P R O T O C O L 

 A 72-HOUR TOXICITY TEST WITH 

THE FRESHWATER ALGA (Pseudokirchneriella subcapitata) 

OECD Proposal for Updating Guideline 201 

EU Directive 92/69/EEC, Method C.3. 
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 A 72-HOUR TOXICITY TEST WITH 

THE FRESHWATER ALGA (Pseudokirchneriella subcapitata) 

SPONSOR: 

 

SPONSOR’S REPRESENTATIVE: 

TESTING FACILITY: 

STUDY DIRECTOR:

LABORATORY MANAGEMENT:

FOR LABORATORY USE ONLY 

Proposed Dates: 

Experimental

Start Date: ________________________ 

Experimental

Termination Date: _________________ 

Project No.: Sponsor Approval Date: 

Test Concentrations: _________________________________________________________ 

Test Substance No.: ___________ Reference Substance No. (if applicable): ____________ 

PROTOCOL APPROVAL

____________________________________ ___________________________ 

STUDY DIRECTOR DATE 

____________________________________ ___________________________ 

LABORATORY MANAGEMENT DATE 

____________________________________ ___________________________ 

SPONSOR’S REPRESENTATIVE DATE 
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INTRODUCTION 

will conduct an algal toxicity test with the freshwater alga, 

Pseudokirchneriella subcapitata, for the Sponsor at the . aquatic toxicology

facility in Easton, Maryland. The study will be performed based on procedures outlined in the working 

draft of OECD Proposal for Updating Guideline 201: Freshwater Alga and Cyanobacteria, Growth

Inhibition Test (1) and Official Journal of the European Communities No. L383 C.3. Algal Inhibition Test

(2), when possible. Raw data for all work performed at and a copy of the final

report will be filed by project number in archives located on the . site or at an

alternative location to be specified in the final report.

OBJECTIVE 

The objective of this study is to determine the toxicity of a test material to the freshwater green

alga, Pseudokirchneriella subcapitata, during a 72-hour exposure period. 

EXPERIMENTAL DESIGN 

The green alga, Pseudokirchneriella subcapitata, will be exposed to a geometric series of at least

five water accommodated fraction (WAF) loading rates and a negative (culture medium) control for 96

hours. WAF loading rates will be selected in consultation with the Sponsor and will be based on

information such as a results of rangefinding toxicity data, known toxicity data, physical/chemical 

properties of the test substance or other relevant information. Target loading rates will not exceed 1000 

mg/L. For this test, the term loading rate means the total amount of test substance added to the dilution

water volume (mg/L) to achieve the respective WAF solution. Generally, each concentration of test 

material used in the definitive test will be at least 50% of the next higher treatment, unless information

concerning the concentration-effect curve indicates that a different dilution factor is more appropriate.

Water samples from each test concentration will be collected for analysis of Total Organic Carbon (TOC).

Test solutions will be inoculated with approximately 10,000 Pseudokirchneriella cells/mL. Six

replicates in the control and three replicates per treatment will be tested. In order to control bias, the 

position of the flasks will be determined by indiscriminate draw at the beginning of the experiment and

daily during the exposure period. No other potential sources of bias are expected to affect the results of the 

study. 
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The response of the algae will be measured in terms of changes in area under the growth curve

(biomass) and growth rate. EbL50 and ErL50 values (based on the theoretical loading rate that produces a

50% reduction in area under the growth curve (biomass) and growth rate, respectively) will be calculated 

at 24, 48, and 72 hours. The 72-hour EL10 and EL20 values will also be calculated for biomass and

growth rate, if applicable. The no observed effect concentration (NOEL) may be selected based upon

evaluation of the statistical results and the dose-response pattern of growth inhibition. 

MATERIALS AND METHODS 

Test Subs tanc e 

Information on the characterization of test, control or reference substances is required by Good

Laboratory Practice (GLP) Standards and Principles. The Sponsor is responsible for providing

verification that the test substance has been characterized according to GLPs prior to its 

use in the study. If verification of GLP test substance characterization is not provided to 

 it will be noted in the compliance statement of the final report.

