Study Title
EFKA 8530 – 72-Hour Acute Toxicity Test with Freshwater Green Alga, Pseudokirchneriella subcapitata , Following OECD Guideline 201

Data Requirements

OECD Guideline Number 201

Author

Katherina A. Softcheck

Study Completed On
28 February 2008

Submitted to
Ciba Specialty Chemicals Company 540 White Plains Road Tarrytown, New York 10591

Performing Laboratory
Springborn Smithers Laboratories 790 Main Street Wareham, Massachusetts 02571-1037

Laboratory Project ID
Springborn Smithers Study No. 13658.6320
Page 1 of 48Springborn Smithers Study No. 13658.6320 Page 4



### KEY STUDY PERSONNEL

The following Springborn Smithers personnel were responsible for the conduct of the work and reporting of the study results.

Katherina A. Softcheck Study Director
Kathleen L. Maguire Assistant Biologist
Leonardo Fernandes Assistant Biologist
Katherine Urann Assistant Biologist
Ashlee Kirkwood Assistant Biologist
Susan M. Hamel Technical Report Writer
Susan P. Shepherd Director, EcotoxicologySpringborn Smithers Study No. 13658.6320 Page 5



### TABLE OF CONTENTS

Page
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT.................................... 2 QUALITY ASSURANCE STATEMENT ................................................................................ 3
KEY STUDY PERSONNEL..................................................................................................... 4
TABLE OF CONTENTS........................................................................................................... 5
SUMMARY........................................................................................................................

....... 7
1.0 INTRODUCTION............................................................................................................ 9
2.0 MATERIALS AND METHODS ..................................................................................... 9
2.1 Protocol............................................................................................................. 9 2.2 Test Substance ................................................................................................ 10 2.3 Test Organism................................................................................................. 10 2.4 Test Dilution Water......................................................................................... 11 2.5 Test Concentrations ........................................................................................ 12 2.6 Test Solution Preparation................................................................................ 12 2.7 Test Initiation.................................................................................................. 13 2.8 Test Monitoring .............................................................................................. 13 2.8.1 Algal Growth ...................................................................................... 13 2.8.2 Test Conditions ................................................................................... 13 2.9 Determination of EC10, EC20, EC50 and NOEC .......................................... 14 3.0 RESULTS AND DISCUSSION .................................................................................... 16
3.1 Preliminary Test.............................................................................................. 16 3.2 Definitive Test ................................................................................................ 16 3.2.1 Evaluation of Test Conditions ............................................................ 16 3.2.2 Biological Results ............................................................................... 17 PROTOCOL DEVIATIONS ................................................................................................... 19
REFERENCES .....................................................................................................................

... 20 Table 1. Composition of algal growth medium (AAP medium) used during the 72-hour toxicity test exposing Pseudokirchneriella subcapitata to EFKA 8530................................ 22 Table 2. The pH, conductivity, temperat ure and light intensity measured during the 72-hour exposure of Pseudokirchneriella subcapitata to EFKA 8530................................ 23Springborn Smithers Study No. 13658.6320 Page 6

Table 3. Cell density of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of exposure to EFKA 8530............................................ 24 Table 4. Yield of Pseudokirchneriella subcapitata after 72 hours of exposure to EFKA 8530...................................................................... 25 Table 5. E y C10, E y C20, E y C50 and No-Observed-Effect Concentration (NOEC) values for EFKA 8530 estimated from results (biomass expressed as yield) after 72 hours of exposure with Pseudokirchneriella subcapitata

................................. 26 Table 6. Calculated growth rates of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of exposure to EFKA 8530. ....................... 27 Table 7. E r C10, E r C20, E r C50 and No-Observed-Effect Concentration (NOEC) values for EFKA 8530 from results (growth rate) after 72 ho urs of exposure with Pseudokirchneriella subcapitata

. ....................................................... 28 Table 8. Acceptance criteria required by the OECD 201 guideline from the results of the 72-hour exposure of Pseudokirchneriella subcapitata to EFKA 8530................................ 29 Figure 1. Algal growth curves (cell density vs. time) for Pseudokirchneriella subcapitata exposed to EFKA 8530.................. 30 Figure 2. Yield (72-hour) for Pseudokirchneriella subcapitata exposed to EFKA 8530....................................................................... 31 Figure 3. Average growth rate (0- to 72-hour) for Pseudokirchneriella subcapitata exposed to EFKA 8530.................. 32 APPENDIX 1 - STUDY PROTOCOL.................................................................................... 33Springborn Smithers Study No. 13658.6320 Page 7



### SUMMARY

EFKA 8530 – 72-Hour Acute Toxicity Test with Freshwater Green Alga, Pseudokirchneriella subcapitata , Following OECD Guideline 201

