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 officia

endorsement, or recommendation.

Case Study 1: Chronic aquatic toxicity teFarnesane, a hydrocarbon with low watesolubility
Problem statement

Is Farnesane, a hydrocarbon with low water solubility, chronicfreshwater organisms? This talk presents:• The rationale for EPA recommended testing.• The technical challenges Amyris and Wildlife International e

& lessons learned.

• How EPA used the data to arrive at the final decision to drop 
Overview

Farnesane and its physical‐chemical properties

(Amyris)

EPA PMN assessment results & testing recommenda

(EPA)

Overall PMN timeline and lessons learned

(Amyris)

Results and implications for final PMN decision

(Amyris/EPA)

Key questions/challenges associated with testing

(Wildlife International)
What is Farnesane?

• Amyris has introduced a renewable diesel fuel and jet fuel 

meets the rigorous performance requirements set for these 

• Farnesane is sold as renewable diesel in metropolitan are

for use in buses. 

• Complies with ASTM D975 Diesel Fuel Specifications and 

qualify under EPA RFS II. 

• Farnesane is in final stages of validation by the US military 

diesel and jet fuel blends.

“Farnesane”

dodecane, 2,6,10‐trimethyl

CAS 3891‐98‐3
Physical chemical properties of farnesane

Water

Solubility

Modeled

(μg/L)

Water Solubility

Experimental

(μg/L)

Log

Kow Log

Koc

Modeled

Henry Law

Constant

Modeled

(atm‐m3/mole)

Half‐life in wate

modeled

5.3 0.25

>7.20

(Experimental)

7.49 (EPIWIN

model)

4.3‐6.5

2.19×101 (Bond

method)

6.58×101 (Group

Method)

Model river: 1.5 h

Model lake: 5.8 d

Graphic courtesy of http://www.silicones.eu/sustainability‐

environment/silicones‐in‐the‐environment

Based on experimental and modeled results:• Farnesane has low water solubility and has

half‐life in water of <21 days

• Volatilization from water to air is likely• May adsorb to soils and sediments

Amyris did not have experimental solubility or 

at time of PMN submission; this would have be

information to have upfront.

Modeled results were obtained from EPI Suite unless otherwise

noted (run on 7/3/14)
Predicted ecotoxicity

Test Organism Test Type Test End Point Predicted (mg/L)

Fish 96‐hr LC50 *Daphnid 48‐hr LC50 *Green Algae 96‐hr EC50 *

Fish ‐ Chronic Value 0.001Daphnid ‐ Chronic Value 0.001Green Algae ‐ Chronic Value 0.010

QSAR Predictions for Farnesane

* No Effects at Saturation

Water solubility estimated to be 0.0053 mg/L

Log

Kow estimated to be 7.49

Farnesane was classified as

a Neutral Organ

In the absence of experimental data, ecotoxicity concentrationpredicted using SAR methods and (Q)SAR models.
Concentrations of concern

• Acute hazard endpoints were predicted to be no effects 

saturation.

• Chronic Concentration of Concern

= Chronic Value/10

=

• EPA does not typically set COCs

<

1 μg/L due to costs or limitations in 

analytical methods to test below

1 μg/L. Therefore, no values

<

1 μg/

lower detection limit for standard analytical equipment) should be re

experimental data on the chemical or appropriate analog strongly sup

μg/L.
Surface water predictions

• In order to predict risk, potential environmen

(surface water) exposure concentrations (PEC

predicted.

• Key exposure endpoints needed from

E

‐FAST 

complete aquatic risk assessment are:

– “Predicted Environmental Concentration” (PEC) a

“Surface Water Concentration” (SWC).

• The PEC is the concentration of the chemical calculated 

receiving waters.

The SWC were predicted to be 24.5 μg/L and 122 μ

processing operations.
Risk characterization

• “Number of Days Exceeded” or “% year exceed

determines the potential for Chronic Risk.

• SWC of 24.5 μg/L and 122 μg/L exceeded the 

COC of

1 μg/L by 47 and 50 days per year, resp

• There was concern for chronic risk because th

exceed the COC

> 20 days.

• Based on the estimated chronic values, farnes

chem properties, and the potential for chronic 

the decision was to recommend chronic ecototesting to confirm hazard concerns.

