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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23rd November 2017 to 10th December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Isomeric mixture of alkenyl succinate with 99.06%. All test solutions and results were corrected for this purity.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Range finding test

The range-finding test was initiated on November 23, 2017. The test consisted of four nominal concentrations of the test item including 0.15, 1.5, 15 and 150 mg/L plus a negative control (i.e., nutrient media with no added test item prepared the day of test initiation). For this test, two replicate 250 mL Erlenmeyer flasks, each containing 49 mL of the test solution (prepared in nutrient medium) and 1 mL of algal inoculum (5 x 10^3 to 1 x 10^4 algal cells/mL), were established for each concentration. Each control replicate flask contained 49 mL of the same batch of algal nutrient medium and 1 mL of the same algal inoculum, without the addition of the test item. The environmental conditions and testing procedures were the same for all flasks.

Definitive Test

The definitive test was initiated on December 7, 2017. The test consisted of eight nominal concentrations of the test item including 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 mg/L plus a negative control. For this test, four replicate 250 mL Erlenmeyer flasks, each containing 49 mL of the test solution (prepared in nutrient medium) and 1 mL of algal inoculum (5 x 10^3 to 1 x 10^4 algal cells/mL), were established for each concentration. Each control replicate flask contained 49 mL of the same batch of algal nutrient medium and 1 mL of the same algal inoculum, without the addition of the test item. The environmental conditions and testing procedures were the same for all flasks.

Test solutions

Vehicle:
no
Details on test solutions:
Individual test solutions of the test item for both range-finding and definitive tests were prepared from stock solutions (1000 mg/L nominal concentration; corrected for purity) prepared in algal nutrient media without the use of any solubilizing agent. All stock solutions were prepared in 500 mL glass volumetric flasks, stirred for approximately 24 hours using a stir bar and stir plate. The stock solutions were then used to prepare the test concentrations. The 1000 mg/L stock solution used for the range-finding test was prepared by adding 0.56909 g of the test item to 500 mL of algal nutrient media in a 500 mL glass volumetric and stirred for 24 h and settled for 1 h. The individual test solutions were prepared by adding the appropriate amount of stock solution with algal nutrient media to achieve a 400 mL volume of the desired test concentration. The 1000 mg/L stock solution used for the definitive test was prepared by adding 0.56914 g of the test item to 500 mL of algal nutrient media in a 500 mL glass volumetric and stirred for 24 h and allowed to settle for 1 h. The individual test solutions were prepared as above for the range-finding test. All test solutions were stirred for 30 minutes using a stir bar and stir plate prior to being dispensed into the individual test vessels (i.e., 250 mL Erlenmeyer flasks covered with Jaece® non-toxic foam plugs).

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata3 used in testing were obtained from an in-house culture. The starter culture was purchased from the University of Waterloo Culture Collection (CPCC 37) (Appendix B). Cultures of P. subcapitata were maintained according to AquaTox SOP # 206 (AquaTox, 2016b). Cultures were aseptically transferred twice weekly (typically from 3 – 7 day old donors) and maintained in temperature and light controlled environments isolated from all testing. The axenic nature of the stock culture was verified by plating on Trypticase Soy Agar (TSA) and Plate Count Agar (PCA). Algal growth curves conducted semi-annually ensured that algae were in an exponential growth phase, suitable for testing.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
23 ± 1 °C
pH:
7.5 ± 0.1 pH of nutrient medium
Nominal and measured concentrations:
Range finding test

Nominal Concentration (mg/L) 150, 15, 1.5, 0.15 0

Definitve Test

Nominal Concentration (mg/L) 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 1.56, 0.781, 0
Details on test conditions:
Table 3. Summary of Test Conditions for Determining Toxicity of X-19933
to P. subcapitata
Parameter Conditions

Test type Static algal flask test

Test species Uni-algal cultures of P. subcapitata (Korschikov) Hindák (current sourceCPCC 37)

Test duration 72 hour

Agitation during testing Continuous shaking of test vessels on a rotary shaker set at 100 rpm

Incubation chamber Incubated in a growth chamber

Temperature 23 ± 1 °C, as recorded daily with a maximum/minimum thermometer

Light quality Cool-white fluorescent

Light intensity Measured at the surface of the liquid in the flasks. 4440 – 8880 Lux for testing

Photoperiod Continuous (24 hours)

Test vessel Clear glass 250-mL Erlenmeyer flasks covered with Jaece® non-toxic foam plugs

Nutrient medium Filter-sterilized

Nutrient medium/test
solution volume 50 mL

pH of the nutrient medium 7.5 ± 0.1

pH of the test solutions pH is measured (but not adjusted) in all concentrations at the beginning and end of the test (pooled replicates)

Age of algal cells used for
testing 3 – 7 days old and in exponential growth

Number of tests concenteation Range-finding test: 4 + a negative control Definitive multiple concentration test: 8 + a negative control

Number of replicates 2 (Range-finding test) 4 (Definitive test)

concentration of
algal inoculum 5 x 10^3 to 1 x 10^4 cells/mL

Measured endpoints Cell number counted daily, at approximately 24, 48, and 72 hours

Cell count methodology Conducted using a haemocytometer and a phase-contrast microscope (at 100 – 200 times magnification)

Observations Changes in cell development or appearance, such as cell clumping, cell morphology, cell colour, cell shape, cell size, etc.
Additional observations, such as sedimentation of the test solution, precipitation of cells, solution appearance / colouration, o r other abnormalities

Calculated endpoints cell yield average specific growth rate, section-by-section growth rate for controls

Statistical endpoints Cell yield =EC10, EC20 and EC50; NOEC and LOEC at 72hours , Average Specific Growth Rate = EC10, EC20, and EC50 (0 – 3 days) and NOEC and LOEC
Reference substance (positive control):
yes

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
11.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
18.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Results with reference substance (positive control):
The 72-hour EC25 1044mg/L falling within the 95% confidence interval (Warning limits (mean ± 2 standard deviations (SD)) and control limits (mean± 3 SD)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
All of the validity criteria for the range-finding and definitive tests were met
Conclusions:
From the results of the definitive test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be 7.90, 11.2, and 18.8 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be <0.781, <0.781 and 8.78 mg/L, respectively. The NOEC and LOEC were estimated to be 6.25 and 12.5 mg/L, respectively for average specific growth rate and <0.781 and 0.781 mg/L, respectively for cell yield.
Executive summary:

The toxicity of the test substance to the freshwater green alga,Pseudokirchneriella subcapitatawas investigated under static test conditions, according to OECD Guideline 201,“Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (OECD, 2011). 

 

With the following exceptions, all components of this study were conducted in accordance with the requirements of OECD Principles of Good Laboratory Practice (GLP) (OECD, 1998):

 

P. subcapitataculture;

• Reference toxicant testing with P. subcapitata; and,

• Ecotoxicological and analytical method development.

 

Both the range-finding and definitive tests met all the validity criteria and the test results were based on measured concentrations at the beginning and end of the test, or more specifically, the nominal concentrations.

 

Results of the range-finding test indicated that the average algal growth estimates for 72-hour EC10is 1.5 -15mg/l , EC2015 -150mg/l and EC50s is 15 -150mg/l expressed here in terms of nominal concentrations.

 

From the definitive test and based on the TWM concentrations, the average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be 7.90, 11.2, and 18.8 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be <0.781, <0.781 and 8.78 mg/L, respectively.

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