## Study Title Sorbic Acid — 96-Hour Toxicity Test with the Freshwater Green Alga, Pseudokirchneriella subcapitata, Following OCSPP Guideline 850.4500

Data Requirement


## OCSPP Guideline 850.4500

Author
Katherina A. Softcheck
Study Completed On 29 May 2015
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 ProjectID Smithers Viscient Study No. 14091.6101

Page 1 of 73

## Smithers Viscient Study No. 14091.6101 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 multinational entities under FIFRA 10(g).

Company: Brenntag North America, Inc.

CompanyAgent: Shawn P. Wiram Date: May 29, 2015 (Typed Name)

Director Safety, Health & Environment J ~



### (Title) (Signature)

## Smithers Viscient Study No. 14091.6101 Page 3



## GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
The data and report for "Sorbic Acid — 96-Hour Toxicity Test with the Freshwater Green Alga, Pseudokfrchneriella subcapitata, Following OCSPP Guideline 850.4500" were produced and compiled in acoordance with all pertinent U.S. Environmental Protection Agency (EPA) Good Laboratory Practices as set forth under the Federal Insecticide, Fungicide and Rodenticide Act (40 CFR, Part 160) and as accepted by OECD Principles of Good Laboratory Practice (OECD, 1998) with the following exceptions: routine dilution water contaminant screening analyses for pesticides, PCBs and toxic metals were conducted at GeoLabs, Inc., Braintree, Massachusetts using standard U.S. EPA procedures and are considered facility records under Smithers Viscient's Standard Operating Procedures. Since the analysis was conducted following standard validated methods, this exception had no impact on the study results.

SMITHERS VISCIENT

~ ~~ 

# Zj Katherina A. §oftcheek Vate 

## Study Director

BRENNTAG NORTH AMERICA,INC.


# $-z5 -► ~

- 

## SPongor Representative Date



## t. SpqgWbr Submitter Date

### Smithers Viscient Study No. 14091.6101 Paee 4


## QUALITY ASSURANCE STATEMENT
The study conduct, raw data and report for °°Sorbic Acid — 96-Hour Toxicity Test with the Freshwater Green Alga, Pseudokirchneriella subcapitata, Following OCSPP Guideline 850.4500" were inspected by the Quality Assurance Unit (QAU) at Smithers Viscient to determine adherence with the study protocol, amendments, laboratory standard operating procedures and the applicable GLP regulations. Dates of study inspections, inspection types, and dates reported to the Study Director and to Management are listed below.


## Inspection Date 8 March 2015 25 March 2015 18 May 2015 29 May 2015 

## Inspection Type 

### Protocol Review In life: Water Quality Data and Drafr Report Final Reported to Study Director/Mana¢ement 8 March 2015 26 March 2015 19 May 2015 29 May 2015

SMITHERS VISCIENT

'POt-. ; 

### h +rw j Zai

~ 

### f Robin Dyer Date Quality Assurance Auditor

## Smithers Viscient Study No. 14091.6101 5



## KEY STUDY PERSONNEL
The following Smithers Viscient personnel were responsible for the conduct ofthe work and reporting of the study results.

Katherina A. Softcheck Kristen L. Soucy Melissa L. Staggs Ashley Beaton
Blake Dinius Nicholas Lyons Regine Felix Huy Le Marjorie E. Dix Gina Giorgio
Heather Grove Study Director Biologist I Biologist I Assistant Biologist Biologist I Assistant Biologist Chemistry Technician II Chemistry Technician II Research Director, Chemistry Technical Report Writer Director of Ecotoxicology

## Smithers Viscient Study No. 14091.6101 Page 6



## TABLE OF CONTENTS Page GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT ..........................................3 QUALITY ASSURANCE STATEMENT ......................................................................................4 KEY STUDY PERSONNEL .......................:...................................................................................5 TABLEOF CONTENTS .................................................................................................................6 SUMMARY.....................................................................................................................................9 1.0 INTRODUCTION ..............................................................................:.................................11 2.0 MATERIALS AND METHODS .........................................................................................11 2.1 Protocol ......................................................................................................11 2.2 Test Substance ...........................................................................................I 1 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 Initiation .............................................................................................15 2.9 Test Monitoring .........................................................................................16 2.9.1 Algal Growth .............................................................................................16 2.9.2 Solution 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 DefinitiveExposure ......................:.......:...............................:..:.......:.........22 3.2.1 Evaluation of Test Conditions ...............:...................................................22 3.2.2 Analytical Results ......................................................................................23 3.2.3 Biological Results ......................................................................................24 PROTOCOL DEVIATION ...........................................................................................................27 REFERENCES..............................................................................................................................28

### Smithers 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 sorbicacid ..................................................................................................30 Table 3. Concentrations of sorbic acid measured in the exposure solutions

during the 96-hour exposure of Pseudokirchneriella subcapitata

. 

## ............ 31 Tatile 4. Cell density ofPseudokirchneriella subcapitata after 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 LowestObserved-Effect Concentration (LOEC) values for sorbic acid calculated from the results (yield) of the 96-hour toxicity test with Pseudokirchneriella .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 LowestObserved-Effect Concentration (LOEC) values for sorbic acid calculated from results (average specifie growth rate) of the 9,6-hour toxicity test with Pseudokirchneriella subcapitata ....................................36 Table 9. Section-by-section growth rates ofPseudokirchneriella 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 LowestObserved-Effect Concentration (LOEC) values for sorbic acid calculated from results (area under the growth curve) of the 96-hour toxicity test with Pseudokirchnertella 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 Pseudokirchneriella 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 ................................................................................44

## Smithers Viscient Study No. 14091.6101 Page 8

APPENDIX 1

- 

## STUDY PROTOCOL ..........................................................................................45 APPENDIX 2

- 

## ANALYTICAL METHODOLOGY ....................................................................61 Table lA. Analytical results for the recovery of sorbic acid from AAP medium

during the method validation study ............................................................69 Figure lA. Representative chromatogram of a 0.0585 mg/L calibration standard

used for quantification during the method validation study with sorbicacid

. 

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

fortified with sorbic acid during the method validation study ...................71 Figure 3A. Representative chromatogram of a control sample during the method

validation study with sorbic acid

. 

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

quantitate sorbic acid in the recovery samples. ......, ..................................73

## Smithers Viscient Studv No. 14091.6101

SUMMARY

Sorbic Aeid — 96-Hour Toxicity Test with the Freshwater Green Alga, Pseudokirchneriella

subcapitata, Following OCSPP Guideline 850.4500 SPONSOR: Brenntag North America, Inc. PROTOCOL TITLE: 96-Hour Toxicity Test with the Freshwater Green Alga, Pseudokirchneriella subcapitata, Following OCSPP Guideline 850.4500 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 Apri12015 (including recovery period) TEST ORGANISM: Pseudokirchnerfella subcapitata, inoculum - three days since previoustransfer Source - Smithers Viscient culture DILUTION WATER: Algal Assay Procedure (AAP) medium TEST CONDITIONS: 96-hour duration, a temperature range of 22 to 24 °C continuous illumination at 51 to 68 μE/m 2 /S, continuous agitation at a shaking rate of 100 rpm NOMINAL TEST CONCENTRATIONS:
TIME-WEIGHTED AVERAGE CONCENTRATIONS: 6.3, 13, 25, 50 and 100 mg/L

7, 3.9, 24, 50 and 110 mg/L
EFFECT CRITERIA: Reduction of 96-hour yield, average specific growth rate (laave) 

## and area under the growth curve (AUGC) relative to the performance of the eontrol data.

## Smithers Viscient Studv No. 14091.6101 Paee 10

RESULTS:

Based on Time-Wei hted Avera e Concentrations m/L EC50 Biological Parameter (95% Confidence Limits NOEC LOEC
96-Hour Yield (6 9 10 10 

### 3.9 24 96-Hour Average Specific 12 Growth Rate 11

— 

### 13 3.9 24 96-Hour Area Underthe 10 Growth Curve 8.5

— 

### 10 3.9 24

## Smithers Viscient StudyNo. 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 alga, Pseudokirchneriella subcapitata. Results of the study are reported as the 96-hour EC50 values 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-Effect 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 Director 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. AII original raw data, the protocol and the 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 protocol entitled "96-Hour Toxicity Test with the Freshwater Green Alga, Pseudokirchneriella subcapitata, Following OCSPP Guideline 850.4500" (Appendix 1). The methods described in this protocol meet the requirements specified in the OCSPP Guideline 850.4500 (U.S. EPA, 2012).



## 2.2 Test Substance The test substance, sorbic acid, was received on 17 February 2015 from GEI Ecology Lab, Denver, Colorado. The following information was provided:

## Smithers Viscient Studv No. 14091.6101 Paee 12

Name: sorbic acid Lot No.: 1401151 CAS No.: I 10-44-1 Purity: 99.5% Expiration Date: 14 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.
Determination of stability and characterization, verification ofthe test substance identity, maintenance of records on the test substance and archival of a sample of the test substance are the responsibility of the Study Sponsor



## 2.3 Test Organism : fhe organism used in this toxicity test was the freshwater green alga, Pseudokirchneriella subcapitata, strain 1648, class Chlorophyceae. The green alga was 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 are listed in Table 1. Representative samples ofthe dilution water source used in the preparation ofthe culture medium were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, 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 Guidelines (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 March 2015 was 0.80 mg/L.

