Study Title

Sorbic Acid

– 96

- Hour Toxicity Test with the

Freshwater Green Alga, Pseudokirchneriella subcapitata

, Following OCSPP Guideline 850.450 0

Data Requirement

OCSPP Guideline 850. 450 0

Author

Katherina A. Softcheck

Study Completed On

2 9 May 2 015

Submitted To

Brenntag North America, Inc.

3111 North Post Road

Indianapolis, Indiana 46226

Performing Laboratory

Smithers Viscient

790 Main Street

Wareham, Massachusetts 02571

- 1037

Laboratory Project ID

Smithers Viscient Study No. 140 91.6101

Page 1 of 73Smithers Viscient Study No. 14091.6101 Page 2



### STATEMENT OF NO DATA CONFIDENTIALITY CLAIM

No claim of confidentiality, on any basis whatsoever, is made for any information contained in

this document. I acknowledge that information not designated as within the scope of FIFRA sec.

10(d)(1)(A), (B), or (C) and which pertains to a registered or previously registered pesticide is

not entitled to confidential treatment and may be released to the public, subject to the provisions

regarding disclosure to mu ltinational entities under FIFRA 10(g).

Company:

Company Agent: Date:

(Typed Name)

(Title) (Signature)Smithers Viscient Study No. 14091.6101 Page 5



### KEY STUDY PERSONNEL

The following Smithers Viscient personnel were responsible for the conduct of the work and

reporting of the study result s.

Katherina A. Softcheck Study Director

Kristen L. Soucy Biologist I

Melissa L. Staggs Biologist I

Ashley Beaton Assistant Biologist

Blake Dinius Biologist I

Nicholas Lyons Assistant Biologist

Regine Felix Chemistry Technician II

Huy Le Chemistry Technician II

Marjorie E. Dix Research Director , Chemistry

Gina Giorgio Technical Report Writer

Heather Grove Director of E cot oxicologySmithers Viscient Study No. 14091.6101 Page 6



### TABLE OF CONTENTS

Page

GOOD LABORATORY PRAC TICE COMPLIANCE STAT EMENT

................................ .......... 3

QUALITY ASSURANCE ST ATEMENT

................................ ................................ ...................... 4

KEY STUDY PERSONNEL

................................ ................................ ................................ ........... 5

TABLE OF CONTENTS

................................ ................................ ................................ ................. 6

SUMMARY

................................ ................................ ................................ ................................ ..... 9

1.0 INTRODUCTION

................................ ................................ ................................ ................ 11

2.0 MATERIALS AND METHOD S

................................ ................................ ......................... 11

2.1 Protocol

................................ ................................ ................................ ...... 11

2.2 Test Substance

................................ ................................ ........................... 11

2.3 Test Organism

................................ ................................ ............................ 12

2.4 Reference Test

................................ ................................ ........................... 13

2.5 Test Dilution Water

................................ ................................ .................... 14

2.6 Test Concentrations

................................ ................................ ................... 14

2.7 Test Solution Preparation

................................ ................................ ........... 14

2.8 Test Ini tiation

................................ ................................ ............................. 15

2.9 Test Monitoring

................................ ................................ ......................... 16

2.9.1 Algal Growth

................................ ................................ ............................. 16

2.9.2 So lution for Algistatic/Algicidal Properties

................................ ............... 16

2.9.3 Test Conditions

................................ ................................ .......................... 17

2.9.4 Analytical Measurements

................................ ................................ ........... 18

2.10 Statistical Analysis

................................ ................................ ..................... 20

3.0 RESULTS

................................ ................................ ................................ ............................. 22

3.1 Preliminary Exposure

................................ ................................ ................. 22

3.2 Definitive Exposure

................................ ................................ ................... 22

3.2.1 Evaluation of Test Conditions

................................ ................................ ... 22

3.2.2 Analytic al Results

................................ ................................ ...................... 23

3.2.3 Biological Results

................................ ................................ ...................... 24

PROTOCOL DEVIATION

................................ ................................ ................................ ........... 27

REFERENCES

................................ ................................ ................................ .............................. 28Smithers Viscient Study No. 14091.6101 Page 7

Table 1. Composition of nutrient medium (AAP medium) used during the

toxicity test exposing Pseudokirchneriella subcapitata to sorbic

acid.

................................ ................................ ................................ ............ 29

Table 2. The pH, conductivity, temperature and light intensity measured

during the 96

- hour exposure of Pseudokirchneriella subcapitata to

sorbic acid.

................................ ................................ ................................ . 30

Table 3. Concentrations of sorbic acid measured in the exposure solutions

during the 96

- hour exposure of Pseudokirchneriella subcapitata.

............ 31

Table 4. Cell density of Pseudokirchneriella subcapitata af ter 24, 48, 72 and

96 hours of exposure to sorbic acid.

................................ .......................... 32

Table 5. Yield for Pseudokirchneriella subcapitata after 24, 48, 72 and

96 hours of exposure to sorbic acid.

................................ .......................... 33

Table 6. EC50, No

- Observed

- Effect Concentration (NOEC) and Lowest

Observed

- Effect Concentration (LOEC) values for sorbic acid

calculated from the results (yield) of the 96

- hour toxicity test with

Pseudokirchneriel la subcapitata

. ................................ .............................. 34

Table 7. Average specific growth rates of Pseudokirchneriella subcapitata

after 24, 48, 72 and 96 hours of exposure to sorbic acid.

.......................... 35

Table 8. EC50, No

- Observed

- Effect Concentration (NOEC) and Lowest

Observed

- Effect Concentration (LOEC) values for sorbic acid

calculated from results (average specific growth rate) of the 96

- hour

toxicity test with Pseudokirchneri ella subcapitata

. ................................ ... 36

Table 9. Section

- by

- section growth rates of Pseudokirchneriella subcapitata

after 24, 48, 72 and 96 hours of exposure to sorbic acid.

.......................... 37

Table 10. Calculated area under the growth curve (AUGC) of

Pseudokirchneriella subcapitata after 24, 48, 72 and 96 hours of

exposure to sorbic acid.

................................ ................................ .............. 38

Table 11. EC50, No

- Observed

- Effect Concentration (NOEC) and Lowest

Observed

- Effect Concentration (LOEC) values for sorbic acid

calculated from results (area under the growth curve) of the 96

- hour

toxicity test with Pseudokirchneriella subcapitata

. ................................ ... 39

Figure 1. Relationship between nominal and time

- weighted average

concentrations of sorbic acid during the 96

- hour static toxicity test

exposing Pseudokirchneriella subcapitata

. ................................ ............... 40

Figure 2. Algal growth curves (cell density vs. time) for Pseudokirchneriella

subcapitata exposed to sorbic acid.

................................ ........................... 41

Figure 3. Yield for Pseudokirchneriell a subcapitata exposed to sorbic acid.

.......... 42

Figure 4. Average specific growth rates (0

- to 96

- hour) for

Pseudokirchneriella subcapitata exposed to sorbic acid.

.......................... 43

Figure 5. Area under the growth curve for Pseudokirchneriella subcapitata

exposed to sorbic acid.

................................ ................................ ............... 44Smithers Viscient Study No. 14091.6101 Page 8

APPENDIX 1

- STUDY PROTOCOL

................................ ................................ .......................... 45

APPENDIX 2

- ANALYTICAL METHODOLO GY

................................ ................................ .... 61

Table 1A. Analytical results for the recovery of sorbic acid from AAP medium

during the method validation study.

................................ ........................... 69

Figure 1A. Representative chromatogram of a 0.0585 mg/L calibration standard

used for quantification during the method validation study with

sorbic acid.

................................ ................................ ................................ . 70

Figure 2A. Representative chromatogram of a 5.00 mg/L recovery sample

fortified with sorbic acid during the method validation study

. .................. 71

Figure 3A. Representative ch romatogram of a control sample during the method

validation study with sorbic acid

. ................................ .............................. 72

Figure 4A. A typical regression analysis for the calibration standards used to

quantitate sorbic acid in the recovery samples.

................................ ......... 73Smithers Viscient Study No. 14091.6101 Page 9



### SUMMARY

Sorbic Acid

– 96

- Hour Toxicity Test with the Freshwater Green Alga

, Pseudokirchneriella

subcapitata , F ollowing OCSPP Guideline 850.450 0

SPONSOR: Brenntag North America, Inc.

PROTOCOL TIT LE: 96

- Hour Toxicity Test with the Freshwater Green Alga

, Pseudokirchneriella subcapitata , F ollowing OCSPP

Guideline 850.450 0

SMITHERS VISCIENT

STUDY NUMBER: 14091.6101

TEST SUBSTANCE: Sorbic acid, Lot No. 1401151

, reported to have a purity

of 99.5 %

, was received from GEI Ecology Lab on

17 February 2015

.

DEFINITIVE

TEST DATES: 23 March to 5 April 2015 (including recovery period)

TEST ORGANISM: Pseudokirchneriella subcapitata , inoculum

- three days

since previous transfer

Source

- Smithers Viscient culture

DILUTION WATER: Algal Assay Procedure (AAP) medium

TEST CONDITIONS: 96

- hour duration, a temperature range of 22 to 2 4 °C

continuous illumination at 51 to 68 μ E/m 2 /S

, continuous

agitation at a shaking rate of 100 rpm

NOMINAL TEST

CONCENTRATION S : 6.3

, 13

, 25

, 50 and 100 mg /L

TIME

- WEIGHTED

AVERAGE

CONCENTRATIONS : 1.7

, 3. 9

, 24

, 50 and 1 10 mg/L

EFFECT CRITERIA: Reduction of 96

- hour yield

, average specific growth rate

( μ ave ) and area under the growth curve (AUGC) relative

to the performance of the co ntrol data

.Smithers Viscient Study No. 14091.6101 Page 10

RESULTS:

Biological Parameter Based on Time

- Weighted Average Concentrations (mg/L)

EC 50

(95% Confidence Limits) NOEC LOEC

96

- Hour Yield 10

( 6.9

– 10 ) 3.9 24

96

- Hour Average Specific

Growth Rate 12

( 11

– 13 ) 3.9 24

96

- Hour Area Under the

Growth Curve 10

( 8.5

– 10 ) 3.9 24Smithers Viscient Study No. 14091.6101 Page 11



### 1.0 INTRODUCTION

The objective of this study was to determine the effect of sorbic acid on the growth of the

freshwater green a lga

, Pseudokirchneriella subcapitata

. Results of the study are reported as the

96

- hour EC 50 val ues and 95% confidence limits for total biomass (expressed as yield), average

specific growth rate and area under the growth curve (AUGC), calculated from the cell density

data. The 96

- hour No

- Observed

- Effect Concentration (NOEC) and Lowest

- Observed

- Effec t

Concentration (LOEC) values for yield, average specific growth rate and area under the growth

curve are also reported.

