodated Fraction (WAF) bottles.– Average recovery 51.5%.– QC samples with 50μg a.i./L: up to 95.8% recovery.LabrecommendationBasedonsolubilitylimits,highestconcentrationforaquatictoxicitytesting:~60μga.i./L,usingDimethylformamide(DMF)assolvent14Challenge #2: Use of solvent?• To conduct flow-through types of studies, it is necessary to prepareconcentrated stock solutions. Due to low solubility, solvent would berequired.– Static ELS tests not advisable because of potential fungal growthand low survival of control organisms due to low dissolved oxygen.• Amyris concern: Is the use ofsolvent environmentally relevant?• EPA concerns:– Solvent may interfere with “the true toxicity” of the PMN substance.– It would be necessary to rule out solvent effects on toxicity.15Resolution of Challenge #2Wildlife International’s rationale for solvent use:• EPA provides list of preferred solvents (OPPTS 850.1300, page 8,section D) and EPA memo allowing for professional judgment.• DMF has been used in thousandsof ecotoxicity tests.• low toxicity to aquatic organisms• efficacy as a solubilizing agent• less microbial growth than some other organic solvent such as acetoneResolution: Wildlife proceeded with the studies using DMF.– The concentration of the solvent inall treatment concentrations wasnot greater than 0.1 mL/L, per guideline requirements.– A solvent control group was includedto the tests to identify potentialimpacts due to the addition of the solvent.16Challenge #3: Recovery challengesRecoveries were good in Fish ELS flow-through study but not in equilibrateddaphnia pre-tests. Why?– Potential factor: Lower flow through rates in daphnia test vs. Fish ELSTo investigate:Confirmed concentration in stock solutions. Stock solutions storedrefrigerated in amber bottles.Changes to speed of mixer didn’t seem to make much difference.Sampled mixing chambers prior to delivery in chambers and checkedchambers.• Wildlife isolated problem to the mixing chamber.– Adsorption?– Volatility? Chambers covered with stainless steel lid, althoughpartially open to accommodate the mixer.17Resolution to Challenge #3• EPA and Amyris identified 2 options:1) Static renewalWildlife to attempt the range-finding portion of a static renewal test.Daphnia were fed algae and renewal occurred on M-W-F.If #1 unsuccessful,2) Conduct Flow-through, recognizing thatrecoveries would not meet the80-120% guideline.In the end:-Static-Renewal: poor recoveries as well.-Proceeded with flow-through– Recoveries were good!– Attributed to increased flow rates from 5 to 11 turnovers18Exposure: algae• 96-hour, Static test• The test substance was administered to the test organism in culturemedium.• The test solutions were prepared by diluting the desired volume of theappropriate stock with culture medium.• Test chambers were sterile, 250-mL Erlenmeyer flasks plugged withfoam stoppers and containing 100 mL of test solution.• Test chambers were positioned onmechanical shaker table in anenvironmental chamber and were shaken continuously atapproximately 100 rpm.19Exposure: fish and daphnia• Fish ELS and Daphnia: Flow-through testing• The delivery of test substance totest chambers began at least 5 daysprior to the test in order to establish equilibrium concentrations of thetest substance.• A continuous-flow diluter was used.• A syringe pump delivered stock solutions to mixing chambers formixing with dilution water prior to delivery to the test chambers.• Test solutions were split to deliver to each replicate test chamber. Theproportion of water split to each replicate was checked prior to testinitiation and approximately weekly to ensure that flow rates varied byno more than ±10% of the mean flowrate of the four replicates.• Each test chamber received at least 11 volume additions (daphnia) and21 volume additions (ELS) of test solution every 24 hours.20Daphnia life cycle21Flow-through system for fish ELS22Summary of resultsTestOrganism Measuredtestconcentrations(μga.i./L)Recoveries NOEC(μg/L)CommentsAlgae 1.6,2.5,3.3,4.4,6.6,9.3(geometric meanmeasured)Testmaterialnotdetectedatendofstudy,likelyduetoadsorptiontoalgae77‐99% 86ResultsarebasedonDay0measuredconcentrations*Fish 11,18,34,44,66(measuredondays1,6,14,21,28,32)66‐94% 66 NosignificanteffectsathighestconcentrationtestedDaphnia 12,20,36,54,77 77‐100%54Reductioninlengthandneonateproductionathighestconcentrationtested(LOEC=77μg/L)23Challenge #4: How did EPA use the test data indecision making?• Lowest NOEC = 54 μg/L (Daphnia reproduction)• EPA reviewed the study reports and determined that the resultsindicated no effects at saturation (NES).• After the ecotoxicity re-evaluation, the risk assessment was revisitedand since the COC changed from 1 μg/L to NES the risk was reevaluated to be low.As a result of the low risk determination after the ecotoxicityexperimental studies were completed, the EPA “dropped” the PMN fromfurther review.24TimelineFiledPMNJuly2011PMNresubmissionDecember 2011AmyrisputsPMNinsuspensiontogenerateaquatictoxdataApril2012FunctionalwatersolubilitytestMay2012DraftprotocolstoEPASeptember2012EPAfeedbackonprotocolsanduseofsolvent(testingalreadyinprogress)January2013Technicaldifficultieswithdaphniarecoveries;consultationwithEPAFebruary2013FinalStudiessubmittedJuly2013PMNdroppedOctober201325In summary• Amyris has contributed to the body of knowledge regarding chronicaquatic toxicity of C15 alkanes.• While toxicity was measurable in Daphnia, it was at a concentrationabove predicted and experimental water solubility.• Considerable effort was required to create test solutions withmeasurable farnesane.– Solvent was required.– Analytical recoveries in flow-through testing were variable betweentests, perhaps due to volatilization, adsorption, and/or degradation.• Testing was not an over-night process.– Collaboration between Wildlife/Amyris/EPA was critical for qualitystudies that EPA could use in decision-making.– Due to high demand/low bandwidth, test scheduling is a challenge.26PresentersKarenEisenreich,Ph.D.ToxicologistUS‐EPA/OPPT/RAD(MC7403M)1200PennsylvaniaAve.,N.W.Washington,DC20460Eisenreich.Karen@epa.gov202‐564‐7843SeanGallagherandHankKruegerManager&DirectorofAquaticToxicologyWildlifeInternational8598CommerceDriveEaston,MD21601sgallagher@wildlifeinternational.com410‐822‐8600FernandoGarciaSeniorDirector,ScientificandRegulatoryAffairs&JanetGardnerChemicalRegulatoryManagerAmyrisInc.http://www.amyris.com/5885HollisSt.Suite100Emeryville,CA94608jgardner@amyris.com1‐510‐601‐465227Parking lot: topics for further discussion• Solvent use28