STUDY SPONSOR: Ciba Specialty Chemicals Company

PROTOCOL TITLE: 72-Hour Acute Toxicity Test with Freshwater Green Alga, Pseudokirchneriella subcapitata , Following OECD Guideline 201, Springborn Smithers Protocol No.: 050107/OECD/Pss.-STA, nominal

SPRINGBORN SMITHERS STUDY NUMBER: 13658.6320

TEST SUBSTANCE: EFKA-8530, Lot No. 15306, tested as 100%, was received from Ciba Specialty Chemicals Company on 31 October 2007

DEFINITIVE EXPOSURE DATES: 29 November to 2 December 2007

TEST ORGANISM: Pseudokirchneriella subcapitata , inoculum - three days since previous transfer Source - Springborn Smithers (Wareham) culture

TEST CONDITIONS: 72-hour duration, 23 to 24 oC, continuous illumination at 420 to 540 footcandles (4500 to 5800 lux), shaking rate of 100 rpm.

DILUTION WATER: Algal Assay Procedure (AAP) medium

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

EFFECT CRITERIA: 0- to 72-hour average growth rate (μ ave ) and biomass expressed as yield relative to the performance of the control data.Springborn Smithers Study No. 13658.6320 Page 8

RESULTS:

Based on Nominal Concentrations (mg/L) Biological Parameter EC10 (95% Confidence Intervals) EC20 (95% Confidence Intervals) EC50 (95% Confidence Intervals) NOEC

0 - 72-Hour Yield <6.3 (NA) a 23 (13 - 33) 59 (50 - 67) 25 0 - 72-Hour Average Growth Rate 45 (37 - 53) 72 (66 - 77) >100 (NA) 25

a NA = Not Applicable. EC value was empirically estima ted, therefore 95% confidence intervals could not be determined. Additional testing to further define the EC50 values was not performed since the highest concentration tested represents the maxi mum concentration requi red by the OECD 201 guideline.Springborn Smithers Study No. 13658.6320 Page 9



### 1.0 INTRODUCTION

The purpose of this study was to determine th e effect of EFKA 8530 on the growth of the freshwater green alga, Pseudokirchneriella subcapitata , formerly Selenastrum capricornutum

. The results are based on nominal concentrations of EFKA 8530 and are reported as the 72-hour EC10, EC20 and EC50 values for biomass, expresse d as yield, and averag e growth rate data
calculated from the 72-hour cell de nsity counts and average grow th rate data, denoted as E y C10,
E y C20 and E y C50 and E r C10, E r C20 and E r C50, respectively, and the No-Observed-Effect Concentration (NOEC) values for total yield and average growth rate.

The study was initiated on 20 November 2007, 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 72-hour acute toxicity test was c onducted from 29 November to 2 December 2007 at Springborn Smithers Laboratories, ( SSL) located in Wareham, Massachusetts. All original raw data, the protocol and the fina l report produced during this study are stored in Springborn
Smithers' archives at the above location.



### 2.0 MATERIALS AND METHODS

2.1 Protocol

The toxicity test was performed according to the Springborn Smithers Laboratories protocol entitled "72-Hour Acute Toxicity Te st with Freshwater Green Alga, Pseudokirchneriella subcapitata , Following OECD Guideline 201", Springbor n Smithers Laboratories Protocol No.: 050107/OECD/Pss.-STA, nominal ( Appendix 1 ). The methods described in this protocol meet the requirements specified in the OECD Guid eline for Testing of Chemicals #201, Freshwater Alga and Cyanobacteria, Gr owth Inhibition Test ( OECD, 2006 ).Springborn Smithers Study No. 13658.6320 Page 10



### 2.2 Test Substance

The test substance, EFKA-8530, was received on 31 October 2007 from Ciba Specialty Chemicals Company, Allentown, Pennsylvania. The following information was provided:

Name: EFKA-8530 Lot No.: 15306 CAS No.: Not Listed Purity: Tested as 100% Recertification Date: 31 January 2009

Upon receipt at Springborn Smithers, the test su bstance (SSL No. 126-09) was stored at room temperature in the original contai ner in a dark ventilated cabinet. Concentrations are presented as mg/L.

Determination of stability and characterizati on, verification of the te st 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 alga used in this toxicity te st was the freshwater green alga, Pseudokirchneriella subcapitata , formerly Selenastrum capricornutum , strain 1648, Class Chlorophyceae. The alga was obtained from University of Texas, Austin, Texas, and was maintained in stock culture at Springborn Smithers.

