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 Project ID

Smithers Viscient Study No. 14091.6101

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

STATEMENT OF NO DATA CONFIDENTIALITY CLAIM

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

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

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

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

regarding disclosure to multinational entities under FIFRA 10(g).

Company: 

Company Agent: Date:

(Typed Name)

(Title) (Signature)


Smithers Viscient Study No. 14091.6101 Page 5

KEY STUDY PERSONNEL

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

reporting of the study results.

Katherina A. Softcheck Study Director

Kristen L. Soucy Biologist I

Melissa L. Staggs Biologist I

Ashley Beaton Assistant Biologist

Blake Dinius Biologist I

Nicholas Lyons Assistant Biologist

Regine Felix Chemistry Technician II

Huy Le Chemistry Technician II

Marjorie E. Dix Research Director, Chemistry

Gina Giorgio Technical Report Writer

Heather Grove Director of 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

TABLE OF CONTENTS.................................................................................................................6

SUMMARY.....................................................................................................................................9

1.0 INTRODUCTION................................................................................................................11

2.0 MATERIALS AND METHODS.........................................................................................11

2.1 Protocol......................................................................................................11

2.2 Test Substance ...........................................................................................11

2.3 Test Organism............................................................................................12

2.4 Reference Test ...........................................................................................13

2.5 Test Dilution Water....................................................................................14

2.6 Test Concentrations ...................................................................................14

2.7 Test Solution Preparation...........................................................................14

2.8 Test 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 Definitive Exposure ...................................................................................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 

sorbic acid. .................................................................................................30

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

during the 96-hour exposure of Pseudokirchneriella subcapitata.............31

Table 4. Cell density of Pseudokirchneriella subcapitata 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 specific growth rate) of the 96-hour 

toxicity test with Pseudokirchneriella subcapitata....................................36

Table 9. Section-by-section growth rates of Pseudokirchneriella subcapitata

after 24, 48, 72 and 96 hours of exposure to sorbic acid. ..........................37

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

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

exposure to sorbic acid...............................................................................38

Table 11. EC50, No-Observed-Effect Concentration (NOEC) and LowestObserved-Effect Concentration (LOEC) values for sorbic acid

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

toxicity test with Pseudokirchneriella subcapitata....................................39

Figure 1. Relationship between nominal and time-weighted average 

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

exposing Pseudokirchneriella subcapitata................................................40

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

subcapitata exposed to sorbic acid. ...........................................................41

Figure 3. Yield for 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 1A. Analytical results for the recovery of sorbic acid from AAP medium 

during the method validation study............................................................69

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

used for quantification during the method validation study with 

sorbic acid. .................................................................................................70

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

fortified with sorbic acid during the method validation study...................71

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

validation study with sorbic acid. ..............................................................72

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

quantitate sorbic acid in the recovery samples. .........................................73
Smithers Viscient Study No. 14091.6101 Page 9

SUMMARY

Sorbic Acid – 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 April 2015 (including recovery period)

TEST ORGANISM: Pseudokirchneriella subcapitata, inoculum - three days 

since previous transfer

Source - Smithers Viscient culture

DILUTION WATER: Algal Assay Procedure (AAP) medium

TEST CONDITIONS: 96-hour duration, a temperature range of 22 to 24 °C

continuous illumination at 51 to 68 μE/m2

/S, continuous 

agitation at a shaking rate of 100 rpm

NOMINAL TEST

CONCENTRATIONS: 6.3, 13, 25, 50 and 100 mg/L

TIME-WEIGHTED

AVERAGE

CONCENTRATIONS: 1.7, 3.9, 24, 50 and 110 mg/L

EFFECT CRITERIA: Reduction of 96-hour yield, average specific growth rate 

(μave) and area under the growth curve (AUGC) relative 

to the performance of the control data.
Smithers Viscient Study No. 14091.6101 Page 10

RESULTS:

Biological Parameter

Based on Time-Weighted Average Concentrations (mg/L)

EC50

(95% Confidence Limits) NOEC LOEC

96-Hour Yield 10

(6.9 – 10) 3.9 24

96-Hour Average Specific

Growth Rate

12

(11 – 13) 3.9 24

96-Hour Area Under the 

Growth Curve

10

(8.5 – 10) 3.9 24
Smithers Viscient Study No. 14091.6101 Page 11

1.0 INTRODUCTION

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

freshwater green 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. All 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 Study No. 14091.6101 Page 12