– The chronic base set: Fish Early

‐Life Stage, Chronic 

and Algae tests were recommended.
Tests conducted:

• Faced with options of SNUR or Consent Order, Amyris put 

suspension and contracted Wildlife International to conduc

1. A 96-HOUR TOXICITY TEST WITH THE FRESHWATER 

(Pseudokirchneriella subcapitata) (U.S. EPA OCSPP Nu

850.4500

– Static test

2. EARLY LIFE-STAGE (ELS) TOXICITY TEST WITH THE 

MINNOW (Pimephales promelas) (OPPTS Number 850

– Flow‐through test

3. A FLOW-THROUGH LIFE-CYCLE TOXICITY TEST WIT

CLADOCERAN (Daphnia magna) (OPPTS Number 850

– Flow‐through test

All tests conducted using measured concentrations and in accordance with

SPECIAL CONSIDERATIONS FOR CONDUCTING AQUATIC LABORATORY STUD

(OPPTS 850.1000) 
1) Would the testing be technically feasible given the low w

solubility of farnesane? Would it be possible to detect farneat μg/L levels of toxicity?2) Should solvent be used to create stock solution for testin3) What was source of inconsistent recoveries?4) How would EPA use the test data in decision making?

Testing questions/challenges
Stability Adsorbt

Degradation

(Hydrolysis and

Phytolysis)

Volatili

Functional Solubility

Is testing feasible?
Challenge #1: Determining water solubility 

• Physical and chemical data indicated that the mat

would be difficult to mix in water.

• Modeled water solubility is 5.3 

μg/L, high Kow, vo

adsorption.

• In literature, farnesane has low water solubility of 

(Parkerton et al., 2007).
Resolution of Challenge #1

• Wildlife International conducted “Functional Water Solubility

determine feasible aquatic toxicity testing concentrations.

• Farnesane has low water solubility.

– Material would not go into solution in initial trial, therefore 

was used at same rate as testing.

– Saturated aqueous solutions of the test substance were 

using 0.1 ml acetone; Nominal concentration of 100 

μg a

clear Water Accommodated Fraction (WAF) bottles.

– Average recovery 51.5%.– QC samples with 50 

μg a.i./L: up to 95.8% recovery.

Lab recommendationBased on solubility limits, highest concentration for aquatic toxicity t

~60 μ

g a.i./L, using Dimethylformamide (DMF) as solvent
Challenge #2: Use of solvent?

• To conduct flow-through types of studies, it is necessary to p

concentrated stock solutions. Due to low solubility, solvent wrequired.

– Static ELS tests not advisable because of potential funga

and low survival of control organisms due to low dissolve

• Amyris concern: Is the use of solvent environmentally releva• EPA concerns: 

– Solvent may interfere with “the true toxicity” of the PMN s

– It would be necessary to rule out solvent effects on toxicit
Resolution of Challenge #2

Wildlife International’s rationale for solvent use:• EPA provides list of preferred solvents (OPPTS 850.1300

section D) and EPA memo allowing for professional judgm

• DMF has been used in thousands of ecotoxicity tests.

• low toxicity to aquatic organisms • efficacy as a solubilizing agent• less microbial growth than some other organic solvent such as aceto

Resolution: Wildlife proceeded with the studies using DMF.– The concentration of the solvent in all treatment concentr

not greater than 0.1 mL/L, per guideline requirements.

– A solvent control group was included to the tests to identi

impacts due to the addition of the solvent.
Challenge #3: Recovery challenges

Recoveries were good in Fish ELS flow-through study but not in daphnia pre-tests. Why?

– Potential factor: Lower flow through rates in daphnia test vsTo investigate:Confirmed concentration in stock solutions. Stock solutions 

refrigerated in amber bottles. 

Changes to speed of mixer didn’t seem to make much diffeSampled mixing chambers prior to delivery in chambers an

chambers.

• Wildlife isolated problem to the mixing chamber.

– Adsorption? – Volatility? Chambers covered with stainless steel lid, althou

partially open to accommodate the mixer. 
Resolution to Challenge #3

• EPA and Amyris identified 2 options:1) Static renewal 

Wildlife to attempt the range-finding portion of a static renewDaphnia were fed algae and renewal occurred on M-W-F.If #1 unsuccessful, 

2) Conduct Flow-through, recognizing that recoveries would n

80-120% 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 turnovers 
Exposure: algae

• 96-hour, Static test• The test substance was administered to the test organism in 

medium. 