The culture medium used was Algal Assay Proc edure (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 so urce used in the preparation of the culture medium were analyzed periodically for the pr esence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed in agreement withSpringborn Smithers Study No. 13658.6320 Page 11
ASTM guidelines ( 2002 ). In addition, a representative sa mple of AAP medium was analyzed monthly for total organic carbon (TOC) concentra tion. The TOC concentration of the sample
collected in November and December 2007 wa s 0.26 and 0.59 mg/L, respectively. 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 either 0.1 N hydrochloric acid or 0.1 N sodium hydroxide. Stock cultures were grown in 250-mL glass flasks each containing 100 mL of medium. The flasks were c overed 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 23 ± 2 o C and continuous illuminati on at the surface of the medium with an intensity range of 4500 to 5900 lux (420 to 550 footcandles). Lighting was supplied by Premira VitaLux ® fluorescent bulbs. Culture flas ks were agitated continuously on an orbital shaker. Temperature was cont rolled using an environmental chamber.

The inoculum used to initiate the toxicity test with EFKA 8530 was taken from a stock culture that had been transferred to fresh medium three days before testing.



### 2.4 Test Dilution Water

The AAP medium used to prepare the exposure so lutions was formulated in the same manner as
the culture medium. Several liters of AAP medi um were prepared using sterile, deionized water and were equilibrated to test temperature. Th e initial pH of this medium was adjusted, if necessary, to 7.5 ± 0.1 prior to use.Springborn Smithers Study No. 13658.6320 Page 12



### 2.5 Test Concentrations

Based on the results of preliminary testing (see Section 3.1 ) conducted at Springborn Smithers and consultation with the Study Sponsor, nominal concentrations of 6.3, 13, 25, 50 and 100 mg/L were selected for the definitive exposure.



### 2.6 Test Solution Preparation

A 100 mg/L stock solution was prepared prior to test in itiation by placing 0.2000 g of
EFKA 8530 in a 2000-mL volumetric flask and bri nging it to volume with AAP medium. The resulting stock solution was observed to be cl ear and soapy with no visible undissolved test substance. Following approximately one hour of mixing with a Teflon ® -coated stir bar and magnetic stir plate, the solution was observed to be slightly soa py, with no visible undissolved test substance. Test solutions were prepared from dilutions of the 100 mg/L stock solution as
follows:

Stock Concentration (mg/L) Volume of Stock Used (mL) Diluted to (mL) with AAP Medium Nominal Concentration (mg/L)
100 500 NA a 100

100 500 1000 50

100 250 1000 25

100 130 1000 13

100 63 1000 6.3

a NA = Not Applicable.
All resulting test solutions were observed to be clear, colorless a nd slightly soapy with no visible
undissolved test substance. Additional untreat ed AAP medium was used for the control.

Replicate 250-mL flasks, three per treatment leve l and six for the control, were conditioned prior to use by rinsing with the appropriate test so lution. One hundred milliliters of the appropriate test solution was then placed in each replicate flas k. Six flasks for the control, as required by the OECD guideline, were maintained under the same conditions as the treatment level vessels butSpringborn Smithers Study No. 13658.6320 Page 13

contained no EFKA 8530. Each test flask was labele d with the test concentr ation, replicate, test species and study number. All test vessels were fitted with stainless steel caps which permitted gas exchange.



### 2.7 Test Initiation

Approximately 45 minutes following preparation of the test solutions and addition to the exposure flasks (100 mL/flask), 2.6 mL of an inoculum of Pseudokirchneriella subcapitata cells, at a density of 38 x 10 4 cells/mL, was aseptically introduced into each flask. This inoculum provided the required cell dens ity of approximately 1.0 x 10 4 cells/mL.



### 2.8 Test Monitoring

2.8.1 Algal Growth

At each subsequent 24-hour interval, a single ce ll count was conducted on each replicate solution
of the treatment levels and the six controls using a hemacytometer (Neubauer Improved) and a compound microscope. One sample was removed from each flask for counting. 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 for each sample until at least 400 algal cells or four fields
were counted. Observations of the health of the algal cells were made at each 24-hour interval.

2.8.2 Test Conditions

The test was conducted in an environmental cham ber designed to maintain the test conditions specified in the protocol: a temperature of 23 ± 2 o C, continuous light intensity of 4500 to 5900 lux (420 to 550 footcandles, see Section 2.3 ) and photosynthetically-a ctive radiation (PAR) range of 60 to 120 μE/m 2 /s. An orbital shaker table 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 flas ks in the environmental chamber. Minimum andSpringborn Smithers Study No. 13658.6320 Page 14
maximum temperatures and the shaking rate of th e orbital shakers were recorded daily. Light intensity was measured at each flask location with a VWR Traceable light meter at 0-hour and at each 24-hour interval during the exposure period. Light intensity was measured in footcandles
and converted to lux based on 1 footcandle = 10.76 lux. Photosyntheti cally-active radiation (PAR) of the test area was measured at test initiation using a Licor Model LI-189SA photometer
and a model LI-190SA radiation sensor. Test fl asks were randomly placed on the shaking table at test initiation based on computer-generated random numbers. Following each observation interval, the test flasks were assigned ne w random positions based on computer-generated
random numbers.