The study was initiated on 25 February 2015 , the day the Study Director signed the protocol, and

was completed on the day the Study D irector signed the final report. The experimental phase of

the 96

- hour definitive test was conducted from 23 March to 5 April 2015 (including recovery

period) at Smithers Viscient ( SMV ) located in Wareham, Massachusetts. All original raw data,

the protoc ol and t he original final report produced during this study are stored in Smithers

Viscient's archives at the above location

.



### 2.0 MATERIALS AND METHODS

2.1 Protocol

Procedures used in this toxicity study followed those described in the Smithers Viscient protoco l

entitled " 96

- Hour Toxicity Test with the Freshwater Green Alga

, Pseudokirchneriella

subcapitata , F ollowing OCSPP Guideline 850.450 0 ” ( Appendix 1 ). The methods described in

this protocol meet the requirements specified in the OC SPP Guideline 850. 450 0

( U.S. EPA, 2012 ).



### 2.2 Test Substance

The test substance, sorbic acid, was received on 17 February 2015 from GEI Ecology Lab

,

Denver, Colorad o

. The following information was provided:Smithers Viscient Study No. 14091.6101 Page 12

Name: sorbic acid

Lot No.: 1401151

CAS No.: 110

- 44

- 1

Purity: 99. 5 %

Expiration Date: 1 4 January 2016

Upon receipt at Smithers Viscient, the test substance (SMV No. 7535 ) was stored at room

temperature in the original container, in a dark ventilated cabinet. Concentr ations were adjusted

for the purity of the test substance.

Determination of stability and characterization, verification of the test substance identity,

maintenance of records on the test substance and archival of a sample of the test substance are

the re sponsibility of the Study Sponsor.



### 2.3 Test Organism

The organism used in this toxicity test was the freshwater green a lga

, Pseudokirchneriella

subcapitata , strain 1648 , class Chlorophyceae . The green a lga w as obtained from the Culture

Collection of Algae at The University of Texas (UTEX) , Austin, Texas, and was maintained in

stock culture at Smithers Viscient

.

The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile,

deionized water. The components used to formulate AAP medium a re listed in Table 1

.

Representative samples of the dilution water source used in the preparation of the culture

medium were analyzed periodically for the presence of pesticides, PCBs and toxic metals by

GeoLabs, Inc., Braintre e, Massachusetts using U.S. EPA standard methods ( U.S. EPA, 1997 )

.

None of these compounds have been detected at concentrations that are considered toxic in any

of the water samples analyzed in agreement with ASTM Gui deline s ( 2007 ). In addition, a

representative sample of AAP medium was analyzed monthly for total organic carbon (TOC)

concentration. The TOC concentration of the sample collected in Ma rch 2015 was 0. 80 mg/L.Smithers Viscient Study No. 14091.6101 Page 13

The U.S. EPA recom mends that the TOC of dilution water used for toxicity tests should be

≤ 2.0 mg/L.

If necessary, the pH of the culture medium was adjusted to pH 7.5 ± 0.1 with dilute hydrochloric

acid (HCl) or sodium hydroxide (NaOH) . Stock cultures were grown in 250

- mL g lass flasks

each containing 100 mL of AAP medium. The flasks were co vered with stainless steel caps

which permitted gas exchange.

The stock cultures were maintained within the following conditions: a shaking rate of

100 ± 10 rpm , a temperature of 24 ± 2 o C and continuous illumination at the surface of the

medium providing photosynthetically

- active radiation (PAR) of approximately

60 μ E/m 2 /S ± 15%, e.g.

, 51 to 69 μ E/m 2 /S

. Lighting was supplied by Premira VitaLux ®

fluorescent bulbs . Culture flasks were agitated continuously on an orbital shaker. Temperature

was controlled using an environmental chamber.

The inoculum used to initiate the toxicity test with sorbic acid was taken from a stock culture

that had been transferred to fresh medium three days before testing.



### 2.4 Reference Test

A reference test was conducted from 13 to 17 April 2015 to evaluate the sensitivity of

Pseudokirchneriella subcapitata to zinc chloride. The reference toxicant, zinc chloride, was

purchased from a commercial supplier and was therefore characterized by its label. Based on the

results of this tes t, the 96

- hour EC 50 value based on cell density was determined to be

0. 0 91 mg Zn/L, with 95% confidence intervals of 0. 0 71 to 0. 1 2 mg Zn/L . Previous reference

testing determined a mean EC 50 value of 0. 09 0 mg Zn/L for P. subcapita ta . The EC 50 value

closel y approximates the results determined in previous testing; therefore, the culture is

considered of appropriate sensitivity for use in toxicity testing.Smithers Viscient Study No. 14091.6101 Page 14



### 2.5 Test Dilution Water

The AAP medium used to prepare the exposure solutions was formulated in the same ma nner as

the culture medium. Several liters of AAP medium were prepared using sterile, deionized water

and were equilibrated to test temperature. The initial pH of this medium was adjusted with dilute

sodium hydroxide or hydrochloric acid , if necessary, t o 7.5 ± 0.1 prior to use.



### 2.6 Test Concentrations

Based on the results of preliminary testing (see Section 3.1 ) and in consultation with the Study

Sponsor, the nominal concentrations selected for the definitive test we re 6.3, 13, 25, 50 and

100 mg / L.



### 2.7 Test Solution Preparation

Prior to exposure initiation , a 100 mg/L primary stock solution was prepared by bringing

0.2009 g of sorbic acid ( 0. 1999 g as active ingredient) to volume in a glass volumetric flask with

2000 mL of AAP medium. The resulting solution was clear and colorless with a large amount of

undissolved material on the bottom of the flask and on the solution surface. The solution was

sonicated for 10 minutes and then mix ed with a stir plate and Teflon ®

- coate d stir bar for

approximately two and a half hours

. Following sonication and mixing

, the solution was

observed to be clear and colorless with no visible undissolved material

. The exposure solution s

were prepared by diluting the 100 mg/L solution as follow s:

Primary Stock Solution

Concentration

(mg / L) Volume of Primary

Stock Used

(L) Final Dilution

Volume with

AAP Medium

(L) Exposure Solution

Concentration

(mg/L)

10 0 0. 50 NA 100

10 0 0. 50 1

. 0 50

10 0 0. 25 1

. 0 25

10 0 0. 13 1

. 0 13

10 0 0.0 63 1

. 0 6.3

NA = N ot Applicable.Smithers Viscient Study No. 14091.6101 Page 15

T he resulting exposure solutions were observed to be clear and colorless with no visible

undissolved test substance following preparation

.

Additional untreated AAP medium was used to prepare the control. Control vessels were

maintained un der the same conditions as the treatment vessels but contained no sorbic acid

.



### 2.8 Test Initiation

Four replicate, sterile 250

- mL Erlenmeyer flasks for the treatment level and control (A, B

, C and

D ) were prepared as required by the OCSPP G uideline 850.4500

. An additional replicate flask

(E) of all treatments and the control was prepared to assess water quality daily throughout the

exposure

. All flasks were conditioned prior to use by rinsing with the appropriate solution

.

One hundred milliliters of the a ppropriate exposure solution was then placed in each replicate

flask. All test vessels were fitted with stainless steel caps which permit gas exchange. Each test

flask was labeled with the study number, concentration, replicate and test species.

In orde r to estimate the impact that the presence of algae biomass had on the test substance

concentration, an additional replicate flask ( F ) of the nominal 25 mg / L test solution w as

prepared. This flask, which was not inoculated with algae , was analyzed at 96 h ours of exposure

for sorbic acid concentration. The result of this analysis was compared with the result for the

nominal 25 mg/L test solution containing algae.

After the test solutions were added to the test flasks (100 mL per flask), a 1. 575

- mL inoculu m of

Pseudokirchneriella subcapitata cells, at a density of approximately 63 × 10 4 cells/mL, was

aseptically introduced into each flask. This inoculum provided the required initial (0

- hour) cell

density of approximately 1.0 × 10 4 cells/mL.Smithers Viscient Study No. 14091.6101 Page 16



### 2.9 Test Monitorin g

2.9.1 Algal Growth

At each subsequent 24

- hour interval, cell counts were conducted on each replicate solution of the

treatment levels and the control vessels using a hemacytometer (Neubauer Improved) and

compound microscope

. Cell counts for each replicate wer e determined using a single sample

removed from each flask. One or more hemacytometer fields, each 0.10 × 0.10 cm in surface

area and 0.010

- cm deep and containing 0.00010 mL of culture, were examined until at least

400 a lga e cells or four fields were coun ted. Observations of the health of the algal cells were

also made and recorded at each 24

- hour interval.

