SRC TR-12-074

HPV2 TEST RULE DATA ADEQUACY REVIEW (FINAL):

TOXICITY TO DAPHNIA AND ALGAE

Acetaldehyde, CASRN 75-07-0

Prepared by:

Jennifer Rayner, Julie Klotzbach, Teresa Manyin, and Laura Morlacci

Chemical, Biological and Environmental Center

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.2.009.001

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Debra Milligan, Project Officer

Meena Sonawane, Work Assignment Manager

David Brooks, Alternate Work Assignment Manager

September 14, 2012
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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates daphnid and algal toxicity study reports, which were submitted 

to EPA on May 18, 2012 by the Acetaldehyde Working Group in response to the C2 

testing requirement for acetaldehyde (CASRN 75-07-0) in the final test rule, “Testing of 

Certain High Production Volume Chemicals; Second Group of Chemicals” (76 FR 1067, 

January 7, 2011; referred to herein as HPV2). Since the log Kow of CASRN 75-07-0 is 

< 4.2,1the C2 requirement consists of (1) Acute Toxicity to Daphnia testing following 

ASTM guideline E 729-96 and (2) Toxicity to Plants (Algae) testing following ASTM 

guideline E 1218-04ε1. The submitted daphnid and algal toxicity study reports are 

evaluated for adequacy herein. No other toxicity data are evaluated in this report. 

B. EVALUATING DATA ADEQUACY

Submitted data are evaluated for adequacy in Section D. Each study was summarized 

according to a standard format and assigned a Klimisch reliability code based on its 

scientific merit and conduct, using professional judgment. To determine whether data 

were adequate to satisfy SIDS endpoints, study details were compared to current standard 

test guidelines to establish whether the study met current testing requirements. Studies 

for a common endpoint are grouped together in the document and an endpoint adequacy

assessment is provided near the top of the first page of the summaries for that endpoint. 

If the data available for the endpoint were determined to be inadequate, a basis for that 

conclusion is supplied. A table summarizing data availability/adequacy for each assessed

endpoint is included on the last page of this document (Section E). 

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST 

RULE

Data submitted by the Acetaldehyde Working Group for Acute Toxicity to Daphnia and 

Toxicity to Plants (Algae) endpoints are considered adequate to satisfy the C2 testing 

requirement. Data have not been submitted to address the F2 testing requirement, which 

consists of a Reproduction/Developmental Toxicity Screening Test following TSCA 

guideline 40 CFR 799.9355; therefore, reproductive and developmental toxicity

endpoints remain as data gaps.

 

1 Log Kow = -0.34; value obtained from Hazardous Substance Databank (HSDB). Available online at 

http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?HSDB as of September 13, 2012.
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D. DATA SUBMITTED FOR C2 TESTING REQUIREMENT

D-1. Acute Toxicity to Aquatic Invertebrates 

Summary of endpoint: Adequate

Study 1: Acute toxicity study in Daphnia magna (Kuhl and Wydra, 2010a)

Title: Final Report: Acute Toxicity of Acetaldehyde to Daphnia magna in a Static 48-

hour Immobilisation Test in Closed Vessel Design

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Acetaldehyde, CASRN 75-07-0

Remarks: Physical state: colorless liquid; Batch No.: ALDE190309; Purity: 99.96%; 

Density: 0.788 g/cm2at 20°C; Solubility: completely miscible in water

METHOD

Method/guideline followed: OECD TG 202

Test type: Daphnia sp., Acute Immobilization Test (Static)

GLP compliant?: Yes

Year study was performed: 2009

Test species: Water flea (Daphnia magna), 3-19 hours old, females, bred at test facility

Analytical monitoring: Yes

Exposure duration: 48 hours

Concentrations tested: Nominal concentrations: 6.25, 12.5, 25, 50, and 100 mg/L;

Measured concentrations (Total Organic Carbon [TOC] method): 7.1, 14.2, 30.3, 58.4, 

and 104 mg/L. The two lowest concentrations were below the limit of quantitation (LOQ) 

for the TOC method; therefore, the measured concentrations for nominal concentrations 

of 6.25 and 12.5 mg/L were estimated by multiplying the nominal concentrations by

114%, which was the overall recovery of the three highest concentrations.

