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Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 March 2018 - 12 March 2018
1 (reliable without restriction)
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
Analytical monitoring:
Details on sampling:
On Day 0, samples were collected from the bottom outlet of the aspirator bottle from the batch solution prepared for each treatment group and the control. Samples on Day 3 (termination) were collected from a pooled sample collected from the three remaining replicate test chambers. Duplicate samples were analyzed for each treatment group; and a single sample was analyzed for the control group. Samples were collected in 20 mL volatile organic analysis (VOA) vials with PTFE-lined screw caps (no headspace) and analyzed immediately. Samples were analyzed using headspace gas chromatography with flame ionization detection. All samples retained were stored under refrigerated conditions.

Details on test solutions:
The individual treatment batch solutions were prepared by adding the appropriate volume of test substance to the dilution media in glass aspirator bottles using a gas-tight glass and stainless syringe. Four liter batch solutions were prepared for the control, 1.5, 4.7 and 15 mg/L treatment groups. The volumes of the batch solutions were 20, 20 and 12 L for the 0.045, 0.14 and 0.46 mg/L treatment groups, respectively. The aspirator bottles were sealed with a PTFE screw plugs. The volume of test substance dispensed into the aspirator bottle was calculated using the nominal test concentration, the quantity of dilution media and the specific density of the test substance. The treatment solutions were mixed with Teflon®-coated stir bars on magnetic stir plates using approximately 8 - 20 % vortex height to static height. The treatment solutions were mixed for approximately 24 hours at test temperature. The control solution was prepared in the same manner with no test substance. At the end of mixing, all solutions were allowed to settle for 1 hour and 15 minutes. At the end of the settling period, the solutions were removed from the mixing vessels through the outlet at the bottom of the aspirator bottles and placed into the nine respective test chambers per treatment group. At test initiation, each test chamber were inoculated with an appropriate volume of algal culture to achieve an initial cell density of approximately 10,000 cells/mL.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Algae were cultured at the testing facility. Initial strain (1648) was provided by UTEX, The Culture Collection of Algae, The University of Texas at Austin, Austin, TX 78712. A liquid culture of algae was received by the testing facility on 27 September 2016.
Culture Methods -Algae were cultured in approximately 150 mL of nutrient media (same as dilution media with the exception of sodium bicarbonate) prepared with deionized water and reagent grade chemicals. Cell counts are performed weekly to ensure that the cells are in log phase of growth and to verify that the culture is axenic. A new culture is started approximately weekly using inoculum from the previous culture. The cultures of P. subcapitata are held under continuous illumination (4440 - 5920 lux) provided by cool-white fluorescent bulbs.
Cell Density and Age at Test Initiation: Initial cell density of algae was approximately 10,000 cells/mL in each replicate. The algae culture was collected from a stock culture in log phase growth at 4 days.

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
not reported
Test temperature:
Dissolved oxygen:
not reported
Details on test conditions:
Test chambers were 50 mL glass Erlenmeyer flasks with PTFE-lined screw caps to prevent contamination, evaporation and/or volatilization. Each chamber contained approximately 64 mL of test solution (no headspace) and an approximately 1.5 cm Teflon®-coated stir bar. All test chambers were placed on a multi-position magnetic stir-plate to aid in dissolution kinetics and to support algal growth in a no head-space environment.Each replicate test chamber was labeled to show study number, treatment group and replicate number. Each replicate was inoculated with algae and placed on a multi-position magnetic stir plate for the duration of the study. Test chambers were randomly assigned daily on the stir plate using a computer-generated randomization schedule (SAS, 2016).

EXPERIMENTAL PROCEDURE-9 replicates for all treatments, initial cell density ~ 10,000 cells/ml, n-hexyl acrylate mg/L test treatment groups (prep'd in algal media): Control, 0.045, 0.14, 0.46, 1.5, 4.7 and 15 mg/L. Nine replicate test chambers were prepared for each treatment group and control group. Three replicate test chambers were sacrificed every 24 ± 1 hours for cell density determination.

Key result
72 h
Dose descriptor:
Effect conc.:
0.35 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Reported statistics and error estimates:
The EC10 and EC50 values were determined based on the percent inhibition relative to the control values for growth rate and yield. The EC10, EC50 values and confidence intervals for average specific growth rate and yield were calculated based on a non-linear regression calculation (Ratkowsky, 1990) using JMP® Version 13 (JMP, 2016).

The NOEC and LOEC values were based on the Dunnett’s test (Dunnett, 1964) determined from the General Linear Model (GLM) procedure of SAS (2016) with percent inhibition of yield or growth rate slope as the dependent variable and concentration as the independent variable. The Shapiro-Wilk test (Shapiro and Wilk, 1965) for normality was used to test if the assumption of normality of the residuals was met; if the residuals were normally distributed the NOEC was based on the estimated values, if they were not normally distributed the NOEC was based on the ranks of the estimated values (SAS, 2016).

Nominal Concentration


Measured Concentrations (mg/L)

0 hour

72 hour

0 (Control)













































Validity criteria fulfilled:

This study was conducted for the Sponsor to evaluate the effects of n-hexyl acrylate on growth of the alga, Pseudokirchneriella subcapitata, in a 72-hour static dose-response test. This study was performed following the OECD 201 guideline (OECD, 2011).

The individual treatment solutions were prepared by adding the appropriate volume of test substance to dilution media in glass aspirator bottles and stirring on magnetic stir plates with a vortex of ranging from 8 - 20% of the static liquid depth overnight. The nominal test concentrations were 0 (control), 0.045, 0.14, 0.46, 1.5, 4.7 and 15 mg/L. The initial measured concentrations were 0.044, 0.13, 0.38, 1.3, 4.5 and 15 mg/L for the 0.045, 0.14, 0.46, 1.5, 4.7 and 15 mg/L treatment groups, respectively. At 72 hours, n-hexyl acrylate was not detected in the control, 0.045, 0.14 and 0.46 mg/L treatment groups. The measured concentrations were 0.26, 3.3 and 12 mg/L for the 1.5, 4.7 and 15 mg/L treatment groups, respectively. All statistical endpoints were based on the initial measured concentrations.

At 72 hours, there were statistically significant treatment-related effects observed when the treatment groups were compared to the control group. The acute toxicity endpoint, EC50 (50% Effect Concentration), is expressed as percent inhibition of growth derived from the average specific growth rate (r) and yield (y) relative to the control. Chronic toxicity endpoints, EC10 (10% Effect Concentration), NOEC (No Observed Effect Concentration) and LOEC (Lowest Observed Effect Concentration) values were determined for average specific growth rate and yield.

All 72-hour endpoints for growth rate and yield are presented below.

Response Variable Based on Initial Measured Concentrations (mg/L)
Growth Rate (r)† ErC10: 0.12 (0.094 – 0.15)
ErC50: 0.35 (0.33 – 0.37)

NOEC: 0.044
LOEC: 0.13

Yield (y) EyC10: 0.067 (0.052 – 0.082)
EyC50: 0.14 (0.13 – 0.15)

NOEC: 0.13
LOEC: 0.38

Values in parentheses ( ) are 95% confidence intervals.

†The preferred observational endpoint in this study is algal growth rate inhibition because it is not dependent on the test design, whereas yield or biomass depends on both growth rate of the test species as well as test duration and other elements of test design (ECHA 2017, OECD 2011).

Description of key information

Key value for chemical safety assessment

EC50 for freshwater algae:
0.35 mg/L

Additional information