## Smithers Viscient StudvNo. 14091.6101 PaQe 13

The U.S. EPA recommends 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 f 

## 0.1 with dilute hydrochloric acid (HCl) or sodium hydroxide (NaOH). Stock cultures were grown in 250-mL glass flasks each containing 100 mL of AAP medium. The flasks were covered with stainless steel caps which permitted gas exchange.
The stock cultures were maintained within the following conditions: a shaking rate of 100 f 

## 10 rpm, a temperature of 24 f 

## 2°C and continuous illumination at the surface of the medium providing photosynthetically-active radiation (PAR) of approximately 60 μE/m Z /S t 15%, e.g., 51 to 69 μE/m Z /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 test, the 96-hour EC50 value based on cell density was determined to be 0.091 mg Zn/L, with 95% confidence intervals of 0.071 to 0.12 mg Zn/L. Previous reference testing determined a mean EC50 value of 0.090 mg Zn/L for P. subcapitata. The EC50 value closely 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 manner 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, to 7.5 f 

## 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 were 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 mixed with a stir plate and Teflon -coated 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 solutions were prepared by diluting the 100 mg/L solution as follows:

Primary Stock Solution Volume of Primary Final Dilution Exposure Solution Concentration

. Stock Used Volume with Concentration (mg/L)

. (1,) AAP Medium (mg/L) (L) 100 0.50 NA 100 100 0.50 1.0 50 100 0.25 1.0 25 100 0.13 1.0 13 100 0.063 1.0 6.3 NA = Not Applicable

### Smithers Viscient Study No. 14091.6101 Page 15



## The 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 under 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 Guideline 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 appropriate 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 order 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 was prepared. This flask, which was not inoculated with algae, was analyzed at 96 hours 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 inoculum of Pseudokirchneriella subcapitata cells, at a density of approximately 63 X 

## 10 4 cells/mL, was aseptically introduced into each flask. This inoculum provided the required initial (0-hour) cell density of approximately 1.0 X 

## 10 4 cells/mL.

### Smithers Viscient Study No. 14091.6101 

## 16



## 2.9 Test Monitoring

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 were determined using a single sample removed from each flask. One or more hemacytometer fields, each 0.10 x 0.10 cm in surface area and 0.010-cm deep and containing 0.00010 mL of culture, were examined until at least 400 algae cells or four fields were counted. 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

- included. 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 ofthe four replicate vessels ofthe nominal 100 mg/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 concentration 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 was microscopically examined every other day to determine if cell growth had resumed. The subculture was discontinued after a substantial increase in cell density (e.g., > l OX) was observed. If an increase in cell density was not observed after nine days, the vessel was terminated.

## Smithers Viscient Study No. 14091.6101 Page 17

2.9.3 Test Conditions 

## The test was conducted in an environmental chamber designed to maintain the test conditions specified in the protocol: a temperature of 24 f 

## 2°C, continuous light intensity at a photosynthetically-active radiation (PAR) range of 51 to 69 μE/m Z /s (approximately equivalent to 3700 to 4900 lux), see Section 2.3. Two orbital shaker tables provided a shaking rate of 100 f 

## 10 rpm.
Temperature was measured continuously with a V WR minimum/maximum thermometer located in a flask of water adjacent to the test 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 photometer 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 V WR Traceable 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 new 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 exposure solutions, the pH of the solutions was also measured at the 24-, 48-, 72- and 96-hour exposure periods. Measurements at test initiation were conducted on the test solutions remaining after the individual test flasks had been filled. The pH of the exposure 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 were respectively composited for pH measurements. Test solution pH was measured with a Yellow Springs Instrument (YSI) Model pH100A 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 initiation (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 removed from the additional replicate flask of the nomina125 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 solutions were centrifuged to remove algae cells to determine the dissolved concentration 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 calculated for eacli treatment level as follows:

CMa 

### =[(CDU — CD4XIn (CDO)— 

### In(CD4)) x 4 Days] / Total Days



## where: C rv ,, a =

CDO — CD4 = 

## ln = Total Days = time-weighted average concentration

measured concentration on day 0 measured concentration on day 4 natural log total number of days, 4

## Smithers 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=Y_fWFx(C-C t J]/yWF where:

Var 

## = variance WF 

## = weighting factor, defined as the time between intervals C = concentration at each interval C w „ 

## = time-weighted average concentration
Quality control (QC) samples (three at 0-hour and six at 96-hour) were prepared with fresh AAP medium at nominal concentrations approximating the test concentration range and remained with the exposure solution samples throughout the analytical process. The three QC samples at 0-hour were processed in the same manner 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 substance 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 system 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 t 5.02% from AAP medium (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 StudyNo. 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. Means and standard deviations for cell density for the treatments 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:

ln X t - ln X O t t - t o

where: μ = specific growth rate (days )

]n = natural logarithm Xo = initial cell density in cells/mL at time to (days) X r = cell density at the specified time interval in cells/mL to = 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 (p, e ) for each replicate was calculated for the entire span of the test period (e.g., 0 to 96 hours) and this data was used for statistical analyses. Per the study guideline, the 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 Studv 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= No, according to the formula:

N, — No + N, + N Z - 2N o N

~ 

## ., +N

~ 

## -2N o A= 2 xt 2 x(tZ t)+...+ 2 x(t

where: A= area under the growth curve (units: x 10" cells x days/mL)

No = calculated number of cells/mL at time to Ni = measured number of cells/mL at t, N„ = measured number of cells/mL at time t„ t, = time of first measurement after beginning of test t„ = time of n` h measurement after beginning of test n= number of ineasurements taken after test initiation
Based on the results of the statistical analysis performed for 96-hour yield, average specific growth rate and 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) was 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-parametric statistical test, e.g., Jonckheere-Terpstra'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% level of certainty was applied.
The 96-hour EC50 values (concentrations of test substance which reduced yield, average growth rate and area under the growth curve by 50%) and the 95% confidence limits 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.

CETIST"' Version 1.8 (Ives, 2013) was used to perform all statistical analyses.



## 3.0 RESULTS

3.1 Preliminary Exposure A preliminary range-finding exposure was conducted at Smithers Viscient at nominal concentrations of 0.10, 1.0, 10, 100 and 1000 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, 170.50, 187.50, 1.00 and 1.13 x 10 4 cells/mL, respectively. The control averaged 186.33 x 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 definitive 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 a$er 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 concentration. 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 63 and 13 mg/L treatment levels and remaining largely unchanged from 

# a pH of approximately 5 in the three high

### Smithers Viscient Study No. 14091.6101 Page 23



## treatment 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 to photosynthesis by the algae. Conductivity of the treatment and control solutions ranged from 90 to 120 μS/cm, increasing slightly as the concentration oftest substance increased. Continuous temperature monitoring established that the temperature ranged from 23 to 24 °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 throughout 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, measured concentrations of each exposure solution closely approximated the nominal concentrations, ranging from 99 to 110% of nominal concentration. The measured concentrations in the 6.3 and 13 mg/L nominal treatment levels declined after 96 hours, and were 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 concentrations 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 27% to 110% of nominal concentrations and defined the treatment levels tested as 1.7, 3.9, 24, 50, and 110 mg/1..
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 24 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 107% 

# (N = 6) of the nominal fortified levels (3.00, 25.0 and 100 mg/L). Analysis of the centrifuged quality control samples resulted in measured concentrations which ranged from 97.8 to 104% (N= 3) ofthe nominal fortified levels (3.00, 25.0 and 100 mg/L). Based on these results, it was established that the appropriate 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 X 

## 10 4 cells/mL. Cell densities in the 1.7, 3.9, 24, 50 and 110 mg/L treatment levels averaged 219.88, 216.79, 2.81, 1.38 and 0.81 x 10' cells/mL, respectively.
Yield is presented in Table 5 and illustrated in Figure 3. The 96-hour yield in the oontrol averaged 218.08 x 10' 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 -0.19.x 10 4 cells/mL, respectively. Based on the results of Shapiro-Wilk's and Bartlett's Equality of Variance Test, this data set passed the requirement for normality and homogeneity of variance; 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 yield were determined to be 3.9 and 24 mg/L, respectively. Based on time-weighted average concentrations, the 96-hour EC50 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 EC50 values for yield are presented in Table 6.

## Smithers Viscient Studv No. 14091.6101 Paee 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 1.36 days ', respectively. The 0- to 96-hour average specific growth rate in the 1.7, 3.9, 24, 50 and 110 mg/L treatment levelsaveraged 1.36, 1.36, 0.26, 0.05 and -0.11 days ', respectively. Based on the results of ShapiroWilk's and Bartlett's Equality of Variance Test, this data set passed the requirements 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 mg/L, respectively. Based on the time-weighted average concentrations, the 96-hour EC50 value for average 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 EC50 values for average specific growth rate are presented in Table 8. Section-by-section growth rates are presented in Table 9.
Area 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 X 

## days/mL. Total AUGC in 1.7, 3.9, 24, 50 and 110 mg/L treatment levels averaged 222.23, 227.30, 3:66, 0.88 and 0.33 x 10 4 cells x days/mL, respectively. Based on the results of Shapiro-Wilk's and Bartlett's Equality of Variance Test, this data set 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 mg/L, respectively. Based on the time-weighted average concentrations, the 96-hour EC50 value for AUGC was determined to be 10 mg/L, with 95% confidence limits of 8.5 to 10 mg/L. The 96-hour NOEC, LOEC and EC50 values for AUGC are presented in Table 11.
The subculture used to evaluate the algistatic/algicidal properties of the test substance was initiated 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 the

## Smithers Viscient Study No. 14091.6101 Page 26

recovery phase had a pH of 

## 7.6 prior to inoculation with cells from the ] 00 mg/L nominal treatment level. The temperature during the recovery phase was maintained at a range of 

## 22 to 24 °C. The photosynthetically-active radiation (PAR) of 

## the test area ranged from 56 to 68 μE/m Z /S over the nine-day test period. A$er nine days, a cell density of 

## 4.00 x 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 substance has an algistatic, rather than algicidal effect on the growth ofPseudoklrchneriella subcapitata 