Test method/conditions remarks: Twenty Daphnia per control and test concentration 

were exposed to the test substance in 4 replicates of 5 animals each for a total of 48 

hours. Visual observation was used to determine the immobility of the Daphnia after 24 
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and 48 hours. Immobility was defined as those animals not able to swim within 15 

seconds after gentle agitation of the test beaker, even if they could still move their 

antennae. 

“The 24-hour and 48-hour EC50 and the 95% confidence limits were calculated by Probit 

analysis. For calculation 0 and 100% values were replaced by 0.1 and 99.9%.” [Kuhl and 

Wydra 2010a, p. 16]

“The NOEC and LOEC after 24 and 48 hours were determined directly from the raw 

data.” [Kuhl and Wydra 2010a, p. 16]

“The software used to perform the statistical analysis was ToxRat Professional, Version 

2.10, ToxRat® Solutions GmbH, 2009.” [Kuhl and Wydra 2010a, p. 16] 

TEST SPECIES:

Name: Daphnia magna (Straus), clone 5

Source: The Daphnia magna were taken from the in-house laboratory culture. 

Maintenance and Acclimatization: Parental Daphnia were cultivated in reconstituted 

water of a similar quality with regards to pH, the constituent salts, and total hardness as 

the test medium used. First brood progeny were not used. The Daphnia in the stock 

culture were fed green algae freshly grown in the laboratory. 

GROWTH MEDIUM AND DILUTION WATER:

Reconstituted water was used for the growth of the Daphnia and the preparation of stock 

and test solutions of the test item.

PRE-EXPERIMENTS AND RANGE-FINDING TEST (NOT GLP COMPLIANT):

“Pre-experiments were performed to determine the solubility of the test item in test water 

and to select suitable methods for the preparation of a stock solution and the dosage of 

the test item into the test media.” [Kuhl and Wydra 2010a, p. 15]

A range-finding test was performed to determine the test concentrations; concentrations 

above the limit concentration (nominal 100 mg test item/mL) were not tested.

PREPARATION OF TEST ITEM CONCENTRATIONS:

“The test media of the nominal test item concentrations of 12.5, 25, 50 and 100 mg/L 

were prepared by adding the appropriate volume of the test item directly into a glass 

flask. The lowest concentration was prepared by dilution of the test medium of nominal

12.5 mg test item/L. All mixing vessels were closed with a glass stopper to prevent 

evaporation of the test item.” [Kuhl and Wydra 2010a, p. 16] 

“In the control, test medium was used without the addition of test item.” [Kuhl and 

Wydra 2010a, p. 16] 

The test media were prepared just before the start of the test. 
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EXPOSURE CONDITIONS:

The test vessels were 110-mL glass beakers with conical glass stoppers. Test vessels 

were filled completely with test solution; headspace was minimized. The pH was 

measured at the beginning of the test and after 48 hours of exposure and ranged from 7.5-

8.0. The total hardness of the reconstituted water was 2.5 mmol/L (equivalent to 250 mg 

CaCO3/L), and the alkalinity was 0.9 mmol/L. During the test, a temperature range of 

20-21°C was maintained in the test vessels. The oxygen concentration ranged from 8.0-

8.7 mg/L. The light intensity was 340-380 lumen/m2, with a photoperiod of 16 hours 

light and 8 hours dark. 

“Duplicate samples from the freshly prepared test media of all test concentrations and the 

control were taken at the start of the test.” [Kuhl and Wydra 2010a, p. 17] 

“For the determination of the stability and of the maintenance of the test item 

concentrations during the test period, two replicates from the actual test media of all test 

concentrations and the control were collected at the end of the test (after 48 hours).” 

[Kuhl and Wydra 2010a, p. 17] 

“The concentrations of the test item were analyzed in the duplicate test media samples 

from both sampling times (0 and 48 hours). From the control samples only one of the 

duplicate samples was analyzed from each of both sampling times.” [Kuhl and Wydra 

2010a, p. 17] 

VALIDITY CRITERIA:

The test was considered valid if (1) the control immobilization rate did not exceed 10% 

and no more than 10% of daphnids showed signs of disease, stress, or unusual behavior; 

and (2) the dissolved oxygen concentration at the end of the test was ≥ 3 mg/L in all 

control and test vessels.