2.9.2 Solution for Algistatic/Algicidal Properties

In order to investigate algistatic/algicidal properties of the test substance, a recovery period was

inc luded. The objective of the recovery period was to determine if algal growth would resume

after inhibition as a result of exposure to the test substance. Following exposure, a sample was

removed from the composite of the four replicate vessels of the nom inal 100 m g/ L test

concentration at exposure termination. A 6

- mL aliquot of the composite sample was then diluted

with 94 mL of freshly prepared AAP medium to yield a subculture with a nominal concentration

of 6. 0 mg / L , slightly less than the lowest conce ntration tested

.

The performance of the subculture was used to determine if the effects of the test substance on

the algae were algistatic, in which case cells would resume growth in the subculture, or algicidal,

in which case no growth would occur in the subculture. The subcultures were incubated for up

to nine days under conditions consistent with those maintained during the definitive exposure.

During this period, the subculture w as microscopically examined every other day to determine if

cell growth had resumed. The subculture w as discontinued after a substantial increase in cell

density (e.g., ≥ 10X) was observed. If an increase in cell density was not observed after nine

days, the vessel w as terminated.Smithers Viscient Study No. 14091.6101 Page 17

2.9.3 Test Conditions

The test was conducted in a n environmental chamber designed to maintain the test conditions

specified in the protocol: a temperature of 24 ± 2 ° C, continuous light intensity at a

photosynthetically

- active radiation (PAR) range of 51 to 69 μE/m 2 /s (approximately equivalent

to 3700 to 4900 lux) , see Section 2.3 . Two orbital shaker tables provided a shaking rate of

100 ± 10 rpm.

Temperature was measured continuously with a VWR minimum/maximum thermometer located

in a flask of water adjacent to the tes t flasks in the environmental chamber. Minimum and

maximum temperatures were recorded daily. Light intensity was measured as

photosynthetically

- active radiation (PAR). The PAR of the test area was measured at test

initiation and at each 24

- hour interval during the exposure period with a LI

- COR Model LI

- 189

photo meter and LI

- COR Model LI

- 190SA radiation sensor. Light intensity was also measured in

lux. Light intensity was measured in four locations around the perimeter of the test area with a

VWR Tracea ble light meter at 0

- hour. Test flasks were randomly placed on the shaker tables in

an environmentally controlled chamber at test initiation based on computer

- generated random

numbers. Following each observation interval, the test flasks were placed in n ew random

positions in the environmental chamber

.

Water quality parameters (pH and conductivity) were measured at test initiation. Additionally,

based on the known acidic nature of the test substance and the expected impact of it on the pH of

the exposur e solutions, the pH of the solutions was also measured at the 24

- , 48

- , 72

- and

96

- hour exposure period s

. Measurements at test initiation were conducted on the test solution s

remaining after the individual test flasks had been filled. The pH of the expo sure solutions at 24,

48 and 72 hours was measured in the additional replicate of each treatment and the control

established for this purpose. At test termination, after cell counts were completed, the replicate

solutions for each treatment and control we re respectively composited for pH measurements.

Test solution pH was measured with a Yellow Springs Instrument (YSI) Model pH100 A pH

meter and conductivity was measured using a YSI Model 3100

- 115V conductivity meter.Smithers Viscient Study No. 14091.6101 Page 18

2.9.4 Analytical Measurements

At test initi ation (0 hour) and test termination (96 hours), a single sample was removed from

each test solution and the control and analyzed for sorbic acid

. Samples analyzed at 0

- hour were

removed from the test and control solutions in the volumetric flasks prior to division into the

replicate test vessels. Samples analyzed at exposure termination ( 96 hours ) were removed from

a composite solution from the replicate test vessels of each test concentration and the control.

At 96 hours of exposure, a sample was remo ved from the additional replicate flask of the

nominal 25 mg/L test concentration ( replicate F ) which did not contain algae and analyzed for

sorbic acid concentration . The result of the analysis was compared with that obtained for the

96

- hour analysis of the nominal 25 mg/L solution containing algae to assess the impact that algae

had on the test substance concentration.

Prior to processing and analysis, all 96

- hour exposure solution s were centrifuged to remove algae

cells to determine the dissolved conce ntration of test substance, as required by the OCSPP

Guideline 850.4500

. Samples removed from the test solutions were centrifuged in Nalgene

centrifuge tubes at 3000 rpm for 15 minutes

.

Based on OCSPP 850.4500 guidance, a time

- weighted average was calcul ated for each treatment

level as follows:



## ( ) ( ) ( ) ( ) 

## [ ] Days Total / Days 4 x C ln C ln / C C C D4 D0 D4 D0 twa

− − =

where:

C

twa = time

- weighted average concentration

C D0 = measured concentration on day 0

C D 4 = measured concentration on day 4

ln = natural log

Total Days = total number of days, 4Smithers Viscient Study No. 14091.6101 Page 19

The variance for this set of data, which is defined as the average of the squared differences from

the mean, is calculated according to the equation below. The standard deviation is the square

root of the variance.

Var = ∑ [WF x (C

- C

twa ) 2 ] / ∑ WF

where:

Var

= variance

WF

= weighting factor, defined as the time between intervals

C

= concentration at each interval

C

twa = time

- weighted average concentration

Q uality control (QC) samples (three at 0

- hour and six at 96

- hour) were prepared with fresh

A A P medium at nominal concentrations approximating the test concentration range and

remained with the exposure solution samples thro ughout the analytical process. The

three QC samples at 0

- hour were processed in the same ma nner as test solution samples. Of the

six QC samples at 96

- hour, three were processed with centrifugation in the same manner as the

test solution samples and three were processed without centrifugation to evaluate the impact of

centrifugation on test subs tance recoveries. Analysis of the QC samples was used to judge the

precision and quality control maintained during the analytical process

.

All samples were analyzed for sorbic acid using automated injection on a high performance

liquid chromatographic sy stem equipped with ultraviolet detection (HPLC/UV) based on

methodology validated at Smithers Viscient. The method validation study was conducted prior

to the initiation of the definitive test and established an average recovery of 99.5 ± 5.02 % from

AAP m edium ( Appendix 2 ). Defined limits for acceptance of QC sample performance were set

at 80.0 % to 120%. Conditions and procedures used throughout the analysis of exposure solution

and QC samples during this study were the same as those used in the method validation

.Smithers Viscient Study No. 14091.6101 Page 20



### 2.10 Statistical Analysis

The cell density in each test flask was calculated for each daily interval by dividing the number

of cells counted by the number of fields examined and multiplying by 10,000 to yield cells/mL.

Me ans and standard deviations for cell density for the treatment s and the control were calculated

from individual replicate values.

Yield was calculated as the biomass (cell density) at each interval of the test minus the initial

biomass at the start of the test.

Average specific growth rate was determined for the daily cell density counts. For each test

solution, the specific growth rate between the inoculation density and the cell density of the

specified time interval was calculated using the following equation:

μ = ln X t - ln X 0

t t - t 0

where :

μ = specific growth rate (days

- 1 )

ln = natural logarithm

X 0 = initial cell density in cells/mL at time t 0 (days)

X

t = cell density at the specified time interval in cells/mL

t 0 = time of test initiation

t t = time of observation interval in days (based on actual time elapsed

between observation intervals)

The average specific growth rate ( μ ave ) for each replicate was calculated for the entire span of the

test period (e.g., 0 to 96 hours) and this data wa s used for statistical analyses. Per the study

guideline

, t he section

- by

- section growth rates during the course of the test (0

- to 24

- , 24

- to 48

- ,

48

- to 72

- , and 72

- to 96

- hours)

, although not used for evaluation, were also calculated for each

replicate and are included with the study results as recommended in the OCSPP study guideline

.Smithers Viscient Study No. 14091.6101 Page 21

The area under the growth curve is determined for each replicate by calculating the area between

the growth curves and the horizontal line N = N 0 , according to the formu la:

A

= N N

2 x t + N + N

- 2N

2 x (t

- t ) +

. . . + N + N

- 2N

2 x (t

- t ) 1 0

1 1 2 0

2 1 n

- 1 n 0

n n

- 1

where:

A

= area under the growth curve (units: × 10 4 cells × days/mL)

N 0 = calculated number of cells/mL at time t 0

N 1 = measured number of cells/mL at t 1

N

n = measured number of cells/mL at time t

n

t 1 = time of first m easurement after beginning of test

t

n = time of n th measurement after beginning of test

n = number of measurements taken after test initiation

Based on the results of the statistical analysis performed for 96

- hour yield, average specific

growth rate an d area under the growth curve, the No

- Observed

- Effect Concentration (NOEC, the

highest test concentration which demonstrated no statistically adverse effects (p ≤ 0.05) when

compared to the control data) and Lowest

- Observed

- Effect Concentration (LOEC , the lowest test

concentration which demonstrated a statistically significant effect when compared to the control

data ) values for each endpoint were determined The data were first checked for normality using

Shapiro

- Wilk’s Test ( U.S. EPA, 2002 ) and for homogeneity of variance using Bartlett's Test

( U.S. EPA, 2002 ). If the data sets passed the tests for homogeneity and normality, then

Williams ’ Multiple Comparison Test ( U.S. EPA, 2002 ) w as used to determine the NOEC and

LOEC values

. If the data did not pass the tests for homogeneity and /or normality, then the

NOEC and LOEC were determined using an appropriate non

- par ametric statistical test,

e.g., Jonckheere

- Terpstr a ’s Step

- Down Test ( U.S. EPA, 2002 ). All statistical determinations

were made at the 95% level of certainty, except in the case of Shapiro

- Wilk’s and Bartlett's

Tests, where the 99% l evel of certainty was applied.

The 96

- h our EC 50 values (concentrations of test substance which reduced yield

, average growth

rate and area under the growth curve by 50% ) and the 95% confidence limit s were also

determined by linear interpolation of response ( percent inhibition compared with the control)Smithers Viscient Study No. 14091.6101 Page 22

versus the time

- weighted average concentration using the ICp method ( Norberg

- King, 1993 ). If

less than the required response was observed (i.e., < 50% response), the EC value was

empirically estimated to be greater than the highest time

- weighted average concentration tested.