## at a nominal concentration of 100 

## mg/L.
The protocol requires the following criteria per the OCSPP Guideline 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 test are summarized below and presented in the following table:

OCSPP Acceptance Criteria for Control Study Results

0- to 96-hour average yield CV < 15%: 16% 0- to 96-hour average specific growth rate CV <_ 15%: 3.0% 96-hour cell density in the control > 100 x 10 ° cells/mL: 219.08 x 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 x 10 4 cells/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 1 /4) 

## only slightly exceeds the criterion (15%) and the concentration-response and NOEC, LOEC and EC50 values for all endpoints were clearly defined. Therefore, this study is considered acceptable and the results accurately reflect the sensitivity of Pseudokirchneriella subcapitata 

## to sorbic acid under testing conditions.

## Smithers Viscient Studv No. 14091.6101 27



## PROTOCOL DEVIATION
No deviations from the protocol occurred during this study.

## Smithers Viscient Studv No. 14091.6101 Page 28 

## REFERENCES
ASTM, 2004. Standard Guide for Conducting Static Toxicity Tests with Microalgae. Standard E-1218-04, American Society for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, Pennsylvania 19428, pp. 293-306. Ives, M., 2013. CETIS, Comprehensive Environmental Toxicity Information SystemT"'. User's Guide. Tidepool Scientific So$ware, McKinleyville, California. Miller, W.E.; J.C. Greene and T. Shiroyama, 1978. The Selendstrum 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 Interpolation 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. OECD, 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)17. 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. Test 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 850.4500: Algal Toxicity EPA 712-C-005. U.S. Environmental Protection Agency. Washington, D.C.

## Smithers Viscient Studv No. 14091.6101 Page 29

Table l. 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

MgC1 2 •6H a ,0 12.16 mg/L CaCl z •2H 2 0 4.41 mg/L MgSO 4 •7H2O 14.7 mg/L K 2 HPO 4 *3H 2 0 1.368 mg/L NaHCO 3 15.0 mg/L

. . . 

### H 3 130 3 185.5 pg/L Na 2 Se0 4 a 1.88 pg/L

MnC1 2 •4H z 0 415.4 μg/L ZnC1 2 3.270 μg/L CoCl z •611 2 0 1.43 μg/L

CuCl z •2H z 0 0.012 pg/L Na 2 Mo0 4 •2H 2 0 7.26 μg/L FeCl 3 •6H z 0 160 gg/L Na 2 EDTA•2H 2 0 300 pg/L ' Additional nutrient required, personal communication. Dr. R.R.L. Guillard, June 1991
If required, pH was adjusted to 7.5 t 0.1 with dilute sodium hydroxide (NaOH) or hydrochloric acid (HCI).

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



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

during the 96-hour exposure of 

## Pseudokirchneriella subcapitata to sorbic acid.

Nominal Concentrafion (mg/L) 0 Hour 24 Hour pg
48 Hour 72 Hour 96 Hour Conductivity (μS/cm) 0 Hour

Control 7.2 7.3 7.4 9.1 9.4 91
6.3 6.7 7.3 7.5 9.1 93 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/Mazimum Temperature ( ° C)
0- 24 Hours 24 - 48 Hours 48 - 72 Hours 72 - 96 Hours
23/24 23/24 23/24 23/24

Light Intensity (PAR)"
Units 0 Hours" 24 Hours 48 Hours 72 Hours 96 Hours
μE/m 2 /S 51 - 66 51 - 63 53 - 62 52 - 64 52 - 64 ' PAR = photosynthetically-active radiation ° The light intensity (lux) of the test area at test initiation ranged from 4300 to 4900

## Smithers Viscient Studv No. 14091.6101 Paee 31

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

during the 96-hour exposure of 

## Pseudokirchneriella subcapitata.

Nominal Measured Concentration (mg/L) Percent Time- Percent Concentration ( 

### mg/L) Decline from Weighted of 

### Nominal 0 Hour 96 Hours °

~ 

### 0 Hour Average"` Control <0.59 <0.027 NA` NA NA

6.3 6.4 0.17 97 1.7 27


### 13 13 0.56 , 96 3.9 30
25 25 23/24 r 7/5 f 24 98
50 51 49 3 50 100
100 110 100 6 110 110

Uncentrifuged Uncentrifuged Centrifuged QCs#1 3.00 2.94 2.94 3.00 (98.9) (98.0) (97.8)
QC#2 26.1 25.8 25.8 

### 25.0 (104) (103) (103)
QC#3 107 105 104 

### 100 (107) (105) (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. ` Crna= KCoo—CDa)/(In(Coo)—In(CDa))x4Days]/TotalDays ° 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. ` NA = Not Applicable. f 

### Result of the additional sample without algae present analyzed at 96 hours of exposure to determine biological uptake/degradation. 9 QC = Quality Control sample. Percent recovery presented in parentheses.

## Smithers Viscient Study No. 14091.6101 

## 32

Table 4. Cell density of 

## Pseudokirchneriella subcapitata after 24, 48, 72 and

96 hours of exposure to sorbic acid.

Time Weighted Average Concentration (mg

~ ) Replicate 24 

### Cell Density (x 10 4 cells/mL) Observation Interval (Hours) 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) ° 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?5 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)

. 24 A 2.00 2.00 1.75 2.50 B 3.50 2.25 2.75 3.75 C 2.25 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) 1.50(0.46) 1.19(0.43) 1.38(0.75) 110 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) 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 0-24 Yield (x 10" cells/mL) Observation Interval (Hours) 0-48 0-72 0-96 Percent Inhibition'" ( 0 /6)
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)" 4.56(1.89) 26.19(8.51) 73.63(10.53) 218.08(35.25) NA` 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 24 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" (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) 0.50(0.46) 0.19(0.43) 0.38"(0.75) 100 110

~ ~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 4 (0.66) 100

e Percent inhibition relative to the control. b Mean, standard deviation (SD) and pereent inhibition were calculated from original raw data, not from the rounded values presented in this table. ` NA = Not Applicable. ° Significantly reduced compared to the wntrol, based on Williams' Multiple Comparison Test.

## Smithers Viscient Study No. 14091.6101 Paee 34

Table 6. EC50, No-Observed-Effect Concentration (NOEC) and LowestObserved-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 EC50 NOEC LOEC 

### EC Value (mg/L): 10 3.9 24 95% Confidence Limits: 6.9 — 10

## Smithers Viscient Study No. 14091.6101 Page 35



## Table 7. Average specif• ic growth rates 

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

Time Weighted Average Concentration

_ (mg2) Replicate 0-24 (0.919 days) Averaee Specific Growth Rate (davs 1)

Observation Interval (Hours)

~ 0-48 0-72 (1.94 days) (2.89 da s) 0-96 (3.95 davs) percent

Inhibition° e

(0 ~ O)
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)" 1.82(0.37) 1.68(0.17) 1.49(0.05) 1.36(0.04) NA` 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) 1.50(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 24 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 4 (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 ° (0.14) 96 110 A -0.31 0.00 -0.24 407

B 0.24 0.47 0.28 0.14 C 0.00 0.00 0.00` -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"(0.21) 108
' Percent inhibition relative to the control. ^ Mean, standard deviation (SD) and percent inhibifion were calculated from original raw data, not Gom the rounded values presentin this table. ` NA =Not Applicable. a Significantly reduced compared to the wntrol, based on Williams' Multiple Comparison Test. ` Growtlf rate values cannot be mathematically calculated from a cell density of zero. Therefore, a grow[h rate value of zero is used for any replicate with zero cell density.

.

## Smithers Viscient Study No. 14091.6101 Page 36



## Table 8. EC50, No-Observed-Effect Concentration (NOEC) and LowestObserved-Effect Concentration (LOEC) values for 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 EC50 NOEC LOEC
EC Value (mg/L): 12 3.9 24 95% Confidence Limits: 11 — 13

## Smithers Viscient Study No. 14091.6101 Paee 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 Section-by-Section Growth Rate (days') Average Replicate Observation Interval (Hours) Concentration 0-24 24-48 48-72 72-96 (mg/L) (0.919 davs) (1.03 davs) (0.949 da s) (1.05 davs) 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)" 1.82(0.37) 1.55(0.19) 1.1(0.44) 1.02(0.20) 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) 1.50(0.16) 1.13(0.50) 0.93(0.19) 24 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) 0.90(0.33) -0.46(0.45) 0.14(0.28) 0.49(0.30) 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 0.00 0.00" O.oO"

D 0.24 0.00 -0.24 -0.66 Mean (SD) 0.04(0.26) 0.24(0.32)

° Mean and standard deviation (SD) were calculated from original raw data, not from the rounded values present in this table. ^ Cmowth rate values cannot be mathemafically 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 38



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



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

Time Area Under the Growth Curve (x 10 cells x davs/mL) Weighted Observation Interval (Nours) Percent Average Replicate Inhibitioni b Concentration 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 23190 C 1.26 12.68 52.06 175.50 241.51 D 1.49 9.99 42.09 137.75 191.33 Mean(SD)" 2.10(0.87) 15.76(5.22) 47.34(4.62) 153.86(19.77) 219.05(22.99) NA` 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 15731 238.03

B 3.56 25.11 57.63 18141 269.72 C 1.03 9.87 46,01 147.33 204.24

. D

. 1.95 1139 48.26 135.20 197.20 Mean (SD) 2.50(1.22) 17.55(7.84) 51.43(5.28) 155.81(20.5) 227.30(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"(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) 0.30(0.31)

~ ,. . 0.88"(0.62) 100

.. 110 A -0.11 -0.13 -0.24

~ . ~ ~. - -0.40 -0.88

. .