Water quality parameters (pH and conductivity) we re measured at test initiation and at the termination of the 72-hour exposure period. Measur ements at test initia tion were conducted on the test solution remaining after the individual test flasks had b een filled. At test termination, after cell counts were completed, the three repl icate solutions for each treatment and the six controls were respectively composited for pH a nd conductivity measurements. Test solution pH was measured with a Jenco Model 60 pH meter, and conductivity was measured with a Yellow Springs Instrument (YSI) Model No. 33 salinity-conductiv ity-temperature meter.



### 2.9 Determination of EC10, EC20, EC50 and NOEC

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 exam ined and multiplying by 10,000 to yield cells/mL.. Means and standard deviations for cell density fo r each treatment and the control were calculated from individual replicate values.

Average specific growth rate (μ ave ) for each replicate flask was cal culated for the period from
test initiation to each observati on time using the following equation:

μ = ln X - ln X
t - t

t 0
t 0Springborn Smithers Study No. 13658.6320 Page 15

where:

μ = specific growth rate (days -1 ) ln = natural logarithm X 0 = initial cell density in cells/mL X t = cell density at the specified time interval in cells/mL t 0 = time of test initiation t t

= time of observation interval in days (i.e., 1, 2, 3)

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

Percent inhibition of the treatment data wa s calculated relative to the control data.

Based on the results of statistical analysis performed for 72-hour total yield and average growth rate, the No-Observed-Effect Concentration ( NOEC), the highest test concentration which demonstrated no statistically adverse effect (p ≤ 0.05) when compared to the control data, was determined. The data were first checked for normality using Shapiro-Wilks' Test ( Weber et al.,
1989 ) and for homogeneity of variance using Bartlett's Test ( Horning and Weber, 1985 ). The data sets passed the tests for homogeneity a nd normality, therefore, Bonferroni’s Test ( Weber et al., 1989 ) was used to determine the NOEC. All statis tical determinations were made at the 95% level of certainty, except in the case of Shapiro-Wilks' and Bartlett's Tests, where the 99% level of certainty was a pplied. TOXSTAT ® version 3.5 ( Gulley et al

. , 1996 ) was used to perform
these calculations.

At test termination, the EC values were calculated for average growth rate (E r C10, E r C20 and
E r C50 values) and yield (E y C10, E y C20 and E y C50 values) by linear regression of response (percent reduction of yield and growth rate as compared with the control) versus the nominal EFKA 8530 concentration ( Norberg-King, 1993 ). The EC10, EC20 and EC50 values are defined
as the concentration of test substance whic h caused a 10%, 20% or 50% reduction, respectively, in average growth rate or yield, comp ared to the control data. TOXSTAT ® version 3.5 ( Gulley et al., 1996 ) was used to perform both the statistica l (NOEC determinations) and EC10, EC20 andSpringborn Smithers Study No. 13658.6320 Page 16
EC50 calculations. If less than the required response was observed (i.e., < 10, < 20 and < 50% response), the EC value was empirically estimated to be greater than th e highest concentration
tested.



### 3.0 RESULTS AND DISCUSSION

3.1 Preliminary Test

A preliminary range-finding exposure was c onducted at Springborn Smithers at nominal EFKA 8530 concentrations of 0.010, 0.10, 1.0, 10 and 100 mg/L, and a control. Two exposure
vessels were established for each concentration and the control. At test termination, cells exposed to all treatment levels tested and the control were obs erved to be normal. Following 72 hours of exposure, cell densities in th e 0.010, 0.10, 1.0, 10 and 100 mg/L treatment levels averaged 111, 106, 106, 107 and 34 x 10 4 cells/mL, respectively. The control averaged
111 x 10 4 cells/mL. Based on these results and c onsultation with the Study Sponsor, nominal concentrations of 6.3, 13, 25, 50 and 100 mg/L were selected for the definitive exposure.



### 3.2 Definitive Test

3.2.1 Evaluation of Test Conditions

The pH, conductivity, temperatur e and light intensity measur ements recorded during the definitive test are presented in Table 2 . The pH of the test solutions ranged from 5.4 to 6.9 at test initiation. At test termination, the test soluti on pH ranged from 6.7 to 9.4. The increase in pH during the exposure is common in st atic algal cultures and is due to photosynthesis by the algae. Conductivity measured at test in itiation and termination in the treatment and control solutions ranged from 70 to 80 μmhos/cm. Continuous te mperature monitoring established that the temperature ranged from 23 to 24 oC during the te st period. Light intens ity of the test area ranged from 420 to 540 footcandles (4500 to 5800 lux) . The PAR of the test area measured at test initiation ranged from 78 to 89 μE/m 2 /s. The shaking rate was maintained throughout the exposure at a constant rate of 100 rpm.Springborn Smithers Study No. 13658.6320 Page 17

3.2.2 Biological Results Cell densities determined at each observation interval are presented in Table 3 and illustrated in Figure 1 . At test termination (72 hours), cells expo sed to the treatment levels tested and the
control were observed to be normal. The 72-hour cell density in the control averaged
146.67 x 10 4 cells/mL. Cell density in the 6.3, 1 3, 25, 50 and 100 mg/L treatment levels averaged 124.97, 136.47, 114.25, 83.58 and 30.25 x 10 4 cells/mL, respectively.