CETIS TM Version 1.8 ( Ives, 2013 ) w as used to perform all statistical analyses.



### 3.0 RESULTS

3.1 Preliminary Exposure

A preliminary range

- finding exposure was conducted a t Smithers Viscient at nominal

concentrations of 0.10, 1. 0, 10 , 10 0 and 10 00 mg/L and a control. Test solutions were prepared

in a similar manner as described for the definitive test. Two replicate exposure vessels were

established for each concentration and the control. Following 96 hours of exposure, cells

exposed to all treatment levels tested and the control were observed to be normal. Cell densities

in the 0.10, 1.0, 10, 100 and 1000 mg/L treatment levels averaged 208.67, 170.5 0

, 187 .5 0

, 1.00

and 1 .13 × 10 4 cells/mL, respectively. The control averaged 186

. 3 3 × 10 4 cells/mL. Based on

these results and in consultation with the Study Sponsor, nominal sorbic acid concentrations of

6.3, 13, 25, 50 and 100 mg/L and a control were selected for the defini tive exposure.



### 3.2 Definitive Exposure

3.2.1 Evaluation of Test Conditions

The pH, conductivity, temperature and light intensity measurements recorded during the

definitive exposure are presented in Table 2

. The pH of the control solution was 7.2 at test

initiation and increased to 9.4 after 96 hours. The pH of the test solutions ranged from 4.2 to 6.7

at test initiation. The solution pH was inversely proportional to the test concentration, decreasing

with increasing test substance conce ntration. At 96 hours of exposure, the pH of the test

solutions ranged from 4.3 to 9.3 , increasing relative to biomass increase in the 6.3 and 13 mg/L

treatment levels and remaining largely unchanged from a pH of approximately 5 in the three highSmithers Viscient Study No. 14091.6101 Page 23

treatmen t levels (25, 50 and 100 mg/L) where nearly 100% inhibition of algal growth was

observed

. The increase in pH relative to biomass increase during the exposure is common in

static algal cultures and is due t o photosynthesis by the algae. Conductivity of th e treatment and

control solutions ranged from 90 to 120 μS/cm , increasing slightly as the concentration of test

substance increased . Continuous temperature monitoring established that the temperature ranged

from 23 to 2 4 o C throughout the exposure period. The photosynthetically

- active radiation (PAR)

of the test area ranged from 51 to 66 μE/m 2 /S throughout the exposure period. Light intensity

(lux) of the test area at test initiation ranged from 4300 to 4900 lux

. The shaking rate was

maintained througho ut the exposure at a constant rate of 100 rpm.

3.2.2 Analytical Results

The results of the analysis for the exposure solutions are presented in Table 3 and illustrated in

Figure 1

.

At test initiation, m easured concentration s of each exposure solution closely approximated the

nominal concentrations , ranging from 9 9 to 1 10 % of nominal concentration

. The measured

concentrations in the 6.3 and 13 mg/L nominal treatment levels declined after 96 hour s , and we re

2.7 and 4.3% of nominal concentrations, respectively

. Measured concentrations in the remaining

treatment levels tested ( 25, 50 and 100 mg/L ) ranged from 94 to 100 % of the nominal test

concentrations . Since measured concentrations in the low concentrat ions decreased substantially

over time , results are calculated as a time

- weighted average of the measured concentrations in

accordance with OCSPP 850.4500 guidance . Based on time

- weighted average concentrations

,

treatment levels ranged from 2 7% to 110 % of nominal concentrations and defined the treatment

levels tested as 1.7, 3.9, 24, 50, and 110 mg/L

.

The analytical result of the 25 mg/L nominal treatment level with algae present was 23 mg/L at

96 hours ( Table 3 ). The equivalent test solution without algae present resulted in a recovery of

2 4 mg/L at 96 hours ; however, this treatment had nearly 100% inhibition of algal growth

.Smithers Viscient Study No. 14091.6101 Page 24

Consequently the impact of algae on the test substance concentration could not be assessed

within this treatment.

Analysis of the uncentrifuged quality control (QC) samples resulted in measured concentrations

which were consistent with the predetermined recovery range, ranging from 98.0 to 1 07 %

(N

= 6) of the nominal fortified levels (3.00

, 25.0 and 100 m g / L). Analysis of the centrifuged

quality control samples resulted in measured concentrations which ranged from 97. 8 to 104 %

(N = 3) of the nominal fortified levels ( 3.00, 25.0 and 100 m g/L ). Based on these results, it was

established that the appropria te precision and quality control was maintained during processing

and that centrifugation of the test samples did not have a negative impact on their quantitation.

3.2.3 Biological Results

Cell densities determined at each observation interval are presented in Table 4 and illustrated in

Figure 2 . The 96

- hour cell density in the control averaged 219.08 × 10 4 cells/mL. Cell densities

in the 1.7, 3.9, 24, 50 and 110 mg/L treatment levels averaged 219.88, 21 6.79, 2.81, 1.38 and

0.81 × 10 4 cells/mL , respectively

.

Yield is presented in Table 5 and illustrated in Figure 3

. The 96

- hour yield in the control

averaged 218.08 × 10 4 cells/mL. The 96

- hour yield in the 1.7, 3.9, 24, 50 and 110 mg/L

treatment levels averaged 218.88

, 215.79 , 1 .81

, 0.38 and

- 0.1 9 × 10 4 cells/mL , respectively

.

Based on the results of Shapir o

- Wilk’s and Bartlett’s Equality of Variance Test, this data set

passed the requirement for nor mality and homogeneity of variance ; therefore

, Williams ’ Multiple

Comparison Test was used to determine treatment

- related effects. A significant reduction was

detected in t reatment levels ≥ 24 mg/L compared to the control data. Therefore, the 96

- hour

NOEC and LOEC values for yield were determined to be 3.9 and 24 m g /L , respectively

. Based

on time

- weighted average concentrations, the 96

- hour EC 50 value for yield was determined to be

10 mg/ L, with 95% confidence limits of 6.9 to 10 mg/L. The 96

- hour NOEC, LOEC and EC 50

values for yield are presented in Table 6

.Smithers Viscient Study No. 14091.6101 Page 25

Average specific growth rates are presented in Table 7 and illustrated in Figure 4 . The 0

- to

96

- hour average specific growth rate in the control averaged 1. 3 6 days

- 1

, respectively. The 0

- to

96

- hour average specific growth rate in the 1.7, 3.9, 24, 50 and 110 mg/L treatment levels

averaged 1. 36 , 1

. 3 6, 0.26

, 0.05 and

- 0.11 days

- 1 , respectively

. Based on the results of Shapiro

Wilk’s and Bartlett’s Equality of Variance Test, this data set passed the requirement s for both

homogeneity of variance and normality ; therefore

, Williams ’ Multiple Comparison Test was

used to determine treatment

- related effects. A significant reduction was detected in treatment

levels ≥ 24 mg/L compared to the control data. Therefore, the 96

- hour NOEC and LOEC values

for average specific growth rate were determined to be 3.9 and 24 m g/L , respectively . Based on

the time

- weighted average concentrations, the 96

- hour EC 50 value for averag e specific growth

rate was determined to be 12 mg/L, with 95% confidence limits of 11 to 13 mg/L. The 96

- hour

NOEC, LOEC and EC 50 values for average specific growth rate are presented in Table 8

.

Section

- by

- section growth rate s are presented in Table 9

.

A re a under the growth curve (AUGC) is presented in Table 10 and illustrated in Figure 5 . The

total AUGC in the control averaged 219.05 x 10 4 cells × d ays/mL. Total AUGC in 1.7, 3.9, 24

,

50 and 1 1 0 mg/L treatment levels averaged 222.23

, 227.30, 3.66

, 0.88 and

0.33 x 10 4 cells × days/mL , respectively

. Based on the results of Shapiro

- Wilk’s and Bartlett’s

Equality of Variance Test, this data se t passed the requirement for normality but failed the

requirement for homogeneity of variance ; therefore

, Jonckheere

- Terpstra ’s Step

- Down Test was

used to determine treatment

- related effects. A significant reduction was detected in treatment

levels ≥ 24 mg/L compared to the control data. Therefore, the 96

- hour NOEC and LOEC values

for AUGC were determined to be 3.9 and 24 m g/L , respectively . Based on the time

- weighted

average concentrations, the 96

- hour EC 50 value for AUGC was determined to be 10 m g/L, with

95% confidence limits of 8.5 to 10 mg/L. The 96

- hour NOEC, LOEC and EC 50 values for

AUGC are presented in Table 11

.

The subculture used t o evaluate the algistatic/algicidal properties of the test substance was

initi ated using cells from the 100 mg/L nominal treatment level. The pH of the 100 mg/L

composite test solution was 4.3 after 96 hours of exposure. The AAP medium used for theSmithers Viscient Study No. 14091.6101 Page 26

recovery phase had a pH of 7.6 prior to inoculation with cells from the 100 mg/L no minal

treatment level. The temperature during the recovery phase was maintained at a range of 22 to

24 o C

. The photosynthetically

- active radiation (PAR) of the test area ranged from 56 to

68 μE/m 2 /S over the nine

- day test period. After nine days, a cell density of 4 .00 × 10 4 cells/mL

was observed in the subculture

, corresponding to an approximately 82

- fold increase in cell

density (see Section 2.9.2 ) . This observation indicates that the test substanc e has an algistatic,

rather than algicidal effect on the growth of Pseudokirchneriella subcapitata at a nominal

concentration of 100 mg/L.