The Sponsor is responsible for all information related to the test substance and agrees to accept 

any unused test substance and/or test substance containers remaining at the end of the study. 

Test Sol ution Pr eparation 

The test substance will be mixed directly with culture medium on a weight:volume basis.

Each WAF will be prepared individually in a 4 L Pyrex® aspirator bottle with tubulation by mixing an

amount of the test substance in approximately 4 L of dilution water using a Teflon®-coated stir bar on a 

magnetic stir plate. Care will be taken to maintain a vortex depth of approximately 30% of the test 

solution height. Following the mixing period, the WAF solutions will then be allowed to settle for 

approximately 1 hour and the test solution will be decanted off the top (or bottom, as appropriate).

Test Organi sm 

The freshwater green alga, Pseudokirchneriella subcapitata will be used in this test. This species

is representative of an important group of algae and was selected for use in the test based upon past use 

and ease of handling in the laboratory. Stock cultures, obtained from the Culture Collection of Algae at 

the University of Texas at Austin or another supplier, will be maintained in culture medium at 

 for a minimum of two weeks prior to use in a toxicity test. Algae used in toxicity tests 
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will be in exponential growth phase, which is defined as the period of growth when algal cells are dividing

at a constant rate.

Just prior to beginning the test, an inoculum of the stock culture will be prepared so that each

milliliter of inoculum contains enough cells to provide an initial cell density of approximately 10,000 

cells/mL in each replicate.

Cul tur e Medium 

Culture medium prepared according to Standard Operating Procedures 

will be used as dilution water. The medium components and concentrations are presented in Table 1 (1,3).

Stock nutrient solutions will be prepared by adding reagent-grade or better chemicals to 

well water purified by reverse-osmosis. Appropriate volumes of the stock nutrient 

solutions will then be diluted with purified well water to prepare the medium. The medium will be filter 

sterilized (0.2 μm) or autoclaved prior to use. The final pH of the medium should be 8.0 ± 0.1. Analyses 

will be performed at least once annually to determine the concentrations of selected organic and inorganic

constituents of the well water and results of the most recent GLP-compliant analyses will be summarized

in the final report.

Specificationsfor acceptable levels of contaminants have not been established for culture medium. 

However, there are no levels of contaminants reasonably expected to be present in the medium that are 

considered to interfere with the purpose or conduct of the study. 

 

Test A ppara tu s 

Test chambers will be sterile, 250-mL Erlenmeyer flasks plugged with foam stoppers and 

containing 100 mL of test solution. Test chambers will be indiscriminately positioned on a mechanical

shaker in an environmental chamber and will be shaken continuously at approximately 100 rpm. Test

chambers will be labeled with the project number, test concentration, and replicate.

Envi ronm ental Condi tions 

Test flasks will be held at 24 ± 2°C under continuous cool-white fluorescent lighting at an

intensity of approximately 6000 lux +/- 20%. Light intensity will be measured at five locations on the
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shaker table at test initiation. Temperature will be measured twice daily in the environmental chamber 

using a calibrated, hand-held thermometer.

The pH of each treatment and the control will be measured at test initiation and termination using

a Fisher Accumet Model 915 pH meter or equivalent. Samples will be collected from the batch of test

solution prepared at test initiation for each treatment and control group for the measurement of pH. At test

termination, pH will be measured in pooled test solution samples collected from the replicates of each

treatment and control group. 

Biol ogi cal Measur em ent s 

Cell densities will be monitored during the test by conducting cell counts using an electronic

particle counter (Coulter Corporation Model ZM) or a hemacytometer and a microscope. One sample will 

be collected from each replicate of the treatment and control groups at approximate 24-hour intervals 

during the exposure period. Appropriate laboratory Standard Operating Procedures will be followed when

performing algal cell density evaluations. If test solutions show signs of precipitates, cell density

enumeration will be performed with a hemacytometer and microscope. If precipitates are not evident, cell

density enumeration may be made using the electronic particle counter. Exponential growth in the

negative control and solvent control will be confirmed at 72 hours.