Cell biomass, expressed as yield, is presented in Table 4 and illustrated in Figure 2 . The 0- to 72-hour yield in the control averaged 145.67 x 10 4 cells/mL. The 0- to 72-hour yield in the 6.3, 13, 25, 50 and 100 mg/L treatment levels averaged 123.97, 135.47, 113.25, 82.58 and
29.25 x 10 4 cells/mL, respectively. Based on the results of Shapiro-Wilks' and Bartlett's Tests, this data set passed the requirements for normality and homogeneity of variance, therefore,
Bonferroni's Test was used to determine treatme nt-related effects. A significant reduction in yield was detected in treatment levels ≥ 50 mg/L compared to the c ontrol data. Therefore, the 72-hour NOEC for yield was determined to be 25 mg/L. The 72-hour E y C10 was empirically estimated to be <6.3 mg/L, the lowest concentration tested. The 72-hour E y C20 was determined to be 23 mg/L, with 95% confidence inte rvals of 13 to 33 mg/L. The 72-hour E y C50 was determined to be 59 mg/L, with 95% confiden ce intervals of 50 to 67 mg/L. The 72-hour E y C10,
E y C20, E y C50and NOEC values determined fo r this test are presented in Table 5

.

Growth rates are presented in Table 6 and illustrated in Figure 3 . The 0- to 72-hour growth rate in the control averaged 1.68 days -1 . The 0- to 72-hour growth rate in the 6.3, 13, 25, 50 and 100 mg/L treatment levels averaged 1.62, 1.66, 1.60, 1.49 and 1.15 days -1 , respectively. Based on the results of Shapiro-Wilks’ and Bartlett’s Te sts, this data set pass ed the requirements for normality and homogeneity of variance, therefore, Bonferroni's Test was used to determine
treatment-related effects. Bonf erroni's Test determined a signi ficant reduction in average growth
rate in treatment levels ≥ 50 mg/L compared to the control data. Based on these results, the 72-hour NOEC was determined to be 25 mg/L. The 72-hour E r C10 was determined to beSpringborn Smithers Study No. 13658.6320 Page 18
45 mg/L, with 95% confidence interval s of 37 to 53 mg/L. The 72-hour E r C20 was determined to be 72 mg/L, with 95% confidence inte rvals of 66 to 77 mg/L. The 72-hour EC r 50 was empirically estimated to be >100 mg/L, the highest concentration tested. The 72-hour E r C10,
E r C20, E r C50 and NOEC values determined fo r this test are presented in Table 7 . Additional testing to further define the EC50 values was not performed since the highest concentration tested represents the maximum concentr ation required by th e OECD 201 guideline.

The following acceptance criteria were required by th e protocol: the cell growth in the control must increase from the initial density (1.0 x 10 4 cells/mL) by more than 16 times after 72 hours of growth. Additionally, the mean coefficient of variation (CV) for s ection-by-section specific growth rates (day 0 to 1, 1 to 2 and 2 to 3) in the control replicates shou ld not exceed 35%. The CV for the average growth rate of the control for the entire test period (0 - to 72-hour growth rate)
should not exceed 7%. The results of this test are presented in the following table:

Acceptance Criteria for Control Study Results
Exceed 16 x 10 4 cells/mL at 72 hours 146.67 x 10 4 cells/mL

Mean daily growth rate CV < 35% 21% 0 - 72-hour growth rate CV < 7% 3.57%

During this study, the 72-hour cell de nsity in the control was 146.67 x 10 4 cells/mL, which
exceeds the above criterion. The me an daily CV for growth rates was 21% and the CV for 0- to 72-hour growth rate was 3.57%, which meet the above criteria. The replicat e daily growth rates, overall growth rates and coefficient of variance for these values are presented in Table 8

.Springborn Smithers Study No. 13658.6320 Page 19



### PROTOCOL DEVIATIONS

No deviations from the protocol occurred during this study.Springborn Smithers Study No. 13658.6320 Page 20