Name: sorbic acid 

Lot No.: 1401151

CAS No.: 110-44-1

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 of the 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

The 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 of the dilution water source used in the preparation of the culture 

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

GeoLabs, Inc., 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 Study No. 14091.6101 Page 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 ± 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 ± 10 rpm, a temperature of 24 ± 2 oC and continuous illumination at the surface of the 

medium providing photosynthetically-active radiation (PAR) of approximately 

60 μE/m2

/S ± 15%, e.g., 51 to 69 μE/m2/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 ± 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 

Concentration

(mg/L)

Volume of Primary

Stock Used

(L)

Final Dilution

Volume with

AAP Medium

(L)

Exposure Solution 

Concentration

(mg/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 × 104 cells/mL, was 

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

density of approximately 1.0 × 104 cells/mL.
Smithers Viscient Study No. 14091.6101 Page 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 × 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 of the four replicate vessels of the 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., ≥ 10X) 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 ± 2 °C, continuous light intensity at a 

photosynthetically-active radiation (PAR) range of 51 to 69 μE/m2/s (approximately equivalent 

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

100 ± 10 rpm.

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

in a flask of water adjacent to the 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 

VWR 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 

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

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

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

had on the test substance concentration.

Prior to processing and analysis, all 96-hour exposure 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 each treatment 

level as follows:

Ctwa = [(CD0 − CD4)/(ln (CD0)− ln(CD4)) x 4 Days] / Total Days

where:

Ctwa = time-weighted average concentration

CD0 = measured concentration on day 0

CD4 = measured concentration on day 4

ln = natural log

Total Days = 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 = ∑ [WF x (C - Ctwa)

2

] / ∑ WF

where:

Var = variance

WF = weighting factor, defined as the time between intervals

C = concentration at each interval

Ctwa = 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 ± 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 Study No. 14091.6101 Page 20

2.10 Statistical Analysis

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

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

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:

μ = ln Xt - ln X0

t

t - t0

where:

μ = specific growth rate (days-1)

ln = natural logarithm

X0 = initial cell density in cells/mL at time t0 (days)

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

t0 = time of test initiation

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

between observation intervals)

The average specific growth rate (μave) 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 Study No. 14091.6101 Page 21

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

the growth curves and the horizontal line N = N0, according to the formula:

A = N N

2

x t +

N + N - 2N

2 x (t - t ) + ... +

N + N - 2N

2 x (t -t ) 1 0

1

1 2 0

2 1

n-1 n 0

n n-1

where:

A = area under the growth curve (units: × 104 cells × days/mL)

N0 = calculated number of cells/mL at time t0

N1 = measured number of cells/mL at t1

Nn = measured number of cells/mL at time tn

t1 = time of first measurement after beginning of test

tn = time of nth measurement after beginning of test

n = number of measurements taken after test initiation

Based on the results of the statistical analysis performed for 96-hour yield, average specific 

growth rate 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.

CETISTM 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 × 104 cells/mL, respectively. The control averaged 186.33 × 104 cells/mL. Based on 

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

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

3.2 Definitive Exposure

3.2.1 Evaluation of Test Conditions

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

definitive exposure are presented in Table 2. The pH of the control solution was 7.2 at test 

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

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

with increasing test substance 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 6.3 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 of test 

substance increased. Continuous temperature monitoring established that the temperature ranged 

from 23 to 24 oC throughout the exposure period. The photosynthetically-active radiation (PAR) 

of the test area ranged from 51 to 66 μE/m2/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/L.

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

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

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) of the 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 × 104 cells/mL. Cell densities 

in the 1.7, 3.9, 24, 50 and 110 mg/L treatment levels averaged 219.88, 216.79, 2.81, 1.38 and 

0.81 × 104 cells/mL, respectively.

Yield is presented in Table 5 and illustrated in Figure 3. The 96-hour yield in the control 

averaged 218.08 × 104 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 × 104 cells/mL, respectively.