RESULTS

Endpoint value(s): Based on nominal concentrations: 48-hour EC50 = 57.4 mg/L; 48-

hour NOEC = 6.25 mg/L; 48-hour LOEC = 12.5 mg/L; basis for effect levels = 

immobility. 

Results remarks: After 48 hours of exposure, no immobilization of the test animals was 

observed in the control and up to and including the nominal test item concentration of 

6.25 mg/L. At the nominal concentrations of 12.5 and 25 mg/L, one animal per group 

(5%) was immobile. At the two highest test item concentrations, 50 and 100 mg/L, 3 

(15%) and 20 (100%) of daphnids were immobile, respectively. 

None of the Daphnia in the control group showed signs of disease or stress, and none 

were immobilized. The dissolved O2 concentration was ≥8.0 mg O2/L in the control and 

test vessels at the end of the test; thus validity criteria were met. 
Final HPV2 Data Adequacy Review (Daphnia and Algae) CASRN 75-07-0

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“At the start of the test 118% of the nominal test concentration was found (average of test 

concentrations of nominal 100, 50 and 25 mg test item/L). After 48 hours test duration, 

110% of the nominal value was determined (average of test concentrations of nominal 

100, 50 and 25 mg test item/L). During the test the Daphnia were exposed to a mean of 

114% of nominal. Therefore, all reported results refer to nominal concentrations.” [Kuhl 

and Wydra 2010a, p. 22]

CONCLUSIONS

The 48-hour EC50 for Daphnia magna was 57.4 mg/L based on nominal concentrations. 

STUDY RELIABILITY

[2] Reliable with restrictions. Measured concentrations could not be obtained for the two 

lowest concentrations (nominal concentrations of 6.25 and 12.5 mg/L) because values 

were below the limit of quantitation for the analytical method (TOC). Due to the

uncertainty in test item concentrations for the 6.25 and 12.5 mg/L groups, which are 

identified as the NOEC and LOEC for this study, this study is considered “reliable with 

restrictions.”

REFERENCE

Kuhl, R., and Wydra, V. 2010a. Final Report: Acute Toxicity of Acetaldehyde to 

Daphnia magna in a Static 48-hour Immobilisation Test in Closed Vessel Design. 

Performing Laboratory: Institut für Biologische Analytik und Consulting IBACON 

GmbH, Rossdorf, Germany. Sponsor: Wacker Chemie AG, Burghausen, Germany.

Text in quotes is taken directly from Kuhl and Wydra, 2012a.
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D-2. Toxicity to Aquatic Plants 

Summary of endpoint: Adequate

Study 1: Toxicity to Pseudokirchneriella subcapitata (Kuhl and Wydra, 2010b)

Title: Final Report (2nd Original): Toxicity of Acetaldehyde to Pseudokirchneriella 

subcapitata in an Algal Growth Inhibition Test in Closed Vessel Design

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Acetaldehyde, CASRN 75-07-0

Remarks: Physical state: colorless liquid; Batch No.: ALDE190309; Purity: 99.96%; 

Density: 0.788 g/cm2at 20°C; Solubility: completely miscible in water

METHOD

Method/guideline followed: OECD TG 201

Test type: Toxicity test to freshwater algae 

GLP compliant?: Yes

Year study was performed: 2009

Test species: Freshwater green algae (Pseudokirchneriella subcapitata, Strain No. 61.81 

SAG; cultured in-house) 

Analytical monitoring: Yes

Exposure duration: 72 hours

Concentrations tested: Nominal: 6.25, 12.5, 25, 50, and 100 mg/L. Mean measured 

concentrations (mean of Total Organic Carbon [TOC] measurements at 0 and 72 hours) 

were 140, 133, 88, and 95% of nominal concentrations at target concentrations of 12.5, 

25, 50, and 100 mg/L, respectively. For the 6.25 mg/L nominal concentration, measured 

concentrations were below the Level of Quantitation (LOQ).

Test method/conditions remarks: The endpoints were growth rate and yield. The algal 

cell densities (spectrophotometrically measured) were calculated by subtracting the 

absorption values of the blanks (control and test concentration media prepared without 

algae) from each of the measured absorption values of the test media (with algae). 

Inhibition of growth rate and yield was determined relative to control cultures. The 72-
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hour EC10 and EC50 were calculated by probit analysis. For the determination of the 72-

hour LOEC and NOEC, the calculated growth rates and yields at each test concentration 

were tested for significant differences compared to the control values by the Williams ttest. All calculations were carried out using the statistics program ToxRat Professional, 

Version 2.10, ToxRat® Solutions GmbH, 2009.