Samples of test solutions will be collected from each replicate per treatment and control group at 

the end of the test. When cell counts are conducted using an electronic particle counter, these samples will 

be pooled within their respective treatments, and subsamples will be removed and examined

microscopically for atypical cell morphology (e.g., changes in shape, size or color). When cell counts are 

conducted using a hemacytometer and a microscope, each replicate sample will be examined 

microscopically for atypical cell morphology. Growth of cells in the flasks also will be assessed for 

aggregations or flocculations of cells and adherence of the cells to the test chamber.

Sam pling for Anal yt ical Measur em ents 

Samples will be collected from the WAF preparation vessels for the control and each test 

concentration at the beginning of the test and from pooled (old) solutions from each test concentrations at 
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the end of the test. Total organic carbon (TOC) will be measured in each sample using a Shimadzu Model

5000 TOC analyzer, or equivalent. 

Condi tions for the Validity of the Test 

The following criteria will be used to judge the validity of the test: 

1) the mean cell density in the control flasks must have increased by a factor of at least 16

within 3 days,

2) the pH of the control should not normally deviate by more than 1.5 units during the test,

and 

3) the coefficient of variation of average specific growth rates in the control replicates 

during the whole test period should not exceed 7%.

Statistical Anal ys es 

Area under the growth curve will be calculated for the treatment and control groups using the

following formula: 

A = ((N1-No)/2)(t1)+((N1+N2-2No)/2)(t2-t1)+...+((Nn-1+Nn-2No)/2)(tn-tn-1) 

Where: 

A = Area under the growth curve

No = Nominal number of cells/mL at to

N1 = Measured number of cells/mL at t1

N2 = Measured number of cells/mL at t2

Nn = Measured number of cells/mL at tn

t1 = Time of first measurement after beginning of test (hours) 

t2 = Time of second measurement after beginning of test (hours) 

tn = Time of nth measurement after beginning of test (hours)

Average specific growth rates will be calculated for the treatment and control groups using the 

following formula: 

n o

n o

t - t

ln N - ln N

μ  
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Where: 

μ = Average specific growth rate 

N0 = Nominal cell density (cells/mL) at to

Nn = Measured cell density (cells/mL) at tn

tn = Time after beginning of test (hours) 

to = Time of beginning of test (hours) 

Percent inhibition will be calculated for each treatment group using the following formula: 

 x 100

Mean Control Response

Mean Control Response - Mean Treatment Response Percent Inhibition  

EbL50 values and ErL50 values will be determined, when possible, using linear interpolation (4) or 

other suitable techniques with treatment response (biomass and growth rate) and exposure concentration 

data. The 72-hour EL10 and EL20 values will also be calculated for biomass and growth rate, if 

applicable. 

A no observed effect loading rate (NOEL) may be selected based on an evaluation of the doseresponse pattern and the results of the statistical analysis of the biomass and growth rate data at 72 hours.

Statistically significant differences between the control and the treatment groups will be identified using 

analysis of variance (ANOVA) and a test to compare treatment mean responses to the control response

(e.g., Dunnett’s test or Bonferroni’s t-test). Data will be assessed for normality and homogeneity of

variances prior to performing the ANOVA. Transformations will be used, when possible, to correct any

condition of non-normality or unequal error variances. If a solvent control is used in addition to the 

negative control, their responses will be compared using Student’s t-test. Data from the two control groups

will be pooled for treatment level analyses if no statistical differences exist. Otherwise, all comparisons 

should be made to the solvent control group. Statistical comparisons for the ANOVA and the comparison

of mean responses will be carried out using TOXSTAT V3.5 software (4) or equivalent.

RECORDS TO BE MAINTAINED 

Records to be maintained for data generated at Wildlife International, Ltd. will include, but not be

limited to: 
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 1. Copy of signed protocol.

 2. Identification and characterization of the test substance, if provided by Sponsor.

 3. Dates of initiation and termination of the test.

 4. Source of algae.

 5. Culture conditions.

 6. Growth measurements.

 7. Calculation and preparation of test concentrations. 

 8. Observations. 

9. The methods used to analyze test substance concentrations and the results of analytical 

measurements.