• The test solutions were prepared by diluting the desired vol

appropriate stock with culture medium. 

• Test chambers were sterile, 250-mL Erlenmeyer flasks plug

foam stoppers and containing 100 mL of test solution. 

• Test chambers were positioned on mechanical shaker table 

environmental chamber and were shaken continuously at approximately 100 rpm. 
Exposure: fish and daphnia

• Fish ELS and Daphnia: Flow-through testing• The delivery of test substance to test chambers began at le

prior to the test in order to establish equilibrium concentratiotest substance.

• A continuous-flow diluter was used.• A syringe pump delivered stock solutions to mixing chambe

mixing with dilution water prior to delivery to the test chamb

• Test solutions were split to deliver to each replicate test cha

proportion of water split to each replicate was checked prior initiation and approximately weekly to ensure that flow rates no more than ±10% of the mean flow rate of the four replica

• Each test chamber received at least 11 volume additions (d

21 volume additions (ELS) of test solution every 24 hours. 
Daphnia life cycle 
Flow-through system for fish ELS
Summary of results

Test Organism Measured test

concentrations (μg a.i./L)

Recoveries NOEC

(μg/L)

Comments

Algae 1.6, 2.5, 3.3, 4.4, 6.6, 9.3

(geometric mean

measured)Test material not

detected at end of study,

likely due to adsorption

to algae

77‐99% 86 Results are ba

measured con

Fish 11, 18, 34, 44, 66

(measured on days

1,6,14,21,28, 32)

66‐94% 66 No significant 

concentration 

Daphnia 12, 20, 36, 54, 77 77‐100% 54 Reduction in le

neonate produconcentration (LOEC = 77 μg/
Challenge #4: How did EPA use the test d

decision making? 

• Lowest NOEC = 54 μg/L (Daphnia reproduction)• EPA reviewed the study reports and determined that the res

indicated no effects at saturation (NES).

• After the ecotoxicity re-evaluation, the risk assessment was 

and since the COC changed from 1 μg/L to NES the risk waevaluated to be low.

As a result of the low risk determination after the ecotoxicity experimental studies were completed, the EPA “dropped” the further review.
Timeline

Filed PMN

July 2011

PMN resubmissionDecember 2011

Amyris puts PMN in suspension to generate

aquatic tox dataApril 2012

Functional water solubility test

May 2012

Draft protocols to EPASeptember 2012

EPA feedback on protocols and use of solvent

(testing already in progress)January 2013

Technical difficulties with daphnconsultation with EPAFebruary 2013

Final Studies submitted

July 2013

PMN droppedOctober 2013
In summary

• Amyris has contributed to the body of knowledge regarding 

aquatic toxicity of C15 alkanes. 

• While toxicity was measurable in Daphnia, it was at a conce

above predicted and experimental water solubility.

• Considerable effort was required to create test solutions wit

measurable farnesane. 

– Solvent was required. – Analytical recoveries in flow-through testing were variabl

tests, perhaps due to volatilization, adsorption, and/or de

• Testing was not an over-night process. 

– Collaboration between Wildlife/Amyris/EPA was critical fo

studies that EPA could use in decision-making.

– Due to high demand/low bandwidth, test scheduling is a 
Presenters

Karen Eisenreich, Ph.D.

ToxicologistUS‐EPA/OPPT/RAD (MC 7403M)

1200 Pennsylvania Ave., N.W.

Washington, DC 20460

Eisenreich.Karen@epa.gov202‐564‐7843

Sean Gallagher and Hank K

Manager & Director of Aqu

Wildlife International8598 Commerce DriveEaston, MD 21601sgallagher@wildlifeinterna

410‐822‐8600

Fernando Garcia

Senior Director, Scientific and Regulatory Affairs

&Janet Gardner

Chemical Regulatory ManagerAmyris Inc. http://www.amyris.com/

5885 Hollis St. Suite 100

Emeryville, CA 94608jgardner@amyris.com

1‐510‐601‐4652 
Parking lot: topics for further discussio

• Solvent use