Based on the results of Shapiro-Wilk’s and Bartlett’s Equality of Variance Test, this data set 

passed the requirement for normality and homogeneity of variance; therefore, Williams’ Multiple 

Comparison Test was used to determine treatment-related effects. A significant reduction was 

detected in treatment levels ≥ 24 mg/L compared to the control data. Therefore, the 96-hour 

NOEC and LOEC values for yield were determined to be 3.9 and 24 mg/L, respectively. Based 

on time-weighted average concentrations, the 96-hour EC50 value for yield was determined to be 

10 mg/L, with 95% confidence limits of 6.9 to 10 mg/L. The 96-hour NOEC, LOEC and EC50

values for yield are presented in Table 6.
Smithers Viscient Study No. 14091.6101 Page 25

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

96-hour average specific growth rate in the control averaged 1.36 days-1

, respectively. The 0- to 

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

averaged 1.36, 1.36, 0.26, 0.05 and -0.11 days-1

, 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 104 cells × 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 104 cells × 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 100 mg/L nominal 

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

24 oC. The photosynthetically-active radiation (PAR) of the test area ranged from 56 to 

68 μE/m2

/S over the nine-day test period. After nine days, a cell density of 4.00 × 104 cells/mL 

was observed in the subculture, corresponding to an approximately 82-fold increase in cell 

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

rather than algicidal effect on the growth of Pseudokirchneriella 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 × 104 cells/mL: 219.08 × 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 × 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 

(16%) 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 Study No. 14091.6101 Page 27

PROTOCOL DEVIATION

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

REFERENCES

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

E-1218-04, 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 SystemTM. User’s 

Guide. Tidepool Scientific Software, McKinleyville, California.

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

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

Corvallis, Oregon.

Norberg-King, Teresa J. 1993. A Linear 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 Study No. 14091.6101 Page 29

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

toxicity test exposing Pseudokirchneriella subcapitata to sorbic acid.

Compound Concentration

NaNO3 25.5 mg/L

MgCl2•6H2O 12.16 mg/L

CaCl2•2H2O 4.41 mg/L

MgSO4•7H2O 14.7 mg/L

K2HPO4•3H2O 1.368 mg/L

NaHCO3 15.0 mg/L

H3BO3 185.5 μg/L

Na2SeO4

a 1.88 μg/L

MnCl2•4H2O 415.4 μg/L

ZnCl2 3.270 μg/L

CoCl2•6H2O 1.43 μg/L

CuCl2•2H2O 0.012 μg/L

Na2MoO4•2H2O 7.26 μg/L

FeCl3•6H2O 160 μg/L

Na2EDTA•2H2O 300 μg/L

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

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

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

bottle test. EPA 600/9-78-018. U.S. Environmental Protection Agency, Corvallis, Oregon.
Smithers Viscient Study No. 14091.6101 Page 30

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

during the 96-hour exposure of Pseudokirchneriella subcapitata to

sorbic acid.

Nominal

Concentration

(mg/L)

pH Conductivity

(μS/cm)

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

Control 7.2 7.3 7.4 9.1 9.4 91

6.3 6.7 7.3 7.5 9.1 9.3 89

13 6.3 7.1 7.4 9.1 9.2 90

25 5.3 5.6 5.6 5.6 6.2 94

50 4.6 4.7 4.7 4.7 4.7 100

100 4.2 4.2 4.2 4.2 4.3 120

Minimum/Maximum Temperature (°C)

0 - 24 Hours 24 - 48 Hours 48 - 72 Hours 72 - 96 Hours

23/24 23/24 23/24 23/24

Light Intensity (PAR)a

Units 0 Hoursb 24 Hours 48 Hours 72 Hours 96 Hours

μE/m2/S 51 - 66 51 - 63 53 - 62 52 - 64 52 - 64

a PAR = photosynthetically-active radiation b The light intensity (lux) of the test area at test initiation ranged from 4300 to 4900.
Smithers Viscient Study No. 14091.6101 Page 31

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

during the 96-hour exposure of Pseudokirchneriella subcapitata.

Nominal

Concentration

(mg/L)

Measured Concentration (mg/L) Percent 

Decline from 

0 Hour

TimeWeighted 

Averagebc

Percent of 

Nominalb

0 Hour 96 Hoursa

Control < 0.59d < 0.027 NAe NA NA

6.3 6.4 0.17 97 1.7 27

13 13 0.56 96 3.9 30

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

50 51 49 3 50 100

100 110 100 6 110 110

Uncentrifuged Uncentrifuged Centrifuged

QCg #1

3.00

3.00

(98.9)

2.94

(98.0)

2.94

(97.8)

QC#2

25.0

26.1

(104)

25.8

(103)

25.8

(103)

QC#3

100

107

(107)

105

(105)

104

(104)

a All 96-hour test and control solutions were centrifuged prior to analysis. b Time-weighted average concentrations and percent of nominal were calculated using the actual analytical 

(unrounded) results and not the rounded (two significant figures) values presented in this table. c Ctwa = [(CD0 − CD4)/(ln (CD0)− ln(CD4)) x 4 Days] / Total Days d 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. e NA = Not Applicable. f Result of the additional sample without algae present analyzed at 96 hours of exposure to determine biological 

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

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

96 hours of exposure to sorbic acid.