. B 0.I1 0.90 1.30 -

~ 1:05 3.37

C 0.00 0.00 -0.47 -0.92 -1.40 D O.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 4 (2.14) 100

° Percent inhibition relative to tlre control. e Mean, standard deviation (SD) and percent inhibition were calculated from original raw data, not from the rounded values presented in this table. ` NA=NotApplicable. d Significantly reduced compared to the control, based on the Jonckheere-Terpstra's Step-Down Test.

## Smithers Viscient Study No. 14091.6101 39

Table 11. EC50, No-Observed-Effect Concentration (NOEC) and LowestObserved-Effect Concentration (LOEC) values for sorbic acid calculated from results (area under the growth curve) of the 96-hour toxicity test with 

## Pseudokirchueriella subcapitata.

96-Hour Results Based on Time-Weighted Average Concentrations 

### EC50 NOEC LOEC EC Value (mg/L): 10 3.9 24 95% Confidence Limits: 8.5 — 10

## Smithers Viscient Study No. 14091.6101 

# m

Figure 1. Relationship between nominal and time-weighted average

concentrations of sorbic acid during the 96-hour static toxicity test exposing 

## Pseudokirchneriella subcapitata.

100 - - - - Theoretica3 iinetvhere time

iveighted average equals nominal concentrations Logregression oFthe actual time weiglhted avuage versus nominal concentrations



# l

10:0 100.0 

### Log Nonrinal Concentration (nig(L) ~ 

## F C

~

~ 0 0 

## W

~ 

## > a

~ 

### 3 ~ H 0 a 10



### 1 I
~ 

### I.0

### Smithers Viscient Study No. 14091.6101 Page 41



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

## Pseudokirchneriella

subcapitata exposed to sorbic acid.



## m 15

.-, 

## a

~ 

### 14

~ ~ V

~ 

### X 13

..~

~

. N

~ ~ 

## Gl 12 a v U en 0 

### `a 11

~ 

## d 

# z lo

24 48 72 96


## Time (Hours)

## Smithers Viscient Study No. 14091.6101 Paee 42

Figure 3. Yield for 

## Pseudokirchneriella subcapitata exposed to sorbic acid.

245



### 195



### a

~ 145 ti


### ~ y V O 

### X v 'b



### ~ 

### 95

45

-5 Control 1.7 3.9 24 50 110



## Time Weighted Average Corlcentration (mglL)

*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 rates (0- to 96-hour) for 

## Pseudokirchneriella

subcapitata exposed to sorbic acid.

1.40

1.20

1.00

~ ti a 0.80 u

~ Y
~

~ 

# ~ 0.60 ) 0 ry V 0.40

r

r *



### Control 1.7 3.9 24 50 110



### Time Weighted Average Concentration (mg/L) 0.20

M



### *Significantly reduced compared to the control, based on Williams' Multiple Comparison Test.

## Smithers Viscient Study No. 14091.6101 Pa€

.,



## Figure 5. Area under the growth curve for 

## Pseudokirchueriella subcapitata

exposed to sorbic acid.

250

200 

# .Y T b v , 150



## ~
~
~ ~ 

# ~ 100


# ~ 0

~. 

### 50 

# i

~

~ 

# 1

~ 

# 1

~

~

~ 

# ~ ~ ~



### * *



### Control 1.7 3.9 24 50 110 Time Weighted Average Concentration (mg/L)

*Significantly reduced compared to the control, based on Jonckheere-Terpstra's Step Down Test.

## Smithers Viscient Study No. 14091.6101 45



## APPENDIX 1 - STUDY PROTOCOL

## Smithers Viscient Study No. 14091.6101 Paee 46 

# o SMITHERS V 1 S C I E N T

TESTPROTOCOL

Title; 98-HourToxicityTestwiththeFreshwaterGreen Alga, Pseudokin:hneriella subcepitata, Following

OCSPP Guidelfne 850.4500 Data Requirement(s): OCSPP Guideline 850.4500 TestSubstance(s): Name: SorbicACid Purity: 99.5% Batch or Lot #: 1401151 Analytical Standard: Name: Purity: Batch or Lot #: Study Sponsor: Address:

- Study Monitor: Email I Phone Number: Sponsor ProtocollProject No. (when applicable): Brenntag North America, Inc. 3111 North Post Road Indianapolis, Indiana 46226 Shawn P. Wiram SWiram@brenntag.com / (317) 454-7226 NA Testing Facility: Smithers Viscient

.

790 Main Street Wareham, Massachusetts 02571 Stutly Dlrector. Katherina A. Softcheck " Smithers Visclent Study No.: 14091.6101
Test Concentretions: Proposed Experimental Dates , Start: March 2015 Terminatfon: Apri12015

~~ 

### Z•ZN•ri S o sor Approval Date 

### ~f/~2Q~f 1~~~ aS ~~ aOlS .-- 

### ~ Study Director Signature Stutly Initlation Date

SmiMrs NWfard LLC 790 Mein Streetll^Jereham, td407571 1 p5Oa•2852550 vraewsndhers

~ clenlcom 2000OUOYenbushROaelSnewC,amp,NC279491PSM.376.0141

## Smithers Viscient Study No. 14091.6101 Paee 47

96-Hour Toxicity Test with the Freshwater Green Alga, Pseudokfrchneriefia subcapitata, Following OCSPP Guideline 850.4500

1.0 OBJECTIVE The objective of this test is to determine the effect of a test substance on the growth of a freshwater green alga. Pseudokirchnerielta subcapitata, formerly Setenastrum capricomutum, under static cond'Rlons, The endpoints of this study are biomass yield, average specific grovAh rate and area under the growth curve, calculated from Gell density data. ResuHs will be reported as the 96-hour EC50 values, as well as 96-hour No-Observetl-Effect Concentration (NOEC) antl Lowest-Observed-Effect Concentration (LOEC) values for each endpoint. The EC value is defined as the test substance concentration that will inhibit yield, growth rate, or area under the growth curve by 50% as compared with the control. Additionally, a recovery phase wlll be initfated after 96 hours of exposure to detennine if the test substance effeds are algistatic or .algicltlal. The methods described in this protocol meet the objectives speciried in the U.S. EPA OCSPP Test Guideline, OCSPP 850.4500 Algal Toxicity (U.S. EPA, 2012).

2.0 MATERIALS AND METHODS . 2.1 Chemical Svstem

.

2.1.1 Test Substance Upon arrival at Smithers Viscient, the test and reference substance(s) wili be received by the Test Material Center. Records will be maintained in accordance with GLP requirements, and a Chain-of-Custotly established. The condilion of the eMernal packaging of the test substance will be recorded and any damage noted. The packaging will be removed, the primary storage container inspected for leakage or damage, and the condition recorded. Any damage will be reported to the Sponsor and/or manufacturer. Each sample will be given a unique sample ID number and stored'under the conditions specified by the Sponsor or manufacturer. The foilowing information should be provided by the Study Sponsor, if applicable; test substance lot or batch number, test substance puriry, water solubility (pH and temperature of solubility determination), vapor pressure, storage stability, methods of analysis of the test substance in water, MSDS, and safe hantlling procedures, end a verfietl expiration or reanalysis date. 2.1.2 Test Substanee Concentretion SelecOon A preliminary test will be conducted generally using five widely spaced test concentrations with two replicate vessels per treatment and control. The highest test concentration should not exeeed the water solubility of the test substance or 1,000 mg/L. The de0nitive test conoentratlons will be selected based on the results of the preliminary test and in consultation with the Study Sponsor. Test concentrations will be selected to produce a sufficient response to determine EC50 values. Atlditionally, an attempt will bemade lo define a No-Observed-Effect Conceritration (NOEC) and Lowest-ObservedEffect Concentration. (LOEC) for the endpoints stated in Section 3.0. Based on the Page2of14

## 48

Smithers Viscient Studv No. 14091.6101

concentration-response, and nature of the endpoints,(yield, growth rate and area under the growth curve), it may not be possible to attain each of these vatues. A negative control,.untreated algal growth medium, will be included in the test. At least five concentrations and a control will be tested. The dilution factor between test concentrations will be preferably two, but will not exceed four, unless the prellminary test indicates the concentration-response is shallow and a wider dilution factor is needed to define the test endpoints. Test substance concentrations wlll be corrected based on the purity of the test substance and expressed on a weight per volume basis (e.g., mg/L). 2.1.3 Limit Test If the highest concentration tested In the preliminary exposure (equivalent to the maximum labei rate as though it were applied to a 15-cm water column, 1,000 mg/L or the limit of water solubllity) results iri growth inhibition of less than 50'/o and indicates the test substance has no toxic effects, a single concentration (limit test) de5nitive test may be conducted. In a Iimit test, if the test substance is a pes8cide, it will be tested at a single concentration, equivalent to or slightly above the maximum label rate applied to a 15-cm water column (e.g., 1 Ib/A = 0.734 mg/L, U.S. EPA, 1986). For other test substances, a maximum concentration of 1,000 mg/L or the maximum solubility concentration will be tested. A negative control (algal growth medium) vrill also be included in the test. 2.1.4 Solvent Control An organic solvent (DMF) may be used as a carrier to solubilize the test substance. In such a case, the concentration of solvent in the test solutions will be equivalent in each treatment level and will not exceed 0.10 mL/L. A solvent control will be included in the test, which consists of untreated algal growth medium plus the concentration of solvent that occurs in the test solutions. An organic solvent will not be used'rfthe test substance is a formulation. 2.1.5 Stock Solution Preoaration The test substance will be weighed on an analytical balance for which a calibralion log is maintained. A Chemical Usage Log wlll atso be maintained in which tlie amount, the date, the intended use and the usets iniGals are recorded each time the test substance is used. A dosing stock solution v+ill be prepared prior to exposure initiation by dissolving an appropriate amount of test substance in algal growth medium or organic solvent. If the test substance is highly acidic and reduces the Initial pH of the stock solution below 5.0, appropriate adjustments to pH may be made if deemed necessary by the Stutly Director. Inilial measurement of the stock solution will be made a@er preparation during the rangefinder, including possibte pH adjustment. During the definitive test, if the selected test concentratlons are in a range expected to elicit similar pH changes, the pH of the stock solution will be measured after preparation to determine if adjustment is warranted and the pH of the test solutions will be measured in-one replicate of each treatment and the control daily thereafter (see Section 2.5.2).