### REFERENCES

ASTM. 2002. Conducting acute toxicity tests with fishes, macroinvertebrates and amphibians. Standard E729-96. American Society for Tes ting and Materials, 100 Barr Harbor drive, West Conshohocken, PA 19428.
Gulley, D.D., Boetler, A.M. and Bergman, H.L. 1996 TOXSTAT ® Release 3.5. University of Wyoming, Laramie, Wyoming.
Horning, W.B. and C.I. Weber. 1985. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to fre shwater organisms. EPA/600/4-85/014. Environmental Monitoring and Support Labor atory, U.S. Environmental Protection Agency, Cincinnati, Ohio.
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. Envi ronmental Protection Agency, Corvallis, Oregon.
Norberg-King, Teresa J. 1993. A Linear Interpol ation Method for Subl ethal Toxicity: The Inhibition Concentration (ICp) Approach. Na tional Effluent Toxicity Assessment Center, Environmental Research Labor atory – Duluth, U.S. Envir onmental protection Agency, Duluth, Minnesota. Technical report 03-93.
OECD, 1998. OECD Series on Principles of Good Laboratory Pr actice and Compliance Monitoring. Number 1. OECD Principles on Good Laboratory Practice (as revised in 1997). Environment Directorate Chemi cals Group and Management Committee. ENV/MC/CHEM(98)17. OECD Paris, France. 41 pp.
OECD, 2006. OECD Guideline for Testing of Chem icals. Freshwater Alga and Cyanobacteria, Growth Inhibition Test. Guid eline #201. Adopted 23 March 2006.

U.S. EPA. 1982. Pesticide Assessment Guidelines, Subdivisi on J, Hazard Evaluation: Nontarget Plants . EPA 540/9-82-020. U.S> EPA, Washington, DC.
Weber, C.I., W.H. Peltier, T.J. Norberg-King, W. B. Horning II, F.A. Kessler, J.R. Menkedick, T.W. Neiheisel, P.A. Lewis, D.J. Kle mm, Q.H. Pickering, E.L. Robinson, J.M. Lazorchak, L.J. Wymer and R.W. Freybe rg (eds.). 1989. Short-term methods for estimating the chronic toxicity of effluents a nd receiving waters to freshwater organisms. 2nd ed. EPA/600/4/89/001. Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH.
Williams, D.A. 1971. A test for differences betw een treatment means when several dose levels are compared with a zero dose control. Biometrics 27: 103-117.Springborn Smithers Study No. 13658.6320 Page 21
Williams, D.A. 1972. A comparison of seve ral dose levels with a zero control. Biometrics 28: 519-531.Springborn Smithers Study No. 13658.6320 Page 22


### Table 1. Composition of algal growth medium (AAP medium) used during the 72-hour toxicity test exposing Pseudokirchneriella subcapitata to EFKA 8530.

Compound Concentration
NaNO 3 25.5 mg/L MgCl 2 •6H 2 O 12.16 mg/L
CaCl 2 •2H 2 O 4.41 mg/L MgSO 4 •7H 2 O 14.7 mg/L

K 2 HPO 4 •3H 2 O 1.368 mg/L

NaHCO 3 15.0 mg/L

H 3 BO 3 185.5 μg/L

Na 2 SeO 4 a 1.88 μg/L
MnCl 2 •4H 2 O 415.4 μg/L
ZnCl 2 3.270 μg/L
CoCl 2 •6H 2 O 1.43 μg/L
CuCl 2 •2H 2 O 0.012 μg/L

Na 2 MoO 4 •2H 2 O 7.26 μg/L
FeCl 3 •6H 2 O 160 μg/L

Na 2 EDTA•2H 2 O 300 μg/L

If required, pH was adjusted to 7.5 ± 0.1 with 0.1 N NaOH or 0.1 N HCl

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

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. Enviro nmental Protection Agency, Corvallis, Oregon.Springborn Smithers Study No. 13658.6320 Page 23


### Table 2. The pH, conductivity, temper ature and light intensity measured during the 72-hour exposure of Pseudokirchneriella subcapitata to EFKA 8530.

Nominal pH Conductivity Concentration (μmhos/cm) (mg/L) 0 Hour 72 Hour 0 Hour 72 Hour

Control 6.9 8.9 80 70

6.3 6.7 9.4 80 80

13 6.7 9.0 80 70

25 6.6 9.0 80 70

50 6.3 7.5 80 70

100 5.4 6.7 80 70

Minimum/Maximum Temperature ( o C)
0 - 24 Hour 24 - 48 Hour 48 - 72 Hour

23/24 23/24 23/24

Light Intensity a
0 Hour 24 Hour 48 Hour 72 Hour

footcandles: 450 - 500 420 -510 430 - 540 420 - 540
lux: 4800 - 5300 4500 - 5400 4600 - 5800 4500 - 5800

a The photosynthetically-active radiation of the test area at test initiation ranged from 78 to 89 μE/m 2 /s.Springborn Smithers Study No. 13658.6320 Page 24


### Table 3. Cell density of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of exposure to EFKA 8530.

Cell Density (x 10 4 cells/mL) Nominal Concentration (mg/L) Observation Interval (Hours) 24 48 72