TEST SPECIES:

Name: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG

Source: The test species was obtained from ‘The Collection of Algal Cultures’ of the 

Institute of Plant Physiology at the University of Gottingen (Germany).

Maintenance and Acclimatization: Exponentially growing stock cultures were cultivated 

in the test facility under standardized conditions according to the test guidelines. 

Preparation of pre-cultures: Pre-cultures were set up three days before the start of a test 

and were grown under identical exposure conditions as the stock cultures.

Test cultures: At test start, 60 mL of the test solution was inoculated with 2500 algae 

cells/mL test medium. 

GROWTH MEDIUM AND DILUTION WATER:

Reconstituted water (OECD medium of OECD TG 201) was used for the growth of the 

algae in the pre-cultures and the preparation of stock and test solutions of the test item. 

Water hardness of the final nutrient medium was calculated to be 24 mg/L as CaCO3. 

For the algae test, the test medium was enriched with 300 mg NaHCO3/L to provide 

sufficient CO2 for algal growth in closed test vessels.

PRE-EXPERIMENTS AND RANGE FINDING TEST: 

“Pre-experiments were performed to determine the solubility of the test item in test water 

and to select suitable methods for the preparation of a stock solution and the dosage of 

the test item into the test media.” [Kuhl and Wydra 2010b, p. 15]

A range-finding test was performed to determine the test concentrations; concentrations 

above the limit concentration (nominal 100 mg test item/mL) were not tested.

PREPARATION OF TEST ITEM CONCENTRATIONS:

The test solutions for nominal concentrations of 50 and 100 mg/L were prepared by

adding 22.5 and 45 μL, respectively, of the test item directly into 350 mL test medium. 

“For the preparation of the lower concentrations, a stock solution of 100 mg test item/L

was prepared by adding 45 μL test item into 350 mL test water by intense stirring for 5 

minutes. Adequate volumes of this stock solution were diluted with test water to prepare 

the test media of the desired test concentrations. All media were prepared in closed 

flasks to avoid evaporation of the test item.” [Kuhl and Wydra 2010b, p. 15] 

The control consisted of test medium without addition of the test item. All test solutions 

were prepared just prior to the introduction of algae.
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EXPOSURE CONDITIONS:

The test vessels were 50-mL Erlenmeyer flasks, filled with ~60 mL of test medium and 

sealed with conical stoppers. During the exposure, test solutions were stirred 

continuously with magnetic stirrers. Test vessels were held at a temperature of 23°C with 

continuous uniform illumination. Temperature was measured and recorded daily in a 

water-filled flask incubated under the same conditions as the test flasks. The mean light 

intensity was 5847 lumen/m2(5560-6000 lumen/m2). Light intensity was measured once 

during the test at positions distributed over the experimental surface of the test media. 

The pH was measured at the beginning (8.1) and the end (8.4-8.6) of the test. 

The experimental design of the study included five test concentrations plus one control. 

Three replicates per concentration and six replicates per control were used. 

Duplicate samples of the test media were taken at the start and at the end of the test (after 

72 hours of exposure) and were analyzed for test substance concentration via a TOC

method. Cell densities in each replicate were measured by spectrophotometrical 

measurement at 24, 48, and 72 hours. The cell densities in a number of samples from one 

control replicate were counted by microscope after 72 hours of test duration to develop a 

regression relationship between cell density and measured absorption.

VALIDITY CRITERIA:

The test was considered valid if (1) the cell density in control cultures increased by a 

factor of at least 16 within 72 hours, (2) the coefficient of variation of sectional (daily) 

growth rates in control cultures did not exceed 35%, and (3) the coefficient of variation of 

average growth between control replicates did not exceed 7%.

RESULTS

Endpoint value(s): Based on nominal concentrations: EC50 (growth rate, yield) >100 

mg/L; EC10 (growth rate, yield) >100 mg/L; NOEC (growth rate, yield) = 100 mg/L; 

LOEC (growth rate, yield) >100 mg/L.