10. Statistical calculations.

11. Test conditions and physical/chemical measurements.

12. Copy of final report. 

FINAL REPORT 

A final report of the results of the study will be prepared by Wildlife International, Ltd. The report

will include, but not be limited to the following, when applicable: 

 1. Name and address of the facility performing the study.

2. Dates upon which the study was initiated and completed, and the definitive experimental start and

termination dates. 

3. A statement of compliance signed by the Study Director addressing any exceptions to Good

Laboratory Practice Standards.

4. Objectives and procedures, as stated in the approved protocol, including all changes to the 

protocol. 

5. The test substance identification including name, chemical abstract number or code number,

strength, purity, composition, and other information provided by the Sponsor.

6. Stability and solubility of the test substance under the conditions of administration, if provided by

the Sponsor.

 7. A description of the methods used to conduct the test. 

8. A description of the test organisms, including the source, scientific name, age or life stage, light

intensity and photoperiod. 

 9. A description of the preparation of the test solutions. 
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10. The methods used to allocate organisms to test chambers and begin the test, the number of

organisms and chambers per treatment, and the duration of the test. 

11. A description of circumstances that may have affected the quality or integrity of the data.

12. The name of the Study Director and the names of other scientists, professionals, and supervisory

personnel involved in the study. 

13. A description of the transformations, calculations, and operations performed on the data, a 

summary and analysis of the biological data and analytical chemistry data, and a statement of the 

conclusions drawn from the analyses. A graph plotting the concentration response curve. 

14. Statistical methods used to evaluate the data.

15. The signed and dated reports of each of the individual scientists or other professionalsinvolved in

the study. 

16. The location where raw data and final report are to be stored. 

17. A statement prepared by the Quality Assurance Unit listing the dates that study inspections were 

made and the dates of any findings reported to the Study Director and Management. 

18. If it is necessary to make corrections or additions to a final report after it has been accepted, such

changes will be made in the form of an amendment issued by the Study Director. The amendment

will clearly identify the part of the final report that is being amended and the reasons for the 

amendment, and will be signed by the Study Director.

CHANGING OF PROTOCOL 

Planned changes to the protocol will be in the form of written amendments signed by the Study

Director and approved by the Sponsor’s Representative. Amendments will be considered as part of the 

protocol and will be attached to the final protocol. Any other changes will be in the form of written

deviations signed by the Study Director and filed with the raw data. All changes to the protocol will be

indicated in the final report.

GOOD LABORATORY PRACTICES 

This study will be conducted in accordance with Good Laboratory Practice Standards for EPA (40

CFR Part 160 and/or Part 792); OECD Principles of Good Laboratory Practices (ENV/MC/CHEM (98)

17); and Japan MAFF (11 NohSan, Notification No. 6283, Agricultural Production Bureau, 1 October

1999). Each study conducted by is routinely examined by the
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Quality Assurance Unit for compliance with Good Laboratory Practices, Standard

Operating Procedures and the specified protocol. A statement of compliance with Good Laboratory

Practices will be prepared for all portions of the study conducted by The

Sponsor will be responsible for compliance with Good Laboratory Practices for procedures performed by

other laboratories (e.g., residue analyses or pathology). Raw data for all work performed at 

 and a copy of the final report will be filed by project number in archives located on the 

 site or at an alternative location to be specified in the final report.
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REFERENCES 

1 OECD. 2004. Working draft of OECD Proposal for Updating Guideline 201: Freshwater Alga

and Cyanobacteria, Growth Inhibition Test. Circulated 9 April 2004. 

2 Official Jour nal of the Eur ope an Com m uni ties. 1992. No. L383. Method C.3. Algal

Inhibition Test. 

3 ASTM Standar d Gui de 1218-90E. 1990. Standard Guide for Conducting 96-Hour Toxicity

Tests with Microalgae.

4 West, Inc. and D. D. Gulley. 1996. TOXSTAT Release 3.5. Western EcoSystems Technology, 

Inc. Cheyenne, Wyoming.
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TABLE 1 

FRESHWATER ALGAL MEDIUM CONSTITUENTS 

Com pone nt Nom inal Conc entration

















The pH of the medium will be adjusted, as necessary, to 8 ± 0.1 using 0.1

N NaOH or 10% HCl.