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 +rwj 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 

Biological Parameter EC50 

(95% Confidence Limits NOEC LOEC 

96-Hour Yield (6 910 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/mZ/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 104 cells/mL, was 

aseptically introduced into each flask. This inoculum provided the required initial (0-hour) cell 

density of approximately 1.0 X 104cells/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: 

Crv,,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-Ct

J]/yWF 

where: 

Var = variance 

WF = weighting factor, defined as the time between intervals 

C = concentration at each interval 

Cw„ = 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 Xt- ln XO 

tt- to 

where: 

μ = specific growth rate (days ) 

]n = natural logarithm 

Xo = initial cell density in cells/mL at time to (days) 

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

to = time of test initiation 

tt= 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, + NZ- 2No N ~ ., +N ~ -2No 

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`hmeasurement 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 104cells/mL, respectively. The control averaged 186.33 x 10 4cells/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 104cells/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 4cells/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 levels 

averaged 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 4cells 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 104cells 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/mZ/S over the nine-day test period. A$er nine days, a cell density of 4.00 x 104cells/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 104 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 104cells/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 

(161/4) 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 

 NaNO3 25.5 mg/L 

MgC12•6Ha,0 12.16 mg/L 

CaCl z •2H20 4.41 mg/L 

MgSO4 •7H2O 14.7 mg/L 

 K2HPO4*3H20 1.368 mg/L 

 NaHCO3 15.0 mg/L 

. . . H3 1303 185.5 pg/L 

 Na2Se04a 1.88 pg/L 

MnC12 •4Hz0 415.4 μg/L 

ZnC12 3.270 μg/L 

 CoClz•61120 1.43 μg/L 

CuClz •2Hz0 0.012 pg/L 

Na2Mo04•2H20 7.26 μg/L 

FeCl3 •6Hz0 160 gg/L 

Na2EDTA•2H20 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/m2/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 23/24r 7/5f 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. 

bTime-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 104 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) -0.194 (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) 0.264(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) 0.334(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 

~ 

yV 

O 

v X 

'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 

)0ry 

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 be 

made 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 
Smithers Viscient Studv No. 14091.6101 48 

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. 

2.2Test 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. 

2.2.4Reference 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 

104 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 pPJm21S 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 4cells•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, - No N, + Ni- 2No N,,, ~ +N„ -2No 

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.Viscient 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.com2600Cuekenbuet,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) 
Smithers'Viscient Study No. 14091.6101 Page 63 

Test Substance 

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

Denver, 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 NAa 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 

sNA = 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 

ALS- 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 <0.00747a NAb 

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 -M11MARlI 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_13MAR152015-03-13 17-55-18\056-1701.D 

Sample Name: mid B 

Acq. Operator : Huy Le 6Seq. 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 W.M MV 13MAR15 2015-03-13 1]-55-18\SORBIC ACID _ _ _ 

Last changed : 3/13/2015 5:38:03 PM by Bertha Baroody 

Analysis Method : D:\CHEM32\2\DATA\14091-6100_MV_13MAR152015-03-13 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 25 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 

r2 = 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