The protocol requires the following criteria per the OCSPP G uideline 850. 4500 : 1) at exposure

termination

, the mean yield and average specific growth rate coefficient of variation (CV) of the

control should not exceed 15 %, and 2 ) the cell density in the control should increase by a factor

of at least 100 times by exposure termination (96

- hours) . The results of this tes t are summarized

below and presented in the following table:

OCSPP Acceptance Criteria for Control Study Results

0

- to 96

- hour average yield CV ≤ 15 % : 1 6 %

0

- to 96

- hour average specific growth rate CV ≤ 1 5 % : 3.0 %

96

- hour cell density in the control ≥ 1 00 × 10 4 cells/mL: 219 .08 × 10 4 cells/mL

At exposure termination, the mean (0

- to 96

- hour) average specific growth rate CV for the

control was 3.0%

, which met the above criterion . Additionally, the 96

- hour cell density in the

control was 219 .08 × 10 4 ce lls/mL, which also meets the above criterion. The mean (0

- to

96

- hour) average yield CV for the control was 16%, which is slightly above the acceptable

criterion of 15%. Although the CV for 96

- hour yield exceeds the criterion, the actual value

(16%) only slightly exceeds the criterion (15%) and the concentration

- response and NOEC,

LOEC and EC50 values for all endpoints were clearly define d

. Therefore, this study is

considered acceptable and the results accurately reflect the sensitivity of Pseudokirchneri ella

subcapitata to sorbic acid under testing conditions.Smithers Viscient Study No. 14091.6101 Page 27



### PROTOCOL DEVIATION

No deviations from the protocol occurred during this study.Smithers Viscient Study No. 14091.6101 Page 28



### REFERENCES

ASTM, 2004. Standard Guide for Conducting Static Toxicity Tests with Microalgae. Standard

E

- 1218

- 04, Am erican Society for Testing and Materials, 100 Barr Harbor Drive, West

Conshohocken, P ennsylvania 19428, pp. 293

- 306.

Ives, M

. , 201 3 . CETIS, Comprehensive Environmental Toxicity Information System TM . User’s

Guide. Tidepool Scientific Software, McKinleyvi lle, California.

Miller, W.E., J.C. Greene and T. Shiroyama, 1978. The Selenastrum capricornutum Printz algal

assay bottle test. EPA 600/9

- 78

- 018. U.S. Environmental Protection Agency,

Corvallis

, Oregon.

Norberg

- King, Teresa J. 1993. A Linear Interpol ation Method for Sublethal Toxicity: The

Inhibition Concentration (ICp) Approach. National Effluent Toxicity Assessment Center,

Environmental Research Laboratory

– Duluth, U.S. Environmental Protection Agency,

Duluth, Minnesota. Technical report 03

- 93.

O ECD, 1998. OECD Series on Principles of Good Laboratory Practice and Compliance

Monitoring. Number 1. OECD Principles of Good Laboratory Practice (as revised in

1997). Environment Directorate Chemicals Group and Management Committee.

ENV/MC/CHEM (98)1 7. OECD Paris, France, 41 pp.

U.S. EPA, 1989. Federal Insecticide, Fungicide and Rodenticide Act (FIFRA); Good Laboratory

Practice Standard; Final Rule (40 CFR, Part 160). Federal Register, 48 (230); 34052

-

34074.

U.S. EPA, 1997. Office of Waste. Tes t Methods for Evaluating Solid Waste, Physical/Chemical

Methods (SW

- 846). February 2007. U.S. Environmental Protection Agency,

Washington, D.C.

U.S. EPA, 2002. Short

- Term Methods for Estimating the Chronic Toxicity of Effluents and

receiving Waters to Freshwater Organisms. Fourth Edition. EPA

- 821

- R

- 02

- 013. Office

of Water (4303T), U.S. Environmental Protection Agency, Washington, D.C.

U.S. EPA

, 2012

. Office of Chemical Safety and Pollution Prevention . Ecological Effects Test

Guideline, OCSPP 850. 45 0 0 : Algal Toxicity EPA 712

- C

- 005

. U.S. Environmenta l

Protection Agency. Washington, D.C.Smithers Viscient Study No. 14091.6101 Page 29



### Table 1. Composition of nutrient medium (AAP medium) used during the

toxicity test exposing Pseudokirchneriella subcapitata to sorbic acid

.

Compound Concentration

NaNO 3 25.5 mg/L

MgCl 2 •6H 2 O 12.16 mg/L

CaCl 2 •2H 2 O 4.41 mg/L

MgSO 4 •7H 2 O 14.7 mg/L

K 2 HPO 4 •3H 2 O 1.368 mg/L

NaHCO 3 15.0 mg/L

H 3 BO 3 185.5 μ g/L

Na 2 SeO 4 a 1.88 μ g/L

MnCl 2 •4H 2 O 415.4 μ g/L

ZnCl 2 3.270 μ g/L

CoCl 2 •6H 2 O 1.43 μ g/L

CuCl 2 •2H 2 O 0. 012 μ g/L

Na 2 MoO 4 •2H 2 O 7.26 μ g/L

FeCl 3 •6H 2 O 160 μ g/L

Na 2 EDTA•2H 2 O 300 μ g/L

a Additional nutrient required, personal communication. Dr. R.R.L. Guillard, June 1991.

If required, pH was adjusted to 7.5 ± 0.1 with dilute sodium hydroxide (NaOH) or hydroch loric acid (HCl).

Source: Miller, W.E., J.C. Greene and T. Shiroyama. 1978 . The Selenastrum capricornutum Printz algal assay

bottle test. EPA 600/9

- 78

- 018. U.S. Environmental Protection Agency, Corvallis, Oregon.Smithers Viscient Study No. 14091.6101 Page 30



### Tabl e 2. The pH, conductivity, temperature and light intensity measured

during the 96

- hour exposure of Pseudokirchneriella subcapitata to

sorbic acid.

Nominal

Concentration

( m g /L ) pH Conductivity

( μS /cm )

0 Hour 24 Hour 48 Hour 72 Hour 96 Hour 0 Hour

Control 7.2 7.3 7.4 9.1 9.4 91

6.3 6.7 7.3 7.5 9.1 9.3 89

13 6.3 7.1 7.4 9.1 9.2 90

25 5.3 5.6 5.6 5.6 6.2 94

50 4.6 4.7 4.7 4.7 4.7 100

100 4.2 4.2 4.2 4.2 4.3 120

Minimum/Maximum Temperature ( ° C)

0

- 24 Hours 24

- 48 Hours 48

- 72 Hours 72

- 96 Hours

23/24 2 3/ 2 4 2 3 /2 4 23/ 24

Light Intensity (PAR) a

Unit s 0 Hours b 24 Hours 4 8 Hours 72 Hours 96 Hours

μE/m 2 /S 51

- 66 51

- 63 53

- 62 52

- 64 52

- 64

a P AR = p hotosynthetically

- a ctive r adiation

b The light intensity ( lux ) of the test area at test initiation ranged from 4300 to 4900

.Smithers Viscient Study No. 14091.6101 Page 31



### Table 3. Concentrations of s orbic acid measured in the exposure solutions

during the 96

- hour exposure of Pseudokirchneriella subcapitata

.

Nominal

Concentration

(mg/L) Measured Concentration (mg/L) Percent

Decline from

0 Hour Time

Weighted

Average bc Percent of

Nominal b

0 Hour 96 Ho urs a

Control < 0.59 d < 0.027 NA e NA NA

6.3 6.4 0.17 97 1.7 27

13 13 0.56 96 3.9 30

25 25 23/24 f 7/5 f 24 98

50 51 49 3 50 100

100 110 100 6 110 110

Uncentrifuged Uncentrifuged Centrifuged

QC g #1

3.00 3.00

(98.9) 2.94

(98.0) 2.94

(97.8)

QC #2

25.0 26.1

(104) 25.8

(103) 25.8

(103)

QC #3

100 107

(107) 105

(105) 104

(104)

a All 96

- hour test and control solutions were centrifuged prior to analysis

. b Time

- weighted average concentrations and percent of nominal were calculated using the actual analytical

(unrounded) results and not the rounded (two significant figures) values presented in this table.

c 

## ( ) ( ) ( ) ( ) 

## [ ] Days Total / Days 4 x C ln C ln / C C C D4 D0 D4 D0 twa

− − =

d C oncentrations expressed as less than values were below the limit of quantitation (LOQ). The LOQ for each

analysis is dependent upon the regression, the area of the low standards and the dilution factor of the controls.

e NA = Not Applicable.

f Result of the additional sample without algae present analyzed at 96 hours of exposure to determin e biological

uptake/degradation.

g QC = Quality Control sample. Percent recovery presented in parentheses.Smithers Viscient Study No. 14091.6101 Page 32



### Table 4. Cell density of Pseudokirchneriella subcapitata after 24, 48, 72 and

96 hours of ex posure to sorbic acid.

Time Weighted

Average

Concentration

( m g/L) Replicate Cell Density (x 10 4 cells/mL)

Observation Interval (Hours)

24 48 72 96

Control A 6.50 35.00 73.00 187.67

B 7.75 33.50 63.25 253.00

C 3.75 23.00 88. 75 246.00

D 4.25 17.25 73.50 189.67

Mean (SD) a 5.56(1.89) 27.19(8.51) 74.63(10.53) 219.08(35.25)

1.7 A 6.50 26.00 78.75 214.50

B 7.75 32.00 58.75 275.50

C 3.50 23.25 86.25 201.50

D 6.75 23.00 88.00 188.00

Mean (SD) 6.13(1.83) 26.06(4.19) 77.94 (13.41) 219.88(38.63)

3.9 A 8.50 39.25 76.25 224.00

B 8.75 42.25 81.25 268.50

C 3.25 18.00 81.00 200.33

D 5.25 19.75 84.00 174.33

Mean (SD) 6.44(2.66) 29.81(12.71) 80.63(3.22) 216.79(40 .00 )

2 4 A 2.00 2.00 1.75 2.50

B 3.50 2.25 2.75 3.75

C 2.2 5 0.75 0.75 2.00

D 1.75 1.25 2.00 3.00

Mean (SD) 2.38(0.78) 1.56(0.69) 1.81(0.83) 2.81(0.75)

50 A 1.00 1.25 1.75 0.75

B 1.50 1.75 0.75 2.25

C 1.25 1.00 1.25 1.75

D 0.25 2.00 1.00 0.75

Mean (SD) 1 .00 (0.54) 1.5 0 (0.46) 1.19(0.43) 1.38(0.75)

1 1 0 A 0.75 1.00 0.50 0.75

B 1.25 2.50 2.25 1.75

C 1.00 1.00 0.00 0.25

D 1.25 1.25 1.00 0.50

Mean (SD) 1.06(0.24) 1.44(0.72) 0.94(0.97) 0.81(0.66)

a Mean and standard deviation (SD) were calculated from original raw data, not from the rounded values

presented in this table.Smithers Viscient Study No. 14091.6101 Page 33



### Table 5. Yield for Pseudokirchneriella subcapitata after 24, 48 , 72 and

96 hours of exposure to sorbic acid.