Time Weighted 

Average 

Concentration

(mg/L)

Replicate

Cell Density (x 104 cells/mL)

Observation Interval (Hours)

24 48 72 96

Control A 6.50 35.00 73.00 187.67

B 7.75 33.50 63.25 253.00

C 3.75 23.00 88.75 246.00

D 4.25 17.25 73.50 189.67

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

1.7 A 6.50 26.00 78.75 214.50

B 7.75 32.00 58.75 275.50

C 3.50 23.25 86.25 201.50

D 6.75 23.00 88.00 188.00

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

3.9 A 8.50 39.25 76.25 224.00

B 8.75 42.25 81.25 268.50

C 3.25 18.00 81.00 200.33

D 5.25 19.75 84.00 174.33

Mean (SD) 6.44(2.66) 29.81(12.71) 80.63(3.22) 216.79(40.00)

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)

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

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

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

96 hours of exposure to sorbic acid.

Time Weighted 

Average Concentration

(mg/L)

Replicate

Yield (x 104 cells/mL) Percent

Inhibitionab

(%)

Observation Interval (Hours)

0 - 24 0 - 48 0 - 72 0 - 96

Control A 5.50 34.00 72.00 186.67

B 6.75 32.50 62.25 252.00

C 2.75 22.00 87.75 245.00

D 3.25 16.25 72.50 188.67

Mean (SD)b 4.56(1.89) 26.19(8.51) 73.63(10.53) 218.08(35.25) NAc

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) 1.81d (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) 0.38d(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) -0.19d(0.66) 100

a Percent inhibition relative to the control. b Mean, standard deviation (SD) and percent inhibition were calculated from original raw data, not from the rounded values 

presented in this table. c NA = Not Applicable. d Significantly reduced compared to the control, based on Williams’ Multiple Comparison Test.
Smithers Viscient Study No. 14091.6101 Page 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 specific growth rates of Pseudokirchneriella subcapitata

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

Time Weighted 

Average

Concentration

(mg/L)

Replicate

Average Specific Growth Rate (days-1) Percent

Inhibitionab

(%)

Observation Interval (Hours)

0 – 24

(0.919 days)

0 – 48

(1.94 days)

0 – 72

(2.89 days)

0 – 96

(3.95 days)

Control A 2.04 1.83 1.48 1.33

B 2.23 1.81 1.43 1.40

C 1.44 1.61 1.55 1.39

D 1.57 1.46 1.49 1.33

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

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) 0.26d(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) 0.05d(0.14) 96

110 A -0.31 0.00 -0.24 -0.07

B 0.24 0.47 0.28 0.14

C 0.00 0.00 0.00e -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) -0.11d(0.21) 108

a Percent inhibition relative to the control. b Mean, standard deviation (SD) and percent inhibition were calculated from original raw data, not from the rounded values 

present in this table. c NA = Not Applicable. d Significantly reduced compared to the control, based on Williams’ Multiple Comparison Test. e Growth rate values cannot be mathematically calculated from a cell density of zero. Therefore, a growth rate value of zero is 

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

Table 8. 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 Page 37

Table 9. Section-by-section growth rates of Pseudokirchneriella subcapitata

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

Time Weighted 

Average 

Concentration

(mg/L)

Replicate

Section-by-Section Growth Rate (days-1)

Observation Interval (Hours)

0 - 24

(0.919 days)

24 - 48

(1.03 days)

48 - 72

(0.949 days)

72 - 96

(1.05 days)

Control A 2.04 1.64 0.77 0.90

B 2.23 1.43 0.67 1.31

C 1.44 1.77 1.42 0.97

D 1.57 1.37 1.53 0.90

Mean (SD)a 1.82(0.37) 1.55(0.19) 1.1(0.44) 1.02(0.20)

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 0.00b 0.00b

D 0.24 0.00 -0.24 -0.66

Mean (SD) 0.04(0.26) 0.24(0.32) -0.27(0.32) -0.13(0.44)

a Mean and standard deviation (SD) were calculated from original raw data, not from the rounded values present in this table. b Growth rate values cannot be mathematically calculated from a cell 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 

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

exposure to sorbic acid.