Results remarks: Mean algal cell densities after 72 hours were 24.076, 22.461, 20.667, 

25.850, 41.760, and 47.183 x 104cells/mL at nominal concentrations of 0, 6.25, 12.5, 25, 

50, and 100 mg/L, respectively. Mean percent inhibition of growth rate, relative to 

controls, after 72 hours was 1.5, 3.6, -1.4, -12.1, and -14.9% at nominal concentrations of 

6.25, 12.5, 25, 50, and 100 mg/L, respectively. Mean percent inhibition of yield, relative 

to controls, after 72 hours was 6.8, 14.3, -7.4, -74.2, and -97.0% at nominal 

concentrations of 6.25, 12.5, 25, 50, and 100 mg/L, respectively.

“The 72-hour EC50 and the 72-hour EC10 for the parameters growth rate and yield could 

not be calculated due to the low effects of the test item. Therefore the 72-hour EC50 and 

the 72-hour EC10 were determined to be higher [than the] nominal 100 mg test item/L, the 

highest concentration tested.” [Kuhl and Wydra 2010b, p. 24]
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“The 72-hour NOEC was determined to be at least nominal 100 mg test item/L for the 

parameters growth rate and yield and the associated 72-hour LOEC was determined to be 

higher than nominal 100 mg test item/L for both parameters.” [Kuhl and Wydra 2010b, p. 

24]

“During the test a stimulating effect on parameters growth rate and yield occurred in the 

concentrations of 50 and 100 mg test item/L over 72 hours. However, according to the 

guideline these stimulating effects are not relevant for the determination of the NOEC 

and the EC50.” [Kuhl and Wydra 2010b, p. 24]

“The microscopic examination of the shape of the algal cells after 72 hours of test 

duration did not show any difference between the algae that had been growing at a 

nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, 

the shape of the algal cells was not obviously affected up to this test concentration, the 

highest concentration tested.” [Kuhl and Wydra 2010b, p. 25]

The following validity criteria of the test were met: The cell density of the control 

cultures increased by a factor of 96 within 72 hours. The mean coefficient of variation of 

sectional (daily) growth rates in control cultures was 17.4% (<35%, the validity criteria). 

The mean coefficient of variation of average growth between control replicates was 3.5% 

(<7%, the validity criteria). 

ANALYTICAL MEASUREMENT OF TEST SUBSTANCE CONCENTRATIONS:

Based on the average TOC concentrations for the 50 and 100 mg/L concentrations,

measured concentrations were 106% of nominal values at 0 hours and 77% of nominal at 

72 hours for the two highest test concentrations combined. Therefore, test substance 

concentrations decreased during the test. However, since no inhibitory effect was 

observed on algal growth or yield and the mean measured concentration of the nominal 

100 mg/L test concentration was >80% at 72 hours, the study authors reported all results 

in terms of nominal concentrations.

Since the measurements at nominal concentrations of 12.5 and 25 mg/L showed a high 

degree of variability (large standard deviation) between duplicate samples, the study

authors concluded that the analytical method did not produce reliable analytical results in 

this range. Concentrations at the nominal concentration of 6.25 mg/L were < LOQ. 

The test media at all concentrations appeared clear throughout the test.

CONCLUSIONS

The 72-hr EC50 was found to be >100 mg/L for growth rate and yield. The 72-hour 

NOEC for growth rate and yield was 100 mg/L. The 72-hour LOEC for growth rate and 

yield was >100 mg/L.
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STUDY RELIABILITY 

[1] Reliable without restrictions. Although measured concentrations could not reliably be 

determined for nomimal concentrations ranging from 6.25 to 25 mg/L, no effects on algal 

growth or yield were observed at the highest concentration tested (100 mg/L). Therefore, 

study reliability is not affected by lack of measured values for the lower nominal 

concentrations.

REFERENCE

Kuhl, R., and Wydra, V. 2010b. Final Report (2nd Original): Toxicity of Acetaldehyde 

to Pseudokirchneriella subcapitata in an Algal Growth Inhibition Test in Closed Vessel 

Design. Performing Laboratory: Institut für Biologische Analytik und Consulting 

IBACON GmbH, Rossdorf, Germany. Sponsor: Wacker Chemie AG, Burghausen, 

Germany.

Text in quotes is taken directly from Kuhl and Wydra, 2010b.
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E. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Aquatic 

Invertebrates

Yes Yes No

Toxicity to Aquatic Plants Yes Yes No