Time Weighted

Average Concentration

(mg/L) Replicate Yield (x 10 4 cells/mL) Percent

Inhibition ab

(% ) Observation Interval (Hours)

0

- 24 0

- 48 0

- 72 0

- 96

Control A 5.50 34.00 72.00 186.67

B 6.75 32.50 62.25 252.00

C 2.75 22.00 87.75 245.00

D 3.25 16.25 72.50 188.67

Mean (SD) b 4.56(1.89) 26.19(8.51) 73.63(10.53) 218.08(35 .25) NA c

1.7 A 5.50 25.00 77.75 213.50

B 6.75 31.00 57.75 274.50

C 2.50 22.25 85.25 200.50

D 5.75 22.00 87.00 187.00

Mean (SD) 5.13(1.83) 25.06(4.19) 76.94(13.41) 218.88(38.63) 0

3.9 A 7.50 38.25 75.25 223.00

B 7.75 41.25 80.25 267.50

C 2.25 17.00 80.00 199.33

D 4.25 18.75 83.00 173.33

Mean (SD) 5.44(2.66) 28.81(12.71) 79.63(3.22) 215.79(40 .00 ) 1

2 4 A 1.00 1.00 0.75 1.50

B 2.50 1.25 1.75 2.75

C 1.25

- 0.25

- 0.25 1.00

D 0.75 0.25 1.00 2.00

Mean (SD) 1.38(0.78) 0.56(0.69) 0.81(0.83) 1.81 d (0.75) 99

50 A 0.00 0.25 0.75

- 0.25

B 0.50 0.75

- 0.25 1.25

C 0.25 0.00 0.25 0.75

D

- 0.75 1.00 0.00

- 0.25

Mean (SD) 0 .00 (0.54) 0.5 0 (0.46) 0.19(0.43) 0.38 d (0.75) 100

1 10 A

- 0.25 0.00

- 0.50

- 0.25

B 0.25 1.50 1.25 0.75

C 0 .00 0.00

- 1.00

- 0.75

D 0.25 0.25 0.00

- 0.50

Mean (SD) 0.06(0.24) 0.44(0.72)

- 0.06(0.97)

- 0.19 d (0.66) 100

a Percent inhibition relative to the control.

b Mean, standard deviation (SD) and percent inhibition were calculated from original raw data, not from the rounded values

presented in this table.

c NA = Not Applicable.

d Significantly reduced compared to the contro l , based on Williams ’ Multiple Comparison Test.Smithers Viscient Study No. 14091.6101 Page 34



### Table 6. EC 50

, No

- Observed

- Effect Concentration (NOEC) and Lowest

Observed

- Effect Concentration (LOEC) values for sorbic acid

calculated from the results (yield) of the 96

- hour toxicity test with

Pseudokirchneriella subcapitata

.

96

- Hour Results Based on Time

- Weighted Average Concentrations

EC 50 NOEC LOEC

EC Valu e (mg/L): 10 3.9 24

95% Confidence Limits: 6.9

– 10Smithers Viscient Study No. 14091.6101 Page 35



### Table 7

. Average specific growth rates of Pseudokirchneriella subcapitata

after 24, 48 , 72 and 96 hours of exposure to sorbic acid.

Time Weighted

Average

Concentration

(mg/L) Replicate Average Specific Growth Rate (days - 1 ) Percent

Inhibition ab

(%) Observation Interval (Hours)

0

– 24

( 0. 919 days) 0

– 48

( 1. 94 days) 0

– 72

(2. 89 days) 0

– 96

( 3. 95 days)

Control A 2.04 1.83 1.48 1.33

B 2.23 1.81 1.43 1.40

C 1.44 1.61 1.55 1. 39

D 1.57 1.46 1.49 1.33

Mean (SD) b 1.82(0.37) 1.68(0.17) 1.49(0.05) 1.36(0.04) NA c

1.7 A 2.04 1.68 1.51 1.36

B 2.23 1.78 1.41 1.42

C 1.36 1.62 1.54 1.34

D 2.08 1.61 1.55 1.33

Mean (SD) 1.93(0.38) 1.67(0.08) 1.5 0 (0.06) 1.36(0.04) 0

3.9 A 2.33 1.89 1.50 1.37

B 2.36 1.93 1.52 1.42

C 1.28 1.49 1.52 1.34

D 1.80 1.53 1.53 1.31

Mean (SD) 1.94(0.51) 1.71(0.23) 1.52(0.01) 1.36(0.05) 0

2 4 A 0.75 0.36 0.19 0.23

B 1.36 0.42 0.35 0.33

C 0.88

- 0.15

- 0.10 0.18

D 0.61 0.11 0.24 0. 28

Mean (SD) 0.9(0.33) 0.19(0.26) 0.17(0.19) 0.26 d (0.07) 81

50 A 0.00 0.11 0.19

- 0.07

B 0.44 0.29

- 0.10 0.21

C 0.24 0.00 0.08 0.14

D

- 1.51 0.36 0.00

- 0.07

Mean (SD)

- 0.21(0.89) 0.19(0.16) 0.04(0.12) 0.05 d (0.14) 96

1 1 0 A

- 0.31 0.00

- 0.24

- 0 .07

B 0.24 0.47 0.28 0.14

C 0.00 0.00 0.00 e

- 0.35

D 0.24 0.11 0.00

- 0.18

Mean (SD) 0.04(0.26) 0.15(0.22) 0.01(0.21)

- 0.11 d (0.21) 108

a Percent inhibition relative to the control.

b Mean, standard deviation (SD) and percen t inhibition were calculated from original raw data, not from the rounded values

present in this table.

c NA = Not Applicable.

d Significantly reduced compared to the control, based on Williams ’ Multiple Comparison Test.

e Growth rate values cannot be math ematically calculated from a cell density of zero. Therefore, a growth rate value of zero is

used for any replicate with zero cell density.Smithers Viscient Study No. 14091.6101 Page 36



### Table 8. EC 50

, No

- Observed

- Effect Concentration (NOEC) and Lowest

Observed

- Effect Concentration (LOEC) values fo r sorbic acid

calculated from results (average specific growth rate) of the 96

- hour

toxicity test with Pseudokirchneriella subcapitata

.

96

- Hour Results Based on Time

- Weighted Average Concentrations

EC 50 NOEC LOEC

EC Value (mg/L): 12 3.9 24

95% Co nfidence Limits: 11

– 13Smithers Viscient Study No. 14091.6101 Page 37



### Table 9

. Section

- by

- section growth rates of Pseudokirchneriella subcapitata

after 24, 48 , 72 and 96 hours of exposure to sorbic acid

.

Time Weighted

Average

Concentration

(mg/L) Replicate Section

- by

- Section Growth Rate (d ays - 1 )

Observation Interval (Hours)

0

- 24

(0. 919 days) 24

- 48

( 1.03 days) 48

- 72

( 0.949 days) 72

- 96

( 1.05 days)

Control A 2.04 1.64 0.77 0.90

B 2.23 1.43 0.67 1.31

C 1.44 1.77 1.42 0.97

D 1.57 1.37 1.53 0.90

Mean (SD) a 1.82(0.37) 1.55(0 .19) 1.1(0.44) 1.02(0.2 0 )

1.7 A 2.04 1.35 1.17 0.95

B 2.23 1.38 0.64 1.46

C 1.36 1.85 1.38 0.80

D 2.08 1.20 1.41 0.72

Mean (SD) 1.93(0.38) 1.44(0.28) 1.15(0.36) 0.98(0.33)

3.9 A 2.33 1.49 0.70 1.02

B 2.36 1.54 0.69 1.13

C 1.28 1.67 1.59 0.86

D 1.80 1.29 1.53 0.69

Mean (SD) 1.94(0.51) 1.5 0 (0.16) 1.13(0.5 0 ) 0.93(0.19)

2 4 A 0.75 0.00

- 0.14 0.34

B 1.36

- 0.43 0.21 0.29

C 0.88

- 1.07 0.00 0.93

D 0.61

- 0.33 0.50 0.38

Mean (SD) 0.9 0 (0.33)

- 0.46(0.45) 0.14(0.28) 0.49(0.3 0 )

50 A 0.00 0.22 0 .35

- 0.80

B 0.44 0.15

- 0.89 1.04

C 0.24

- 0.22 0.24 0.32

D

- 1.51 2.03

- 0.73

- 0.27

Mean (SD)

- 0.21(0.89) 0.54(1.01)

- 0.26(0.64) 0.07(0.79)

110 A

- 0.31 0.28

- 0.73 0.38

B 0.24 0.68

- 0.11

- 0.24

C 0.00 0. 0 0 0.00 b 0.00 b

D 0.24 0.00

- 0.24

- 0.66

Me an (SD) 0.04(0.26) 0.24(0.32)

- 0.27(0.32)

- 0.13(0.44)

a Mean and standard deviation (SD) were calculated from original raw data, not from the rounded values present in this table.

b Growth rate values cannot be mathematically calculated from a cell densi ty of zero . Therefore, a growth rate value of zero

is used for any replicate with zero cell density

.Smithers Viscient Study No. 14091.6101 Page 38



### Table 10. Calculated area under the growth curve (AUGC) of

Pseudokirchneriella subcapitata after 24, 48, 72 and 96 hours of

exposure to sorbic acid

.