Time 

Weighted 

Average 

Concentration

(mg/L)

Replicate

Area Under the Growth Curve (× 104 cells × days/mL)

Percent

Inhibitionab

(%)

Observation Interval (Hours)

0 - 24 24 - 48 48 - 72 72 - 96 0 - 96

Control A 2.53 20.24 50.28 136.43 209.48

B 3.10 20.12 44.94 165.75 233.90

C 1.26 12.68 52.06 175.50 241.51

D 1.49 9.99 42.09 137.75 191.33

Mean(SD)b 2.10(0.87) 15.76(5.22) 47.34(4.62) 153.86(19.77) 219.05(22.99) NAc

1.7 A 2.53 15.63 48.73 153.61 220.51

B 3.10 19.35 42.09 175.24 239.78

C 1.15 12.68 50.99 150.71 215.53

D 2.64 14.22 51.70 144.52 213.08

Mean (SD) 2.36(0.84) 15.47(2.85) 48.38(4.38) 156.02(13.36) 222.23(12.11) -1

3.9 A 3.45 23.45 53.83 157.31 238.03

B 3.56 25.11 57.63 183.41 269.72

C 1.03 9.87 46.01 147.33 204.24

D 1.95 11.79 48.26 135.20 197.20

Mean (SD) 2.50(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) 3.66d(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) 0.88d(0.62) 100

110 A -0.11 -0.13 -0.24 -0.40 -0.88

B 0.11 0.90 1.30 1.05 3.37

C 0.00 0.00 -0.47 -0.92 -1.40

D 0.11 0.26 0.12 -0.26 0.23

Mean (SD) 0.03(0.11) 0.26(0.46) 0.18(0.79) -0.13(0.84) 0.33d(2.14) 100

a Percent inhibition relative to the control. b Mean, standard deviation (SD) and percent inhibition were calculated from original raw data, not from the rounded values presented in this 

table. c NA = Not Applicable. d Significantly reduced compared to the control, based on the Jonckheere-Terpstra’s Step-Down Test.
Smithers Viscient Study No. 14091.6101 Page 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 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: 8.5 – 10
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Figure 1. Relationship between nominal and time-weighted average 

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

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

subcapitata exposed to sorbic acid.
Smithers Viscient Study No. 14091.6101 Page 42

Figure 3. Yield for Pseudokirchneriella subcapitata exposed to sorbic acid.

*Significantly reduced compared to the control, based on Williams’ Multiple Comparison Test.
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Figure 4. Average specific growth rates (0- to 96-hour) for Pseudokirchneriella

subcapitata exposed to sorbic acid.

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

Figure 5. Area under the growth curve for Pseudokirchneriella subcapitata

exposed to sorbic acid.

*Significantly reduced compared to the control, based on Jonckheere-Terpstra’s Step Down Test.
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APPENDIX 1 - STUDY PROTOCOL
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APPENDIX 2 - ANALYTICAL METHODOLOGY
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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 ± 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: 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

2. Balance: Mettler Toledo AG285

3. Laboratory equipment: 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)
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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: sorbic acid 

Lot No.: 1401151

CAS No.: 110-44-1

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.

PROCEDURES

Preparation of Stock Solutions

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

Primary 

Stock ID

Amount of 

Substance 

Weighed 

(g), Net 

Weight

Amount of 

Substance 

Weighed (g), 

as Active 

Ingredient

Stock 

Solvent

Final

Volume

(mL)

Primary Stock 

Concentration

(mg/L)

Primary Stock Use

7535A 0.1005 0.1000

Acetonitrile

100 1.00 Secondary stock for 

calibration standards

7535B 0.1007 0.1002 100 1.00

High-level recovery 

samples and secondary 

stocks for low- and midlevel recovery samples

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

Fortifying 

Stock ID

Fortifying

Stock 

Concentration

(mg/L)

Volume of 

Fortification

(mL)

Final 

Volume 

(mL)

Stock 

Solvent Stock ID

Stock 

Concentration

(mg/L)

Stock Use

7535A 1.00 0.500 50.0

Acetonitrile

7535A-1 10.0 Calibration 

standards

7535B

1.00 0.500 50.0 7535B-1 10.0 Low -level 

recovery samples

1.00 5.00 50.0 7535B-2 100 Mid-level recovery 

samples
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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 Fortification and Preparation

Preparation of Recovery Samples:

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

concentrations of 0.0100, 5.00 and 100 mg/L. 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.
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Sample ID Stock Concentration

(mg/L)

Fortification 

Volume

(mL)

Final Volume

(mL)

Fortified 

Concentration 

(mg/L)

Control

A, B & C NAa NA 16.0 0.00

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

a NA = Not Applicable.

