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 48 


Springborn 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, Ecotoxicology 
Springborn 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, temperature and light intensity 

measured during the 72-hour exposure of 

Pseudokirchneriella subcapitata to EFKA 8530................................ 23
Springborn 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. EyC10, EyC20, EyC50 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. ErC10, ErC20, ErC50 and No-Observed-Effect 

Concentration (NOEC) values for EFKA 8530 from results 

(growth rate) after 72 hours 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....................................................................................33
Springborn 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 (μave) 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 estimated, 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 maximum concentration required by the OECD 201 

guideline. 
Springborn Smithers Study No. 13658.6320 Page 9 

1.0 INTRODUCTION 

The purpose of this study was to determine the 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, expressed as yield, and average growth rate data 

calculated from the 72-hour cell density counts and average growth rate data, denoted as EyC10, 

EyC20 and EyC50 and ErC10, ErC20 and ErC50, 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 conducted from 29 November to 2 December 2007 at 

Springborn Smithers Laboratories, (SSL) located in Wareham, Massachusetts. All original raw 

data, the protocol and the final 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 Test with Freshwater Green Alga, Pseudokirchneriella 

subcapitata, Following OECD Guideline 201", Springborn Smithers Laboratories Protocol No.: 

050107/OECD/Pss.-STA, nominal (Appendix 1). The methods described in this protocol meet 

the requirements specified in the OECD Guideline for Testing of Chemicals #201, Freshwater 

Alga and Cyanobacteria, Growth 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 substance (SSL No. 126-09) was stored at room 

temperature in the original container in a dark ventilated cabinet. Concentrations are presented 

as mg/L. 

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 alga used in this toxicity test 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 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. None of these compounds have been detected at 

concentrations that are considered toxic in any of the water samples analyzed in agreement with 
Springborn Smithers Study No. 13658.6320 Page 11 

ASTM guidelines (2002). 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 November and December 2007 was 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 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 23 ± 2 o

C and continuous illumination 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 flasks were agitated continuously on 

an orbital shaker. Temperature was controlled 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 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, 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 initiation by placing 0.2000 g of 

EFKA 8530 in a 2000-mL volumetric flask and bringing it to volume with AAP medium. The 

resulting stock solution was observed to be clear 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 soapy, 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 NAa 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 and slightly soapy with no visible 

undissolved test substance. Additional untreated AAP medium was used for the control. 

Replicate 250-mL flasks, three per treatment level and six for the control, were conditioned prior 

to use by rinsing with the appropriate test solution. One hundred milliliters of the appropriate 

test solution was then placed in each replicate flask. Six flasks for the control, as required by the 

OECD guideline, were maintained under the same conditions as the treatment level vessels but 
Springborn Smithers Study No. 13658.6320 Page 13 

contained no EFKA 8530. Each test flask was labeled with the test concentration, 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 104

 cells/mL, was aseptically introduced into each flask. This inoculum 

provided the required cell density of approximately 1.0 x 104

 cells/mL. 

2.8 Test Monitoring 

2.8.1 Algal Growth 

At each subsequent 24-hour interval, a single cell 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 chamber 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-active radiation (PAR) 

range of 60 to 120 μE/m2

/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 flasks in the environmental chamber. Minimum and 
Springborn Smithers Study No. 13658.6320 Page 14 

maximum temperatures and the shaking rate of the 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. Photosynthetically-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 flasks 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 new random positions based on computer-generated 

random numbers. 

Water quality parameters (pH and conductivity) were measured at test initiation and at the 

termination of the 72-hour exposure period. Measurements at test initiation were conducted on 

the test solution remaining after the individual test flasks had been filled. At test termination, 

after cell counts were completed, the three replicate solutions for each treatment and the six 

controls were respectively composited for pH and 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-conductivity-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 examined and multiplying by 10,000 to yield cells/mL.. 

Means and standard deviations for cell density for each treatment and the control were calculated 

from individual replicate values. 

Average specific growth rate (μave) for each replicate flask was calculated for the period from 

test initiation to each observation time using the following equation: 

μ = ln X - ln X

t - t

t 0

t 0
Springborn Smithers Study No. 13658.6320 Page 15 

where: 

μ = specific growth rate (days-1) 

ln = natural logarithm 

X0 = initial cell density in cells/mL 

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

t0 = time of test initiation 

tt = 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 was 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 and normality, therefore, Bonferroni’s Test (Weber et 

al., 1989) was used to determine the NOEC. All statistical 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 applied. 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 (ErC10, ErC20 and 

ErC50 values) and yield (EyC10, EyC20 and EyC50 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 which caused a 10%, 20% or 50% reduction, respectively, 

in average growth rate or yield, compared to the control data. TOXSTAT® version 3.5 (Gulley et 

al., 1996) was used to perform both the statistical (NOEC determinations) and EC10, EC20 and 
Springborn 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 the highest concentration 

tested. 

3.0 RESULTS AND DISCUSSION 

3.1 Preliminary Test 

A preliminary range-finding exposure was conducted 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 observed to be normal. Following 

72 hours of exposure, cell densities in the 0.010, 0.10, 1.0, 10 and 100 mg/L treatment levels 

averaged 111, 106, 106, 107 and 34 x 104 cells/mL, respectively. The control averaged 

111 x 104 cells/mL. Based on these results and consultation 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, temperature and light intensity measurements 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 solution pH ranged from 6.7 to 9.4. The increase in pH 

during the exposure is common in static algal cultures and is due to photosynthesis by the algae. 