Control A 7.00 33.25 107.50 B 4.00 28.50 147.00 C 4.25 16.50 157.33 D 4.00 30.00 127.50 E 7.75 26.25 179.33 F 5.00 29.50 161.33 Mean (SD) a 5.33(1.64) 27.33(5.77) 146.67(25.68) 6.3 A 5.50 25.75 141.67

B 4.25 35.25 140.00 C 4.50 22.00 93.25 Mean (SD) 4.75(0.66) 27.67(6.83) 124.97(27.48) 13 A 2.25 30.25 125.25 B 5.25 32.50 158.67 C 4.75 32.75 125.50 Mean (SD) 4.08(1.61) 31.83(1.38) 136.47(19.22) 25 A 3.75 23.75 115.25

B 5.25 19.75 106.75 C 3.50 16.50 120.75 Mean (SD) 4.17(0.95) 20.00(3.63) 114.25(7.05) 50 A 1.50 14.00 87.75

B 1.25 11.25 79.50 C 2.50 12.50 83.50 Mean (SD) 1.75(0.66) 12.58(1.38) c 83.58(4.13) 100 A 1.25 7.75 29.50

B 4.25 6.50 31.50 C 2.50 7.25 29.75 Mean (SD) 2.67(1.51) b 7.17(0.63) cd 30.25(1.09)

a Mean and standard deviation (SD) were calculated from original raw data, not from the rounded values presented in this table. b Cells were observed to be slightly bloated.

c Cells were observed to be bloated. d Cell fragments were observed.Springborn Smithers Study No. 13658.6320 Page 25



### Table 4. Yield of Pseudokirchneriella subcapitata after 72 hours of exposure to EFKA 8530.

Yield (x 10 4 cells/mL) Observation Interval (Hours) Nominal Concentration (mg/L) 0-24 0-48 0-72 72 Hour Percent Inhibition ab Control A 6.00 32.25 106.50 B 3.00 27.50 146.00 C 3.25 15.50 156.33 D 3.00 29.00 126.50 E 6.75 25.25 178.33 F 4.00 28.50 160.33 Mean (SD) b 4.33(1.64) 26.33(5.77) 145.67(25.68) NA c 6.3 A 4.50 24.75 140.67

B 3.25 34.25 139.00 C 3.50 21.00 92.25 Mean (SD) 3.75(0.66) 26.67(6.83) 123.97(27.48) 15 13 A 1.25 29.25 124.25 B 4.25 31.50 157.67 C 3.75 31.75 124.50 Mean (SD) 3.08(1.61) 30.83(1.38) 135.47(19.22) 7 25 A 2.75 22.75 114.25

B 4.25 18.75 105.75 C 2.50 15.50 119.75 Mean (SD) 3.17(0.95) 19.00(3.63) 113.25(7.05) 22 50 A 0.50 13.00 86.75

B 0.25 10.25 78.50 C 1.50 11.50 82.50 Mean (SD) 0.75(0.66) 11.58(1.38) 82.58(4.13) d 43 100 A 0.25 6.75 28.50

B 3.25 5.50 30.50 C 1.50 6.25 28.75 Mean (SD) 1.67(1.51) 6.17(0.63) 29.25(1.09) d 80

a Percent inhibition relative to the control. b Mean, standard deviation (SD) and perc ent inhibition are 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 Bonferroni’s' Test.Springborn Smithers Study No. 13658.6320 Page 26



### Table 5. E y 

### C10, E y 

### C20, E y 

### C50 and No-Observed-Effect Concentration (NOEC) values for EFKA 8530 es timated from results (biomass expressed as yield) after 72 hours of exposure with Pseudokirchneriella subcapitata

.

72-Hour Results E y C10 E y C20 E y C50 NOEC
EC value (mg/L): <6.3 23 59 25 95% Confidence Intervals: NA a 13 - 33 50 - 67

a NA = Not Applicable. EC value was empirically estima ted, therefore 95% confidence intervals could not be determined

.Springborn Smithers Study No. 13658.6320 Page 27


### Table 6. Calculated growth rates of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of ex posure to EFKA 8530.

Growth Rate (days -1 ) Observation Interval (Hours) Nominal Concentration (mg/L) 0-24 0-48 0-72 Percent Inhibition ab Control A 2.19 1.85 1.58 B 1.56 1.77 1.68 C 1.63 1.48 1.71
D 1.56 1.80 1.64
E 2.30 1.73 1.75
F 1.81 1.79 1.72 Mean(SD) b 1.84(0.33) 1.74(0.13) 1.68(0.06) NA c
6.3 A 1.92 1.72 1.67

B 1.63 1.88 1.67 C 1.69 1.63 1.53 Mean(SD) 1.75(0.15) 1.74(0.13) 1.62(0.08) 4 13 A 0.91 1.80 1.63
B 1.87 1.84 1.71 C 1.75 1.84 1.63 Mean(SD) 1.51(0.52) 1.83(0.02) 1.66(0.05) 1 25 A 1.49 1.67 1.60