Dilution of Recovery Samples:

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

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

of 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.

Sample ID

Fortified

Concentration 

(mg/L)

Sample 

Volume 

(mL)

Final 

Volumea

(mL)

Sample 

Volume

Final 

Volumeb

(mL)

Dilution 

Factor

Control

A, B & C 0.00 16.0 20.0 NAc NA 1.25

Low

A, B & C 0.0100 40.0 50.0 NA NA 1.25

Mid

A, B & C 5.00 16.0 20.0 0.150 10.0 83.3

High

A, B & C 100 16.0 20.0 0.0400 50.0 1560

a Diluted with acetonitrile. b Diluted with 20:80 acetonitrile:purified reagent water (v:v). c NA = Not Applicable.
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ANALYSIS

Instrumental Conditions

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

following instrumental conditions:

Column: Phenomenex Synergi Hydro-RP 4 μm, 250 mm × 4.6 mm

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

Mobile Phase (B): 100% acetonitrile

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

0.00 95.0 5.0

1.00 95.0 5.0

15.00 0.00 100.0

19.00 0.00 100.0

20.00 95.0 5.0

Run Time: 20.00 minutes

Equilibration Delay: 7.00 minutes

Flow Rate: 1.00 mL/min

Injection Volume: 300 μL

Wavelength: 275 nm

Retention Time: approximately 11.5 minutes

Preparation of Standard Curve

Two sets of calibration standards were analyzed with each sample set: one set prior to 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 of the line (equation 1) was algebraically manipulated to give equation 2. The 

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

intercept from the linear regression analysis, the detector response, and the dilution factor of the 
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recovery sample. Equations 2 and 3 were then used to calculate measured concentrations and 

analytical results.

(3) A DC x DF

m

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

(1) y mx b

=

=

= +

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 quantitation (LOQ) was calculated using the following equation:

INST CNTL

LS

INST

(5) LOQ LOQ x DF

m

A - b (4) LOQ

=

=

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
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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 1A. Representative chromatograms from the analysis of a calibration 

standard, a recovery sample and a control sample are presented in Figure 1A, Figure 2A and 

Figure 3A, respectively. A typical regression analysis for sorbic acid is presented in Figure 4A.
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Table 1A. Analytical results for the recovery of sorbic acid from AAP medium 

during the method validation study.

Sample

ID

Fortified Concentration

(mg/L)

Dilution

Factor

Analytical Result

(mg/L)

Percent of Fortified 

(%)

Control A 0.00 1.25 < 0.00747a NAb

Control B 0.00 1.25 < 0.00747 NA

Control C 0.00 1.25 < 0.00747 NA

Low A 0.0100 1.25 0.00982 98.2

Low B 0.0100 1.25 0.0100 100

Low C 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 calculated using the actual analytical (unrounded) results and not the rounded values 

presented in this table.
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Figure 1A. Representative chromatogram of a 0.0585 mg/L calibration 

standard used for quantification during the method validation study 

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

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

method validation study with sorbic acid.

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

of approximately 11.5 minutes.
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Figure 4A. A typical regression analysis for the calibration standards used to 

quantitate sorbic acid in the recovery samples.

0

20

40

60

80

100

120

140

160

180

0.000 0.020 0.040 0.060 0.080 0.100 0.120 0.140

Response

Concentration

Linear Regression Analysis

Linear Regression Analysis

r

2 = 0.99992

y = 1400.898x - 1.7835

Standard

Concentration

(mg/L)

Standard

Response

Area

0.00585 6.60295

0.0117 14.45008

0.0293 39.57698

0.0585 79.97351

0.0878 120.20045

0.117 162.83325

0.00585 6.56322

0.0117 14.59786

0.0293 39.57217

0.0585 79.97565

0.0878 120.43200

0.117 162.79735