Page 3 of 14

## Smithers Viscient Study No. 14091.6101 Page 49

2.1.6 Test Solution Prenaration Each test substance wncentra6on will be prepared by diluting an appropriate aliquot of the stock solution to volume (e.g., 1000 mL) directly into algal growth medium in an intermediate vessel and mixing thoroughly. Test vessels will be conditioned by rinsing with the appropriate test soiution. The test solution will then be aliquoted among the replicate test vessels. The solu0on remaining in the intermediate vessel will be used to conduct initial water quality measurementa (see Section 2.5.2). Based on OCSPP 850.1000 guidance, stock solution(s) in algal growth medium andlor test solutions may be flitered prior to use if undissolved test substance is observed after preparation or'rf solubility Is unknown at the nominal concentrations to be tested.
2.2 Test Svstem 2.2.1 S ecies Pseudokirchnedetla subcapitata, formerly Se/enastrum capricomufum will be the alga used in this study. Algai cultures are maintainetl at Smithers Viscient, and purchased from a commercial supplier on an as•needed basis. The particular strain of the test species will be identified In the raw data and the final report. 2.2.2 J ustification of Test System Pseudokirchnedelia subcapitata has been selected from the list of candidate species published in the guideline referenced above. The test organism is sultabie for toxicity testing based on its sensitivity to chemical challenges and the extensive data base for this common test species. 2.2.3 Origin and Acclimation The test organism, Pseudoklrchneriel/a subcapltata, will be obtained from laboratory cultures maintained at Smdhers Viscient. Cultures will be maintained In AAP medium (Table 1) in an environmental chamber at contlitions slmilar to testing conditions.Cultures will be transferred twice weekly Into fresh medium to provide three- to sevenday-old cultures for test inoculations. Cultures will be maintained under the required test

. conditions for at least the period of Gme from the Iast transfer.
2.2.4 Reference Testina Pedodically (approximately 2X per year), a reference test will be conducted with Pseudokirchnedella subcapitata from the (aboratory culture. The reference substance will be zinc chloride. The results of the reference test will be used to establish the reliabilHy of the test conditions. . 2.3 PhvsicaLSystem
" 2.3.1 Test Vessels Test vessels will be 250-m1 Edenmeyer flasks fitted with stainless steel caps that permit gas exchange. To provide the proper surface-to-volume ratio, the amount of solution In Page 4 of 14

## Smithers Viscient Study No. 14091.6101 Page 50

each 250-mL flask will be 100 mL. Each test vessel will be labeled with the test concentration, replicate, test species and study number. 2.3.2 Glassware Preuaration AII glassware used in testing will be thoroughly washed with detergent and rinsed with tap water. This will be followed by rinsing with a 10% solution (by volume) of nftric edd followed by a rinse with acetone, and a final dnse with deionized water. Test vessels, e.g. Erlenmeyer flasks and caps, wlll be autoclaved before use. The cleaned glassware will be stored in closed cabinets. 2.3.3 Replicatlon and Control of Bias Four repllcate flasks will be established for each treatment and contml. Additlonal replication may be needed for analytical purposes. Test flasks wlll be randomly positioned based on computer-generated random numbers, on an orbital shaker table in an environmentally-controlied chamber. The test flasks will be assigned new rantlom positions after each observation interval. " 2.3.4 Estimate of Bloloaical Uatake/Dearadation In order to estimate the impact the presence of algal biomass has on the test substance concentration, an additional replicate will be initlated at an intermediate test concentration. Thls vessel will not be inoculated with algae. The solution will be analyzed at exposure termination and the results compared to the analytical result of the equivalent concentration that contained algae. . 2.3.5 Algal Growth Medium The growth medium to be used in this stutly will be Algal Assay Procetlure (AAP). Nutrient stock solutions used in the prepamtion of AAP medium will be prepared by adding appropriate amounts of nutrients to sterile, deionized water. The nutrient stock solutions will be stored in amber glass bottles in the dark at approximately 4'C to minimize potential photochemical changes, and will be renewed at least every six months. The nutdent stodc solutlons will be added to sterile, deionized water and the pH of each batch of inedium will be adjusted, if necessary, to pH 7.5 t 0.1 with dilute hydrochloric acid or sodium hydroxide before use. Periodic analysis of representative samples from the water source will be conducted using U.S. EPA standard methotls (ASTM, 2007) in accordance with 8mithers Viscient's standard operating praoetlures, to ensure the absence of potential toxicants, including pesticides, PCBs and selected toxic metals, at concentrations which may be harmful to the test organism. The concentration of total organio- carbon (TOC) will be measured in a representative sample of AAP medium on a monthly basis. Dilution water wlll be considered acceptable for testing if the TOC is <2.0 mg/L.

Page 5 of 14

## Smithers Viscient Study No. 14091.6101 Page 51

2.4 Testinu Procedure 2.4.1 Inoculation Algae will be transferred aseptically from the stock culture to each test vessel, with the inoculum volume calculated to yieid an initial culture density of approximately 1.0 x 10 4 cells/mL. The total inoculum volume should typically be Iess than 2 mL. 2.4.2 Temoerature ' The test solution temperature will be maintalned at 24 t 2'C by controlling the air temperature within the environmental chamber. Test solution tempemture wiil be continuously monitored with a minimum/maximum thermometer In an additional vessel contalning water placed adjacent to the test vessels. Minimum and maximum temperatures will be recorded daily. 2.4.3 Photoneriod The test vessels will be maintained under lighting consistent with OCSPP 850.4500 guideline requirements: continuous fluorescent illumination sufficient to provide photosynthetically-active radiation (PAR) of approxlmately 60 pPJm 2 1S t 15%, i.e., 51 to 69 pFJm'/S using fluorescent bulbs positioned above the test area. This range is equivalent to approximately 4300 lux, e.g., 3700 - 4900 lux: Radlation (PAR) of the test area will be measured at the solution surface and monitored daily. Light intensity (lux) of the test area will be measured at initiation. 2.4.4 Aaitation

. Test vessels will be agitated cohtinuously during the exposure using an orbital shaker table maintained at 100 t 10 rpm. The agitation rate of the orbital shaker(s) wiil be monitored daily. 2.4.5 Exnosure Initiation and Terminatio

- The test will be initlated when all test and control s6lu5ons have been inoculated with algae. The exposure phase will be terminated following the 96-hour observation interval and a recovery phase may be initiated (see Section 2.5.4). 2.5 Samolinp and Observations 2.5.1 Samolinn and Analysis of Test Solutions At the Sponsor's request, algal medium samples of an appropriate volume will be taken from each treatment and the controls on the day of exposure initiation and on the day of termination for analysis of test substanoe concentration. At exposure initiation (0-hour), a sample from each test solution will be collected prior to division into replicate vessels. At exposure termination (96-hours), test solution samples wili be collected from the composite of replicate test vessels from each test concentration and control. The test vessel initiated to estimate the biological impact of algal growth on the test substance will also be sampied at exposure termination (see Section 2.3.4). In order to determine the dissolved concentratlon, samples removed at 96 hours will be either centrifuged or Page 6 of 14

## Smithers Viscient Study No. 14091.6101 Page 52

filtered to remove algal biomass prior to determination of test substance concentration, as described in OCSPP 850.4500 guideline. The effects of biomass removal by centfifugation or flttration on test substance concentrations will be discussed in the final report. A sample of the stock solution(s) used to prepare the test solutlons will be sampled and analyzed for test substance concentration if deemed necessary by the Study Director. - A minimum of three quality control (QC) samples will be prepared at each sampling

- Interval, stored if necessary and analyzed with the set of study samples. The OC samples will be prepared in algal growth medium at test substance concentmtlons slmilar to the treatment level range. At exposure termination (96 hours), three additional QC samples will be prepared and processed (i.e, centrifuged or fiHered) in a similar manner as the 96 hour exposure solution samples. Results of these analyses indicale the accuracy of the analytical methotl for measudng test substance concentration at each sampling period. The analytical method used to measure test substance conoentrations in the exposure solutions will be validated by Smithers Viscient at the expeoted nominal concentration range prior to exposure initiation. 2.5.2 Water Qualitv Measurements The pH in the test solutlons will be measured in representative samples of each test solution concentration and control(s) at exposure initiation and termination. In addition, conductivity in the test solutions will be measured at exposure Initiation. At exposure initiation, water quality will be measured in the bulk solutions in the intermediate vessels. At exposure termination water quality will be measured in a composite sample of the replicate solutlons for each treatment level and control(s). 2.5.3 Bioloaical Observations and Monitoring Algal density will be monitored in each test vessel at 24, 48, 72, and 96 hours after the start of the exposure. Algal density will be determined by cell counts using a Neubauer Improved hemacytometer and a compound microscope. Generally, one sample will be taken from each test vessel, and one count will be made on each sample. On occasion when a cell count appears unusually high or low relative to others, a second count may be made on that solution and the counts will be averaged. If the Study Director elects to exclude one of the two counts, an explanation will be provided in the data. One or more hemacytometer fields, each 0.10 X 0.10 cm in surface area antl 0.010 cm deep, containing 0.00010 mL of culture, will be counted for each sample until at leest 400 cells orfourfields are counted. , At the time of each density determination, visual observaBons will be made of any unusual appearances of the algal cells including cell size, shape, color, occurrence of flocculation, adherence to glass walls and/or aggregation. Observations of the test solutions (e.g., precipitation, oladty, material adhering to the sides of the test vessels) will also be made daily during the exposure. 2.5.4 Determination of Alnistatic or Alaicidal Effects The following procedure may be used to differentiate between algistatic and algicidal effects of the test substanoe at exposure termination. An algisiatic effect is Page 7 of 14