Conductivity measured at test initiation and termination in the treatment and control solutions 

ranged from 70 to 80 μmhos/cm. Continuous temperature monitoring established that the 

temperature ranged from 23 to 24 oC during the test period. Light intensity 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/m2/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 exposed 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 104 cells/mL. Cell density in the 6.3, 13, 25, 50 and 100 mg/L treatment levels 

averaged 124.97, 136.47, 114.25, 83.58 and 30.25 x 104

 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 104 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 104 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 treatment-related effects. A significant reduction in 

yield was detected in treatment levels ≥ 50 mg/L compared to the control data. Therefore, the 

72-hour NOEC for yield was determined to be 25 mg/L. The 72-hour EyC10 was empirically 

estimated to be <6.3 mg/L, the lowest concentration tested. The 72-hour EyC20 was determined 

to be 23 mg/L, with 95% confidence intervals of 13 to 33 mg/L. The 72-hour EyC50 was 

determined to be 59 mg/L, with 95% confidence intervals of 50 to 67 mg/L. The 72-hour EyC10, 

EyC20, EyC50and NOEC values determined for 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 Tests, this data set passed the requirements for 

normality and homogeneity of variance, therefore, Bonferroni's Test was used to determine 

treatment-related effects. Bonferroni's Test determined a significant 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 ErC10 was determined to be 
Springborn Smithers Study No. 13658.6320 Page 18 

45 mg/L, with 95% confidence intervals of 37 to 53 mg/L. The 72-hour ErC20 was determined 

to be 72 mg/L, with 95% confidence intervals of 66 to 77 mg/L. The 72-hour ECr50 was 

empirically estimated to be >100 mg/L, the highest concentration tested. The 72-hour ErC10, 

ErC20, ErC50 and NOEC values determined for 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 concentration required by the OECD 201 guideline. 

The following acceptance criteria were required by the protocol: the cell growth in the control 

must increase from the initial density (1.0 x 104 cells/mL) by more than 16 times after 72 hours 

of growth. Additionally, the mean coefficient of variation (CV) for section-by-section specific 

growth rates (day 0 to 1, 1 to 2 and 2 to 3) in the control replicates should 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 104 cells/mL at 72 hours 146.67 x 104 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 density in the control was 146.67 x 104 cells/mL, which 

exceeds the above criterion. The mean 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 replicate 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 Testing 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 freshwater organisms. EPA/600/4-85/014. 

Environmental Monitoring and Support Laboratory, 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. 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 on Good Laboratory Practice (as revised in 

1997). Environment Directorate Chemicals Group and Management Committee. 

ENV/MC/CHEM(98)17. OECD Paris, France. 41 pp. 

OECD, 2006. OECD Guideline for Testing of Chemicals. Freshwater Alga and Cyanobacteria, 

Growth Inhibition Test. Guideline #201. Adopted 23 March 2006. 

U.S. EPA. 1982. Pesticide Assessment Guidelines, Subdivision 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. Klemm, Q.H. Pickering, E.L. Robinson, J.M. 

Lazorchak, L.J. Wymer and R.W. Freyberg (eds.). 1989. Short-term methods for 

estimating the chronic toxicity of effluents and 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 between 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 several 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

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

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

Table 2. The pH, conductivity, temperature 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 Intensitya

 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/m2

/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 104 Nominal cells/mL) 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 104 cells/mL) 

Observation Interval (Hours) Nominal 

Concentration 

(mg/L) 0-24 0-48 0-72 72 Hour 

Percent Inhibitionab 

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) NAc

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 percent 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. EyC10, EyC20, EyC50 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. 

72-Hour Results EyC10 EyC20 EyC50 NOEC 

 

EC value (mg/L): <6.3 23 59 25 

95% Confidence Intervals: NAa 13 - 33 50 - 67 

 

a

 NA = Not Applicable. EC value was empirically estimated, 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 exposure to EFKA 8530. 

Growth Rate (days-1)

Observation Interval (Hours)

Nominal 

Concentration 

(mg/L) 0-24 0-48 0-72 Percent 

Inhibitionab

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) NAc

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. ErC10, ErC20, ErC50 and No-Observed-Effect Concentration 

(NOEC) values for EFKA 8530 from results (growth rate) after 

72 hours of exposure with Pseudokirchneriella subcapitata. 

72-Hour Results ErC10 ErC20 ErC50 NOEC 

 

EC value (mg/L): 45 72 >100 25 

95% Confidence Intervals: 37 - 53 66 - 77 NAa 

 

a

 NA = Not Applicable. EC value was empirically estimated, 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 (section-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 72

Natural 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/L
Springborn 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 104 cells/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 rate (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 PROTOCOL 
Springborn Smithers Study No. 13658.6320 Page 34 
Springborn Smithers Study No. 13658.6320 Page 35 
Springborn Smithers Study No. 13658.6320 Page 36 
Springborn Smithers Study No. 13658.6320 Page 37 
Springborn Smithers Study No. 13658.6320 Page 38 
Springborn Smithers Study No. 13658.6320 Page 39 
Springborn Smithers Study No. 13658.6320 Page 40 
Springborn Smithers Study No. 13658.6320 Page 41 
Springborn Smithers Study No. 13658.6320 Page 42 
Springborn Smithers Study No. 13658.6320 Page 43 
Springborn Smithers Study No. 13658.6320 Page 44 
Springborn Smithers Study No. 13658.6320 Page 45 
Springborn Smithers Study No. 13658.6320 Page 46 
Springborn Smithers Study No. 13658.6320 Page 47 
Springborn Smithers Study No. 13658.6320 Page 48