Ti me

Weighted

Average

Concentration

(mg/L) Replicate Area Under the Growth Curve (× 10 4 cells × days/mL)

Percent

Inhibition ab

(%) Observation Interval (Hours)

0

- 24 24

- 48 48

- 72 72

- 96 0

- 96

Control A 2.53 20.24 50.28 136.43 209.48

B 3.10 20.12 44.94 165.75 233.90

C 1.26 12.68 52.06 175.50 241.51

D 1.49 9.99 42.09 137.75 191.33

Mean(SD) b 2.1 0 (0.87) 15.76(5.22) 47.34(4.62) 153.86(19.77) 219.05(22.99) NA c

1.7 A 2.53 15.63 48.73 153.61 220.51

B 3.10 19.35 42.09 175.24 239. 78

C 1.15 12.68 50.99 150.71 215.53

D 2.64 14.22 51.70 144.52 213.08

Mean (SD) 2.36(0.84) 15.47(2.85) 48.38(4.38) 156.02(13.36) 222.23(12.11)

- 1

3.9 A 3.45 23.45 53.83 157.31 238.03

B 3.56 25.11 57.63 183.41 269.72

C 1.03 9.87 46.01 147.33 204.24

D 1.95 11.79 48.26 135.20 197.20

Mean (SD) 2.5 0 (1.22) 17.55(7.84) 51.43(5.28) 155.81(20.5) 227.3 0 (33.43)

- 4

24 A 0.46 1.03 0.83 1.19 3.50

B 1.15 1.92 1.42 2.37 6.87

C 0.57 0.51

- 0.24 0.40 1.25

D 0.34 0.51 0.59 1.58 3.03

Mean (SD) 0.63(0.36) 0.99(0.66) 0.65(0.69) 1.38(0.82) 3.66 d (2.35) 98

50 A 0.00 0.13 0.47 0.26 0.87

B 0.23 0.64 0.24 0.53 1.64

C 0.11 0.13 0.12 0.53 0.89

D

- 0.34 0.13 0.47

- 0.13 0.13

Mean (SD) 0 .00 (0.25) 0.26(0.26) 0.33(0.18) 0.3 0 (0.31) 0.88 d (0.62) 100

1 1 0 A

- 0.11

- 0.13

- 0.24

- 0.40

- 0.88

B 0.11 0.90 1.30 1.05 3.37

C 0.00 0.00

- 0.47

- 0.92

- 1.40

D 0.11 0.26 0.12

- 0.26 0.23

Mean (SD) 0.03(0.11) 0.26(0.46) 0.18(0.79)

- 0.13(0.84) 0.33 d (2.14) 100

a Percent inhibition relativ e to the control.

b Mean, standard deviation (SD) and percent inhibition were calculated from original raw data, not from the rounded values pres ented in this

table.

c NA = Not Applicable.

d Significantly reduced compared to the control, based on the Jonck heere

- Terpstra ’s Step

- Down Test

.Smithers Viscient Study No. 14091.6101 Page 39



### Table 11. EC 50

, No

- Observed

- Effect Concentration (NOEC) and Lowest

Observed

- Effect Concentration (LOEC) values for sorbic acid

calculated from results (area under the growth curve) of the 96

- hour

toxicity test with Pseudok irchneriella subcapitata

.

96

- Hour Results Based on Time

- Weighted Average Concentrations

EC 50 NOEC LOEC

EC Value (mg / L): 10 3.9 24

95% Confidence Limits: 8.5

– 10Smithers Viscient Study No. 14091.6101 Page 40



### Figure 1. R elationship between nominal and time

- weighted average

concentra tions of sorbic acid during the 96

- hour static toxicity test

exposing Pseudokirchneriella subcapitata

.Smithers Viscient Study No. 14091.6101 Page 41



### Figure 2. Algal growth curves (cell density vs. time) for Pseudokirchneriella

subcapitata exposed to sorbic acid

.Smithers Viscient Study No. 14091.6101 Page 42



### Figure 3

. Y ield for Pseudokir chneriella subcapitata exposed to sorbic acid

.

*Significantly reduced compared to the control, based on Williams ’ Multiple Comparison Test.Smithers Viscient Study No. 14091.6101 Page 43



### Figure 4

. Average specific growth rate s (0

- to 96

- hour) for Pseudokirchneriella

subcapitata exposed to sorbic acid

.

*Significantly reduced compared to the control, based on William s ’ Multiple Comparison Test.Smithers Viscient Study No. 14091.6101 Page 44



### Figure 5

. Area under the growth curve for Pseudokirchneriella subcapitata

exposed to sorbic acid

.

*Significantly reduced compared to the control, bas ed on Jonckheere

- Terpstra ’s Step Down Test.Smithers Viscient Study No. 14091.6101 Page 45



### APPENDIX 1

- STUDY PROTOCOLSmithers Viscient Study No. 14091.6101 Page 46Smithers Viscient Study No. 14091.6101 Page 47Smithers Viscient Study No. 14091.6101 Page 48Smithers Viscient Study No. 14091.6101 Page 49Smithers Viscient Study No. 14091.6101 Page 50Smithers Viscient Study No. 14091.6101 Page 51Smithers Viscient Study No. 14091.6101 Page 52Smithers Viscient Study No. 14091.6101 Page 53Smithers Viscient Study No. 14091.6101 Page 54Smithers Viscient Study No. 14091.6101 Page 55Smithers Viscient Study No. 14091.6101 Page 56Smithers Viscient Study No. 14091.6101 Page 57Smithers Viscient Study No. 14091.6101 Page 58Smithers Viscient Study No. 14091.6101 Page 59Smithers Viscient Study No. 14091.6101 Page 60Smithers Viscient Study No. 14091.6101 Page 61



### APPENDIX 2

- ANALYTICAL METHODOLOGYSmithers Viscient Study No. 14091.6101 Page 62

S UMMARY

Methodology was validated ( 13 March 2015 ) to quantify the amount of sorbic acid present in

recovery s amples prepared in Algal Assay Procedure ( AAP ) medium (a freshwater algal

medium). All samples were initially diluted with acetonitrile to a composition of 20:80

acetonitrile:AAP medium (v:v)

. The mid

- and high

- level recovery samples were further diluted

into the calibration standard range with 20:80 acetonitrile:purified reagent water (v:v). All

samples were analyzed by automated injection on a high performance liquid chromatographic

system with ultraviolet detection (HPLC /UV ).

This m ethod was validate d by fortification of AAP medium with sorbic acid at concentrations of

0. 0100

, 5.00 and 100 mg/L

. Recoveries averaged 99.5 ± 5.02 %, with a limit of quantitation

(LOQ) of 0.00 747 mg/L. Defined limits for acceptance of quality control sample performance in

subsequent studies were set at 80.0 to 120%.

EXPERIMENTAL

Equipment

1. Instrument: Hewlett

- Packard Series 1100 quaternary solvent pump

equipped with a Hewlett

- Packard Series 1100 autosampler,

a Hewlett

- Packard Series 1100 vacuum degasser, a

Hewlett

- Pac kard Series 1100 variable wavelength detector

and an Agilent ChemStation version B .0 4 .0 2 for data

acquisition

2. Balance: Mettler Toledo AG285

3. Laboratory equipment: disposable glass pipets, volumetric flasks, graduated

cylinders, positive displaceme nt pipets, autosampler vials

and amber glass bottles with Teflon ®

- lined caps

Reagents

1. Acetonitrile: EMD, reagent grade

2. Phosphoric acid: EMD, reagent grade

3. Purified reagent water: Prepared from a Millipore Milli

- Q ® Direct 8 water

purificatio n system (meets ASTM Type II requirements)Smithers Viscient Study No. 14091.6101 Page 63

Test Substance

The test substance, sorbic acid, was received on 17 February 2015 from GEI Ecology Lab

,

Denver, Colorad o

. The following information was provided:

Name: sorbic acid

Lot No.: 1401151

CAS No.: 110

- 44

- 1

Purity: 99. 5 %

Expiration Date: 1 4 January 2016

Upon receipt at Smithers Viscient, the test substance (SMV No. 7535 ) was stored at room

temperature in the original container, in a dark ventilated cabinet. Concentrations were adjusted

for the purity of the test substance.

PR OCEDURES

Preparation of Stock Solutions

P rimary stock solutions were typically prepared as per the table below:

Primary

Stock ID Amount of

Substance

Weighed

(g), Net

Weight Amount of

Substance

Weighed (g),

as Active

Ingredi ent Stock

Solvent Final

Volume

(mL) Primary Stock

Concentration

(mg/L) Primary Stock Use

7535A 0. 1005 0.1000

Acetonitrile 100 1.00 Secondary stock for

calibration standards

7535B 0.1007 0.1002 100 1.00 High

- level recovery

samples and s econdary

stocks f or low

- and mid

level recovery samples

Secondary stock solutions were typically prepared as per the table below:

Fortifying

Stock ID Fortifying

Stock

Concentration

(mg/L) Volume of

Fortification

(mL) Final

Volume

(mL) Stock

Solvent Stock ID Stock

Concent ration

(mg/L) Stock Use

7535A 1.00 0.500 50.0

Acetonitrile 7535 A

- 1 10.0 Calibration

standards

7535B 1.00 0.500 50.0 7535 B

- 1 10.0 Low

- level

recovery samples

1.00 5.00 50.0 7535B

- 2 100 Mid

- level recovery

samplesSmithers Viscient Study No. 14091.6101 Page 64

All primary and secondary stock solut ions were stored refrigerated in amber glass bottles fitted

with Teflon ®

- lined caps.