## Smithers Viscient Study No. 14091.6101

demonstrated by resumed growth after dilution to a non-affected concentration, where an algicidal e(fect is demonstrated by the lack of growth after dllulion. A sample is taken from the composite of a solution containing growth-inhibited algae, .i.e., the lowest concentration that completely inhibits algal growth. If growth ie not completely inhibited at any concentretion, the highest level that inhibited growth is usetl. A treatment level where zero cells are observed in all replicate test vessels at test termination should not be used to initiate the recovery phase. Sufficient fresh, untreated algal medium (AAP) is added to the sample to dilute the test substance to a conoentration that does not affect algal growth. This subculture is incubated under test conditions for up to nine days. A microscopic observation of the culture is made every other day to determine iP growth has resumed. The subculture is dlscontinued either when a substantial increase (e.g., 2:10X) In cell density is observed, or if no growth is observetl after nine days. The recovery pedod will not be performed if <50% inhibition of cell density is observed in the highest concentratlon tested at exposure tennination. 2.5.5 Validitv Criteria The following validity criteria are included based on OCSPP 550.4500 Guideline requirements: • At exposure termination the control coeffcient of variation (CV) for mean yleld and growth rate should be less than 15%.

- • Cell denslly in the controls should increase by a factor of at least 100 times by

exposure terminalion (96 hours). • The concentration of solvent in the range used is not to affect growth of the test species. • The controls are not to be contaminated with the test substance. • The lowest concentration level is lower than the 96-hour EC50 values for yield, growth rate, and area under the growth curve. If any of these criteria are -not met, an evaluation of the impaat on the study results will be provided and, in consultation with theStudy Sponsor it will`be determined whether a repeat test is required.

3.0 STATISTICS The test endpoints for the exposure phase are biomass yielci (final cell density — initial cell density), average specific growth rate (p.) and area under the growth cunre, calculated from the cell density counts. These data are calculated as means and standard deviations for the replicate control(s) and exposure vessels. CoefOcient of varlance values will be determined for yield and growth rate per section 2.5.5. - Per recent discussions with the U.S. EPA, if a solvent is used as a carrierfor the test substance, a t-Test will be used to compare the results of the solvent control to the control data. If the data Page 8 of 14 

## 53

## Smithers Viscient Studv No. 14091.6101 Paee 54

are similar, the negative (blank) control will be used for further analyses. If the data are not simitar, the results will be tliscussed with the Study Sponsor to determine the impact on the study results and whether a repeat test Is required. Yietd is calculated as biomass (cell density) at each interval of the test mtnus the initial biomass at the start of the test.

. Average specific growth mte is determined from the daily Gell density counts. For each test solution, the speciflc growth rate between the inoculation tlensity and the cell density of the specified time interval will be calculated using the toilowing equation:

]n X„ -Jn X„ o

~ = %

- t.o where: p = specific growth rate (days

- '),

In = naturallogarithm,
X = celt density (oells/mL) at the beginning of the specified time interval, t. (expressed as days),

Xno = caII density (celis/mL) at the previous time Interval, (e.g., tno) (expressed as

days), t, = time at observation interval n in days, based on actual time t„ o = fime at the previous observation interval (e.g., test inRiation) in days, and based on the actual time elapsed between observatlon intervals

The average specific growth rate (p a .) for each replicate is caiculated for the entire span of the test period (e.g., 0 to 96 hours). The 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) are also calculated for each replicate and are included with the study results. The area under the growth curve, in units of units: x 10 4 cells•days/mL, is detennined for each repliaate by calculating the area between the growth curves antl the hodzontal line N= N o , according to the formula:

N, - N o N, + N i

- 2N o N,,,

~ +N„ -2N o A= 2 xt
~ + 2 x(tz-t,)+... + Z x(to

- t,.

~ )

Page 9 of 14

## Smithers Viscient Study No. 14091.6101 Page 55

where: A= area under the growth curve

No = calculated number of cells/mL at time to Nr = measured number of oellslmL at t, N. = measured number of cellslmL at time t. tr = time of frst measurement after beginnirng of test t, = time of no' measurement aRer beginning of test n= number of ineasurements taken after exposure initiation
Results of a single concentration limit test will be reponed as the percent inhibition of each endpoint (biomass yield, average specific growth rate and area under the growth curve) as compared to the control data. Additionally, a t-Test will be perrormed to determine if the treatment data are statistically similar to the control data. , The 96-hour EC50 values (the concentmtions of test substance which reduced biomass yield, average specinc growth rate, and area under the growth curve by 50%) and 95% confidenoe limits will be determined by linear interpolation of response (percent inhibitlon of each endpoint, 

### as compared w(ith control) versus exposure concentration and will be calculated using the ICp method (Norberg-King, 1993).
The 96 hour data will also be evaluated to determine a No-Observed-Effect Concentration (NOEC) and a Lowest-Effect Concentration (LOEC) for each endpolnt, If possible. Tbe NOEC is defined as the highest test concentration which demonstrates no significant adverse effect on yield, growth rete or area under the growth curve when compared to the control data. The LOEC is defined as the lowest test concentration which demonstrates a signiricant advenre effect on yield, growth rate or area under the growth curve when compared to the corrtrol data. If an NOEC concentration is statistically determined to be less than the lowest concentration tested, an ECO5 will be determined as arepresentative NOEC value based on OCSPP guitlance. Prior to compadson using the appropriate statistical test, e.g., Dunnett's Test, Williams' Test, Jonckheere-Terpstra Step-Down Test, the data will be evaluated for normality and homogeneity of variance. A computer program (CETIS-Comprehensive Envlronmental Toxicity Information System; Ives, M., 2013), will be used to calculate the EC values and 95% confidence limits. If less than the required response is observed (i.e., <50% response), the EC value will be empidcally estimated to be greater than the highest concentraflon tested.

4.0 RECORDS TO 6E MAINTAINED
Records to

- be maintained wlll include, but will not be Iimited to, conespondence and other documents relating lo the Interpretatlon and evaluation of data as well as all raw data and documentation generated as a resufl of the study.

Page 10 of 14

## Smithers Viscient StudyNo. 14091.6101

5.0 REPORTING The raw data generated at Smithers Visclent will be peer-reviewed and the final report will be reviewed by the Study Director. AII values wiil be reported to various levels of significance depending on the accuracy of the measuring devices employed during any one prooess. The 4uality Assurance Unit will inspect the fnal report to confrm that the methods, procedures, and observations are aocurately and completely described, that the reported results accurately and completely reflect the raw data generated at Smithers Viscient and to confirm adherence with the study protocol. A single copy of the draft report will be submitted to the Sponsor for review. The report will be finalized according to Standard Operating Procedures. The final report will meet the formatting requirements of EPA's PR Notice 2011=3. AII reports will include, butwill not be Iimitetl to, the following information: . • The study number from Smithers Viscient and Sponsor Study number (if any). • Laboratory and site, dates of testing and personnel involved in the study, i.e., Program Coordinator (if applicable), Study Director and Principal Investigator. • Identification of the test substance which may irtclude chemical name, additional . designations (e.g., trade name), chemical designation (CAS number), empirical formula, molecular structure, manufacturer, lot or batch number, water solubility, vapor pressure, degree of purity of test substance (percent teat chemical) (Sponsor supplied, if available). • Scientific name of the test organism, strain, source, and culturing infonnatlon. • Range-finding study results.

. • Test container volume, test solution volume, and inoculum culture density. • Descdption of exposure solution prepamtion scheme. • Description of test conditions and medium. • Criteda for determination of toxic effects and general observatlons on non-quantifiable effeas. • A table of cell density measurements for each daily interval, and tables for yield, calculated specific growth rates (p), avemge specific growth rates (p•,•) and area under the growth curve for the 96-hour test period. • Data on testtemperatures, conductivity, pH and illumination. • The 96-hour EC50 values and 95% confidence limits for yield, average specific growth mte and area under the growth curve and calculation methods used. • The 96-hour NOEC and LOEC values for yield, average specific growth rate and area untler the growth curve, if possible, and calculation methods used.

Page 11 of 14 

## 56

### Smithers.V 

## iscient StudyNo. 14091.6101 Page 57

• Concentration-response curves of yield, average speciflc growth rates and area under lhe growth curve for each concentration. • Analytical results of test concentration measurements and QC samples, and means of measuretl concentration of the test substance, as well as nominal test concentrations. • A description of the methotl(s) used to analyze for test substance and ff an extraction was neoessary for analysis, a description of the method used to separate the test substance from the aqueous phase and recovery data. • Results of the algistatio/algicidal phase, if applicable. • Final protocol and protocol amendments (if any).

. • Good Laboratory Practice (GLP) compliance statement, signed by the Study Director. • Date(s) of Quality Assurance reviews, and dates reported to the Study Director and management, signed by ihe Quality Assurance Unit. • Copy of the study prot000l and study amendments, if any. • Location of the protocol, raw data and flnal report.

6.0PROTOCOLCHANGES AII amendments to the approved protocol must be documented in writing and signed by both the Study Director and the Sponsor's contact or representative. Protocol amendments and deviations must include the reasons for the change and the predicted impact of the change on the results of the study, H any.