B 1.87 1.58 1.58 C 1.41 1.48 1.62 Mean(SD) 1.59(0.24) 1.58(0.10) 1.60(0.02) 5 50 A 0.46 1.39 1.51

B 0.25 1.28 1.48 C 1.03 1.33 1.49 Mean(SD) 0.58(0.40) 1.34(0.06) 1.49(0.02) d 11 100 A 0.25 1.08 1.14

B 1.63 0.99 1.16 C 1.03 1.05 1.15 Mean(SD) 0.97(0.69) 1.04(0.05) 1.15(0.01) d 32

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 Bonferroni’s Test.Springborn Smithers Study No. 13658.6320 Page 28



### Table 7. E r 

### C10, E r 

### C20, E r 

### C50 and No-Observed-Effect Concentration (NOEC) values for EFKA 8530 fr om results (growth rate) after 72 hours of exposure with Pseudokirchneriella subcapitata

.

72-Hour Results E r C10 E r C20 E r C50 NOEC
EC value (mg/L): 45 72 >100 25 95% Confidence Intervals: 37 - 53 66 - 77 NA a

a NA = Not Applicable. EC value was empirically estima ted, therefore 95% confidence intervals could not be determined.Springborn Smithers Study No. 13658.6320 Page 29


### Table 8. Acceptance criteria required by the OECD 201 guideline from the results of the 72-hour exposure of Pseudokirchneriella subcapitata to EFKA 8530.

Growth Rate (days -1 )
Interval (Days) 0-24 Hour (0.889 days) 24-48 Hours (1.00 days) 48-72 Hours (1.07 days) Mean (SD) % CV of Daily Growth Rate 0-72 Hour (2.96 days)
Control A 2.19 1.55 1.10 1.61(0.55) 34.16 1.58 B 1.56 1.96 1.53 1.68(0.24) 14.29 1.68 C 1.63 1.35 2.11 1.70(0.38) 22.35 1.71 D 1.56 2.01 1.35 1.64(0.33) 20.12 1.64 E 2.30 1.22 1.80 1.77(0.54) 30.51 1.75 F 1.81 1.77 1.59 1.72(0.12) 6.98 1.72 Mean % CV of section-by-section growth rate:

Mean (SD) of overall 0-72 hour growth rates: 21% 1.68(0.06) % CV of 0-72 hour overall growth rate: 3.57%

NOTE: Guideline validation criteria require: 1) daily (s ection-by-section) growth rate CV to be < 35%, and 2) overall growth rate CV to be <7%.Springborn Smithers Study No. 13658.6320 Page 30


### Figure 1. Algal growth curves (cell density vs. time) for Pseudokirchneriella subcapitata exposed to EFKA 8530.



### Time (Hours) 0 24 48 72Natural Log Cell Density (cells/mL)

6 8 10 12 14 Control 6.3 mg/L 13 mg/L 25 mg /L 50 mg/L 100 mg/LSpringborn Smithers Study No. 13658.6320 Page 31


### Figure 2. Yield (72-hour) for Pseudokirchneriella subcapitata exposed to EFKA 8530.



### Nominal Concentration (mg/L)Yield (x 104cells/mL)



### 0 20 40 60 80 100 120 140 160

* *

Control 6. 3 13 25 50 100

* Significantly reduced compared to the control, based on Bonferroni's Test.Springborn Smithers Study No. 13658.6320 Page 32


### Figure 3. Average growth ra te (0- to 72-hour) for Pseudokirchneriella subcapitata exposed to EFKA 8530.



### Nominal Concentration (mg/L)Growth Rate (days-1)



### 0.00 0.40 0.80 1.20 1.60

* *

Control 6.3 13 25 50 100

* Significantly reduced compared to the control, based on Bonferroni's Test.Springborn Smithers Study No. 13658.6320 Page 33



### APPENDIX 1 - STUDY PROTOCOLSpringborn Smithers Study No. 13658.6320 Page 34Springborn Smithers Study No. 13658.6320 Page 35Springborn Smithers Study No. 13658.6320 Page 36Springborn Smithers Study No. 13658.6320 Page 37Springborn Smithers Study No. 13658.6320 Page 38Springborn Smithers Study No. 13658.6320 Page 39Springborn Smithers Study No. 13658.6320 Page 40Springborn Smithers Study No. 13658.6320 Page 41Springborn Smithers Study No. 13658.6320 Page 42Springborn Smithers Study No. 13658.6320 Page 43Springborn Smithers Study No. 13658.6320 Page 44Springborn Smithers Study No. 13658.6320 Page 45Springborn Smithers Study No. 13658.6320 Page 46Springborn Smithers Study No. 13658.6320 Page 47Springborn Smithers Study No. 13658.6320 Page 48