Mobile Phase and Liquid Reagent Solution Preparation

A 0.05% phosphoric acid in purified reagent water mobile phase solution was typically prepared

by adding 0.500 mL of concentrated phosphoric acid to 1 .00 L of purified reagent water . The

solution was mixed well using a stir bar and a stir plate for five minutes then degassed under

vacuum with sonication for ten minutes.

A 20:80 acetonitrile:purified reagent water (v:v) liquid reagent solution was typically prepared

by combining 100 mL of acetonitrile and 400 mL of purified reagent water. The solution was

mixed well using a stir bar and stir plate

.

Preparation of Calibration Standards

A confirmation of the concentratio n of the 10.0 mg/L calibration standard dosing stock was

performed after its use in this testing. It was determined that the actual measured stock

concentration of 7535A

- 1 (used to prepare the calibration standards) was 11.7 mg/L. This

discrepancy from n ominal concentration was likely due to an error during stock preparation.

Calibration standards were prepared in 20:80 acetonitrile:purified reagent water (v:v) by

fortifying with the 11.7 mg/L secondary stock solution to yield concentrations of 0.005 85

,

0.01 12 , 0.02 93 , 0.0 585, 0.0878 and 0.117 mg/L.

Sample Fortification and Preparation

Preparation of Recovery Samples:

The recovery samples were prepared by fortifying AAP medium with sorbic acid to obtain

concentrations of 0.0100, 5.00 and 100 mg/L. Eac h concentration level was prepared in

triplicate. In addition, three samples were left unfortified to serve as controls and were diluted in

the same fashion as the low concentration recovery samples. This procedure is detailed in the

following table.Smithers Viscient Study No. 14091.6101 Page 65

Sa mple ID Stock Concentration

(mg/L) Fortification

Volume

(mL) Final Volume

(mL) Fortified

Concentration

(mg/L)

Control

A, B & C NA a NA 16.0 0.00

Low

A, B & C 10.0 0.0 4 00 40.0 0.0 1 00

Mid

A, B & C 100 0.800 16.0 5.00

High

A, B & C 100 0 1.60 16.0 100

a NA = Not Applicable.

Dilution of Recovery Samples:

To minimize the potential for losses of the test substance, the aqueous test samples were not

sub

- sampled prior to dilution. The first dilution with acetonitrile was performed by the addition

of acetonitri le to the entire volume of the aqueous sample in the container in which it was

fortified to a final composition of 20:80 acetonitrile:AAP medium (v:v). The mid

- and

high

- level recovery samples were subsequently diluted into the calibration standard range with

20:80 acetonitrile:purified reagent water (v:v). The dilution procedure is outlined in the table

below.

Sample ID Fortified

Concentration

(mg/L) Sample

Volume

(mL) Final

Volume a

(mL) Sample

Volume Final

Volume b

(mL) Dilution

Factor

Control

A, B & C 0.00 16.0 2 0.0 NA c NA 1.25

Low

A, B & C 0.0100 40.0 50.0 NA NA 1.25

Mid

A, B & C 5.00 16.0 2 0.0 0.150 10.0 83.3

High

A, B & C 100 16.0 2 0.0 0.0400 50.0 1560

a Diluted with acetonitrile

. b Diluted with 20:80 acetonitrile:purified reagent water (v:v)

.

c NA = Not Applicable.Smithers Viscient Study No. 14091.6101 Page 66

ANALYSIS

Instrumental Conditions

The high performance liquid chromatographic (HPLC) analysis was conducted utilizing the

following instrumental conditions:

Column: Phenomenex Synergi Hydro

- RP 4 μm

, 250 mm × 4.6 mm

Mobile Phase (A): 0.05% phosphoric acid in purified reagent water

Mobile Phase (B): 100% acetonitrile

Gradient: Time (min.) Solvent A (%) Solvent B (%)

0.00 95 .0 5 .0

1.00 95 .0 5 .0

1 5 .00 0.00 100.0

1 9 .00 0.00 100.0

20 .00 95 .0 5. 0

Run Time: 20

. 00 minutes

Equilibratio n Delay: 7 .00 minutes

Flow Rate: 1. 0 0 mL/min

Injection Volume: 3 00 μL

Wavelength: 275 nm

Retention Time: approximately 11.5 minutes

Preparation of Standard Curve

Two sets of calibration standards were analyzed with each sample set : one set prior to ana lysis of

the recovery samples, and the second set immediately following the analysis of the recovery

samples. Injection of recovery samples and calibration standards onto the chromatographic

system was performed by programmed automated injection.

CALCULA TIONS

A calibration curve was constructed by plotting the analyte concentration (mg/L) of the

calibration standards against the peak area of the analyte in the calibration standards. The

equation of the line (equation 1) was algebraically manipulated to g ive equation 2. The

concentration of test substance in each recovery sample was calculated using the slope and

intercept from the linear regression analysis, the detector response, and the dilution factor of theSmithers Viscient Study No. 14091.6101 Page 67

recovery sample. Equations 2 and 3 were th en used to calculate measured concentrations and

analytical results.

DF x DC A (3) m b)

- (y DC(x) (2) b mx y (1)

= = + =

where:

x = analyte concentration

y = detector response (peak area) from the chromatogram

b

= y

- intercept from the regression analysis

m = slope from the regressio n analysis

DC (x)

= detected concentration (mg/L) in the sample

DF

= dilution factor (final volume of the sample divided by the original sample

volume)

A

= analytical result (mg/L), concentration in the original sample

The limit of quantitation (LOQ) was calculated using the following equation:

CNTL INST LS

INST

DF x LOQ LOQ (5) m b

- A LOQ (4)

= =

where:

A LS = mean detector response (peak area) of the low concentration calibration

standard (two injections)

b

= y

- intercept of the linear regression

m = slope of the linear regress ion

LOQ INST = limit of quantitation at the instrument

DF CNTL = dilution factor of the control samples (smallest dilution factor used)

LOQ

= limit of quantitation reported for the analysisSmithers Viscient Study No. 14091.6101 Page 68

RESULTS AND DISCUSSION

The mean recovery was 99.5 % with a sta ndard deviation of 5.02 %. The LOQ was

0.00 747 mg/L . The LOQ will vary from one analysis interval to another, since it is dependent

upon the regression of the calibration standards, the peak area of the low standards and dilution

factor of the controls. Defined limits for acceptance of quality control (QC) sample performance

in subsequent studies were set at 80.0 to 120%.

Analytical results for the recovery of sorbic acid from AAP medium (a freshwater algal medium)

are presented in Table 1A . Representative chromatograms from the analysis of a calibration

standard, a recovery sample and a control sample are presented in Figure 1A

, Figure 2A and

Figure 3A , respectively. A typical regression analysis for sorbic acid is presented in Figure 4A

.Smithers Viscient Study No. 14091.6101 Page 69



### Table 1A. Analytical results for the recovery of sorbic acid from AAP medium

during the method validation study.

Sample

ID Fort ified Concentration

(mg/L) Dilution

Factor Analytical Result

(mg/L) Percent of Fortified

(%)

Control A 0.00 1.25 < 0.00 747 a NA b

Control B 0.00 1.25 < 0.00 747 NA

Control C 0.00 1.25 < 0.00 747 NA

Low A 0.0 100 1.25 0.00982 98.2

Low B 0.0 100 1 .25 0.0100 100

Low C 0.0 100 1.25 0.0102 102

Mid A 5.00 83.3 4.64 92.8

Mid B 5.00 83.3 4.67 93.3

Mid C 5.00 83.3 4.72 94.5

High A 100 1560 103 103

High B 100 1560 104 104

High C 100 1560 106 106

Mean: 99.5

SD: 5.02

% CV: 5.05

N: 9

a Concentrations expressed as less than values were below the limit of quantitation (LOQ). The LOQ for each

analysis is dependent upon the regression, the area of the low standards and the dilution factor of the controls.

The limit of quantitation for the method validation was 0.00 747 mg/L

. b NA = Not Applicable

. Note: Results were calculated using the actual analytical (unrounded) results and not the rounded values

presented in this table.Smithers Viscient Study No. 14091.6101 Page 70



### Figure 1A. Representative chromatogram of a 0.0 585 mg/L calibration

standard used for quantification during the method validation study

with sorbic acid.Smithers Viscient Study No. 14091.6101 Page 71



### Figure 2A. Representative chromatogram of a 5.00 mg/L recovery sample

fortified with sorbic acid during the method validation study

.Smithers Viscient Study No. 14091.6101 Page 72



### Figu re 3A. Representative chromatogram of a control sample during the

method validation study with sorbic acid

.

Note: N o peak equivalent to retention time for sorbic acid

. Sorbic acid is ordinarily detected at a retention time

of approximately 11.5 minute s.Smithers Viscient Study No. 14091.6101 Page 73



### Figure 4A. A typical regression analysis for the calibration standards used to

quantitate sorbic acid in the recovery samples.

0 20 40 60 80 100 120 140 160 180

0.000 0.020 0.040 0.060 0.080 0.100 0.120 0.140Response

Concentration Linear Regression Analysis

Linear Regression Analysis

r 2

= 0.99992

y = 1400.898 x

- 1.7835

Standard

Concentration

(mg/L) Standard

Response

Area

0. 00585 6.60295

0.0117 14.45008

0.0293 39.57698

0.0585 79.97351

0.0878 120.20045

0.117 162.83325

0.00585 6.56322

0.0117 14.59786

0.0293 39.57217

0.0585 79.97565

0.0878 120.43200

0.117 162.79735