7.0 GOOD LABORATORY PRACTICES AII test procedures, documentation, records and reports will comply with the U.S. Envlronmental Protection Agency's Good.Laboratory Practices as set forth under the Federal Insecticide, Fungicide and Rodenticide Act (40 CFR, Part 160).

8.0 REFERENCES ASTM, 2007. Standard practice for conducting acute toxicity tests with fishes, macroinvertebrates and amphibians. Standard E729-96. American Society for Testing and Materials, 100 Barr Harbor Drive, West Conshohocken, Pennsylvania. Ives, Michael. 2013. CETIS, Comprehensive Environmental Toxicity Information System

~ , User's Guide. Tdepool Scientiflc Software, McKinleyville, California.

Page 12 of 14

## Smithers Viscient Study No. 14091.6101 Page 58

. Miiler, W.E., J.C. Greene and T. Shiroyama. 1978. The Se/enastrum caprioornutum Printz algal

assay bottle test. EPA 600/9-78-018. U.S. Environmental Protection Agency, Corvallis Oregon.

. Norberg-King, Teresa J. 1993. .A Linear Interpolation Method for Sublethal Toxicity: The Inhibitlon Concentration (ICp) Approach. National Effluent Toxicity Assessment Center, . Environmental Research Laboratory — Duluth, U.S. Environmental protection Agency,

Duluth, Minnesota. Technical report 03-93. U.S. EPA. 1982. Pesticide Assessment Guidelines, Subdivision J, Hazard Evaluation: Nontarget Plants. EPA 540/9-82-020, U.S. EPA, Washington, DC, U.S. EPA. 1986. Hazard Evaluatlon Division Stantlard Eval'uation Procedure. EPA-540/9-85- 001. U.S. EPA, Washington, D.C.

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

Pradice Standards; Final Rule (40 CFR, Part 160); FR: 8/17/89; pp. 34052. U.S. Environniental Protection Agency, Washington, D.C. U.S. EPA, 1996. Office of Chemical Safety and Pollution Prevention. Ecological Effects . Guideline, OCSPP 850.1000. Special Consideration for Conducting Aquatic Laboratory Studies. "Public Draft". EPA 712-C-96-113. April 1996. U.S. Environmental Protection

. Agency, Washington, D.C. U.S. EPA. 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Watera to Freshwater Organisms. Fourth Edition. Office of Water, U.S. . Environmental Protection Agency, Washington, D.C. EPA/821/R-02-013. U.S. EPA. 2012. Office of Chemical Safety and Pollution Prevention. Ecological Effeots Test Guideline, OCSPP B50.4500: Algal Toxicity. EPA 712-C-12-006. U.S. EPA, . Washington, DC. Williams, D.A. 1971. A test for differences between treatment means when several dose levels

. are compared with a zero iiose control. Biometrics 27: 103-117. . Williams, D.A. 1972. A compadson of several dose levels with a zero controL .. Biometrics 28:

519-531.

.. . . . .. . .. . ... .

. . . .

Page 13 of 14

### Smithers Viscient StudyNo. 14091.6101 Page 59

TABLEI COMPOSITION OF ALGAL ASSAY PROCEDURE (AAP) MEDIUM

Compound Final Concentration

NaNOa 25.5 mg/L MgC6•6Hz0 12.16 mg/L

. CaC12•2H20 4.41 mg/L MgSO4•7Hz0 14.7 mg/L K2HPO4•3H2O 1.368 mg/L NaHCO3

. 15.0 mg/L

- HaBOa 185.5 pg/L

MnCl2•4H20 415.4 pg/L ZnClz 3.270 pg/L CoClz•6Hx0 1.43 pg/L CuC12•21-120 0.012 pg/L

- Na2M004•2H20 7.28 pg/L

. FeCls•6H20 16D pg/L

Naz EDTA•21-120 300 pg/L

, NazSeOa ° 1.88 pg/L Final medium pH ls adjusted to 7.5 3 0.1 with 0.10N NaOH or HCI.

a Additlonal nutrient required, personal communication, Dr. R.R.L. Guillard, June 1991.
SOURCE: Miller, W.E., J.C. Greene and T. Shiroyama. 1978, The Selenastrum

capricomutum Printz algal assay bottle test. EPA 600/948-018. U.S. Environmental Protection Agency, Corvallis, Oregon.

Page 14 of 14

## Smithers Viscient Study No. 14091.6101 Page 60



# 0 SMITHERS V 1 S C I E N T



### PROTOCOLAMENDMENT ' AmendmentNo.: 1 - ProtocotTille: 90-Hour Twdc'ny TesbwHh the Freshwater Green Alga, paeudokirchnenella subcepitete, Foilowing OCSPP Guidellne 850.4500 Study 6ponsur: Brenntag North America, Inc. TeslSuhstance: SorblcAcid Study No.: 14091.5107 Amendment: ProtocolCovorPage The foliowing Infonne0on is being adtled as required to the protocol cover page: AnalyticslStandard: Name: SorhicAcid Pudty: 99.6%

. Batch or Lot #: 1401151 Test Concentm0ona: 0(control), 8.3, 13, 25, 50 and 100 mglL
None of the above changes will hae a nega8ve impaot on the stutly.

, ApprovalSignatures:

2~ cs ~ da ~ ~~1`~kr~ ~3,~1~• n2oiJ"

, thenna A: Softcheck Effac6ve Date Smi VscienlStudyDirector

_ 

### 3-3t_1 ~i~ ' B SponsorRBpresentative Date

.

Page 1 of 1 

# I SmHMn Vaclent LLC T80 Mein Slreet (Wamhum, trLA02fi]1 I p 50a2S6.P550 wxw.smilhersvbGaM.com 2600Cuekenbuet,RoztllamwCamp,NC2]9481p

~ 0a9]6.Ot41

## Smithers Viscient Study No. 14091.6101 Paee 61

APPENDIX 2 - ANALYTICAL METHODOLOGY

## Smithers Viscient StudyNo. 14091.6101 Page 62

SUMMARY Methodology was validated (13 March 2015) to quantify the amount of sorbic acid present in recovery samples 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 method was validated by fortification of AAP medium with sorbic acid at concentrations of 0.0100, 5.00 and 100 mg/L. Recoveries averaged 99.5 t 5.02%, with a limit of quantitation (LOQ) of 0.00747 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:

2. Balance: 3. Laboratory equipment: Hewlett-Packard Series 1100 quaternary solvent pump equipped with a Hewlett-Packard Series 1100 autosampler, a Hewlett-Packard Series 1100 vacuum degasser, a Hewlett-Packard Series 1100 variable wavelength detector and an Agilent ChemStation version B.04.02 for data acquisition Mettler Toledo AG285 disposable glass pipets, volumetric flasks, graduated cylinders, positive displacement 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 purification system (meets ASTM Type II requirements)

### Smither 

## s'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, Colorado. The following information was provided:

Name: Lot No.: CAS No.: Purity: Expiration Date: 

## sorbic acid 1401151 110-44-1 

### 99.5% 

## 14 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.



## PROCEDURES Preparation of Stock Solutions Primary stock solutions were typically prepared as per the table below:

Amount of Amount of Primary Substance Substance Stock Final Primary Stock Stock ID Weighed Weighed (g), Solvent Volume Concentration Primary Stock Use

(g), Net as Active (mL) (mg/L) Wei ht In redient 7535A 0.1005 0.1000 100 1.00 Secondary stock for ' Acetonitrile calibration standards

. High-level recovery 7535B

~ 0.1007 . 0.1002 100 1.00 samples and secondary

-. . . stocks for low- and midIevel recovery sam les


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

Fortifying Volumc of Final Stock Fortifying Stock Stack Stock 

### ID Concentration Fortification Volume Solvent StocklD Concentration StockUse

(mg/L) (~ ) (

~ ) (mg

~ )

7535A 1.00 0.500 50.0 7535A-1 10.0 Calibration

Acetonitrile standards 1.00 0.500 50.0 753513-1 10.0 Low-level 7535B - covery sam les

1.00 5.00 50.0 753513-2 100 Mid-level recovery sam les

## Smithers Viscient Study No. 14091.6101 Page 64

All primary and secondary stock solutions 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 concentration 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 nominal 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 0.00585, 0.0112, 0.0293, 0.0585, 0.0878 and 0.117 mg/L.
Sample FortiScation 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. Each 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

Stock Concentration Fortitication Ftnal Volume Fortified Sample ID 

### (mg

~ ) Volume 

### (mL) Concentration (mL (m /L) Control NA a NA 16.0 0.00 A,B&C Low A,B&C 10.0 0.0400 40.0 0.0100

~ Mid A,B&C 100 0.800 16.0 5.00 High A,B&C 1000 1.60 16.0 100

s 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 acetonitrile 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.

- Fortified Sample Final -- Final SampleID Concentration Volume Volume° Sample Volume° Dilution

(mg/L) (mL)

~~ (mL) Volume 

### (mL) Factor Control 0.00 16.0 20.0 NA` NA 1.25 A,B&C Low 0.0100 40.0 50.0 NA NA 1.25 A,B&C Mid 5.00 16.0 20.0 0.150 10.0 83.3 A,B&C High 100 16.0 20.0 0.0400 50.0 1560 A,B&C " lliluted with acetonitrile. b Diluted with 20:80 acetonitrile:purified reagent water (v:v). ` NA = Not Applicable.

## Smithers Viscient Study No. 14091.6101 Paee 66

ANALYSIS Instrumental Conditions The high performance liquid chromatographic (HPLC) analysis was conducted utilizing the following instrumental conditions:

Column: Mobile Phase (A): Mobile Phase (B): Gradient:

Run Time: Equilibration Delay: Flow Rate: Injection Volume: Wavelength: Retention Time: Phenomenex Synergi Hydro-RP 4 μm, 250 mm x 4.6 mm 0.05% phosphoric acid in purified reagent water 100% acetonitrile Time (min.) Solvent A(%) Solvent B(%) 

## 0.00 95.0 5.0 1.00 95.0 5.0 15.00 0.00 100.0 19.00 0.00 100.0 20.00 95.0 5.0 20.00 minutes 7.00 minutes 1.00 mL/min 300 μL 275 nm approximately 11.5 minutes
Preparation of Standard Curve Two sets of calibration standards were analyzed with each sample set: one set prior to analysis 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.

CALCULATIONS 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 ofthe line (equation 1) was algebraically manipulated to give equation 2. The concentration oftest 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 the

## Smithers Viscient Study No. 14091.6101

recovery sample. Equations 2 and 3 were then used to calculate measured concentrations and analytical results. (1) y=mx+b

(2) DC(x) = (y b)



## m (3)A=DCxDF

where:

x = analyte concentration y = detector response (peak area) from the chromatogram b = y-intercept from the regression analysis m = slope from the regression 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 quantitafion (LOQ) was calculated using the following equation:

(4) LOQINST A LS - b

- 

## m (5) LO`<-LOQINSTxDFCNTL

where: ALS = 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 regression LOQINST = limit of quantitation at the instrument DFCNTL = dilution factor of the control samples (smallest dilution factor used) LOQ = limit of quantitation reported for the analysis 67

## Smithers Viscient Study No. 14091.6101 Page 68

RESULTS AND DISCUSSION The mean recovery was 99.5% with a standard deviation of 5.02%. The LOQ was 0.00747 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 1 A. Representative chromatograms from the analysis of a calibration standard, a recovery sample and a control sample are presented in Figure lA, 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 Fortified Concentration Dilution Analytical Result Percent of Fortified ID (mg/L) Factor (mg/L) (%)
ControlA 0.00 1.25 <0.00747 a NA b ControlB 0.00 1.25 <0.00747 

### NA ControlC 0.00 1.25 <0.00747 NA
Low A 0.0100 1.25 0.00982 98.2 LowB 0.0100 1.25 0.0100 100 LowC 0.0100 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 0.00747 mg/L. b 

### NA 

### = Not Applicable. Note: Results were calculaCed using the actual analytical (unrounded) results and not the rounded values presented in this table.

## Smithers Viscient Study No. 14091.6101 Page 70



## Figure lA. Representative chromatogram of a 0.0585 mg/L calibration

standard used for quantification during the method validation study with sorbic acid.

Data File D:\CHEM32\2\DATA\14091-6101

- M

11MARlI 2015-03-13 17-55-18\045-0601.D Sample Name: standard 4

Acq. Operator .: Huy Le Seq. Line : 6 Acq . Instrument : Inetrument 2 Location : Vial 45 Injection Date : 3/13/2015 8:56:39 PM Inj : 1

Inj Volume : 300.0 μl Acq. Method : D:\CHEM32\2\DATA\14091-6100_MV_13MAR15 2015-03-13 19-55-19\SORHIC ACID_ W.M Last changed : 3/13/2015 5:38:03 PM by Bertha Baroody Analyeis Method : D:\CBEM32\2\DATA\14091-6100_MV_13MAR15 2015-03-13 17-55-18\SOREIC ACID_ W.M (Sequence Method) Last changed : 3/14/2015 7:19:34 PM by Huy Le Method Info : Sorbic Acid, Phenomenex Synergi Hydro-RP 4μm 250 x 4.6mm, gradient analyeie, solvent A: 0.052 H3PO4 in PRW, Solvent B: 100% ACN, 1 mL/min, 295nm, ambient
ECM Server : http://se1007/eel ECM Operator : Huy Le ECM Path : Chemistry\Sorbic Acid\14091-6100\Data\19091-6100_MV_13MAR15 2015-03-13 19-55-18.SC.SSIzip ECM Version : 2

mAU VWD1 A, WavelenglE=275 nm (14091E100_MV_13MAR15 2015-0}131]-55-18WG5A601.D)

35
20
15

0 5~ I 

# ~ ~
0

_ 0 YS 5 7.5 ^ ~ 10 12.5 15 17.5 min
---------------- __a-a-a---se_- = 

# _ Area Percent Report

Sorted By

. Signal Multiplier: ~ ~ . . 1.0000 Dilution: 1.0000 Ose Multiplier & Dilution Factor with ISTDs

Signal 1: VWDl A, Wavelength=275 nm

. Peak RetTime Type Width Area Height Area # [min] [min] mAU *s [mAU ] i 

### ---- I ------- I ---- I ------- I ---------- I ---------- I -------- ~ 1 11.510 BB 0.0922 79.97351 12.98747 100.0000
Totals : 79.97351 12.98747

*** End of Report ***

Irtstrument 2 3/14/2015 7:15:18 PM Hny Le Page 1 oE 1

## 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.

Data File D:\CHEM32\2\DATA\14091-6100_MV_13MAR15 2015-03-13 17-55-18\056-1701.D Sample Name: mid B

Acq. Operator : Huy Le 6 Seq. Line 17 Acq. Inetrument : Instrument 2 Location : Vial 56 Injection Date : 3/14/2015 3:15:10 AM Inj : 1 Inj Volume : 300.0 μl Acq. Method : D:\CHEM32\2\DATA\14091-6100 MV 13MAR15 2015-03-13 1]-55-18\SORBIC ACID W.M

_ _ _ Last changed : 3/13/2015 5:38:03 PM by Bertha Baroody Analysis Method : D:\CHEM32\2\DATA\14091-6100_MV_13MAR15 2015-03-13 17-55-18\SORBZC ACID_ DV.M (Sequence Method) ' Last changed : 3/14/2015 7:14:39 PM by Huy Le Method Info : Sorbic Acid, Phenomenex Synergi Hydro-RP 4Nm 250 x 4.6m, gradient analysie, Solvent A: 0.05i H3PO4 in PRW, 8olvent B: 1001 ACN, 1 mL/min, 275nm, ambient

ECM Servez : http://es1007/8e1 ECM Operator : Huy Le ECMPath : Chemistry\Sorbic Acid\14091-6100\Data\14091-6100_MV 13MAR15 2015-03-13 15-55-18.SC.SSIZip ECM VeYBion : 2

~ - VWD1A,Wevelengih=2]5nm(14091-6100_MV_13MAR152015-03-1317-55-181056-1]01.D) mAU 26

20

15

10

5

0 0 2 5 5 7.5 10 12.5. 15 17.6 .mm



### ____—____:___ 

# _____________________ Area Percent Report ^

Sorted By

. Signal 

### Multiplier:

. 1.0000 Dilution:

. 1.0000 use Multiplier & Dilution £actor with ISTDs Signal 1: VWD1 A, Wavelength=275 nm

- 

### Peak RetTime Type Width Area Height Area # [min] [min] mA0 -s (mAu ] E 

## ---- I ------- I ---- I ------- I ---------- I ---------- I-------- 1 11.511 BB 0.0920 76.68466 12.49796 100.0000

Totals : 76.68466 12.49796

----------- End of RePOrt

InetYLLment 2 3/19/2015 7:15:53 PM HUy Le Page 1 of 1

### Smithers Viscient Study No. 14091.6101 Page 72



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

method validation study with sorbic acid.

Data File D:\CHEM32\2\DATA\14091-6100_MV_13MAR15 2015-03-13 17-55-19\050-1301.D Sample Name: control e

Acq. Operator : Huy Le Seq. Line : 11 Aeq. lnetrument aInetrllrt:ERC 2 LOCation . Vial 50 IRjectiGn Date : 3/13/2015 11:49:46 PM Inj : 1 Inj Volume : 300.0 μl ACq. M2thOd : D:\CHEM32\2\DATA\14091-6100_MV_13MAR15 2015-03-13 17-55-18\SORBIC ACID_ W.M Laet changed : 3/13/2015 5:38:03 PM by Pertha Baroody Analyeie Method : D:\CHEM32\2\DATA\14091-6300_MV_13MAR15 2015-03-13 19-55-19\60RBIC ACID W.M (Seqvence Method) ' Laet changed : 3/14/2015 9:19:34 PM by Huy Le Method Info : Sorbic Acid, Phenomenex Synergi Hydro-RP 44m 250 x 4.6mm, gradienC analy619, Solvent A: 0.055 H3P09 in PRW, SO1Vent D: 3005 ACN, 1 mL/min, 2'15nm, ambient

ECM SerVeT : http://se300'1/9e1 ECM OpeYatOr : Huy Le ECM Path : Chcmistry\Sorbic ACid\14091-6100\Data\14091-6100_MV_13MAR15 2015-03-13 19-55-18.SC.SSIzip ECM Version : 2 

### VWOtA,Wevelen010=2]5nm(1400b8100_MV_13MAR152015-03431]-55-18W50-110L0) mAU 25 20 15- 10- 5 0 0 2.5 5 7.5 10 125 15 t]5 min

Area Percent Report

Sorted By Signal Multiplier:

. 1.0000 Dilution: 1.0000 Dse Multiplier & Dilution Factor with ISTDe N. peaks fcund

.*: End of Report ***

Instrument 2 3/14/2015 7:15:35 PM Huy Le Page 1 of 1



### Note: No peak equivalent to retention time for sorbic acid. Sorbic acid is ordinarily detected at a retention time 

## of approximately 11.5 minutes.

## 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.

190 Linear Regression Analysis

160
140
120 

### 8 1m
p 80
60
40
20
0 0.000 0.020 0.040 0.060 6.0e0 0.100 0.120 0.140 Concentration 

### Linear Regression Analysis

r 2 = 0.99992 y = 1400.898x - 1.7835

Standard Standard Concentration Response 

### (mg/L) 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