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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-03-09 to 2012-03-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples of the test solutions were collected at approximately 0 and 72 hours to measure concentrations of the test substance in the 37 and 73 ng/L test solutions. Only the 37 and 73 ng/L test solutions were evaluated due to limitations with the analytical method. At test initiation, samples were collected from the 37 and 73 ng/L treatment groups and control groups prior to addition of the algae. The stock solutions prepared in N,N-dimethylformamide were also analyzed to verify that the lest solutions were properly dosed. At test termination, the remaining biological replicates from the 37 and 73 ng/L treatment groups and control groups were pooled and then sampled. The 73 ng/L abiotic replicate was sampled at test termination to determine the stability of the test substance under the conditions of the study. All samples were collected in glass volumetric flasks and analyzed immediately without storage.
Vehicle:
yes
Details on test solutions:
A primary stock was prepared by dissolving 0.0200g of the test substance in 1000 mL of N,N-dimethylformamide (DMF) to achieve a nominal concentration 20 μg/L. The primary stock was sonicated for five minutes and inverted to mix and appeared clear and colorless alter mixing. A secondary stock was prepared by diluting 3.65 mL of the primary stock in 100 mL of DMF to achieve a nominal concentration of 730 ng/mL. The secondary stock was serially diluted to prepare additional stock solutions at nominal concentrations of 45.63, 91.25, 182.5 and 365.0 ng/mL.. The stocks were also inverted to mix. The appropriate volume of each stock was added with Hamilton gas-tight syringes into 4 liter, glass aspirator bottles containing freshwater algal medium and the test solutions were stirred for approximately thirty minutes. The aspirator bottles were calibrated to minimize headspace prior to use. The test solutions were then decanted from mid-depth via a tube on the aspirator bottles into the test vessels. The solvent control solution contained 0.1 mL DMF per liter of medium which was the same concentration of DMF present in each of the test concentrations.

The negative control solution consisted of freshwater algal medium without the addition of test substance or solvent. The test and control solutions appeared clear and colorless and were otherwise unremarkable.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Source: Original algal cultures were obtained from the University of Toronto Culture Collection and had been maintained in culture medium at the test laboratory. Algal cells used in this test were obtained from cultures that had been actively growing in culture medium for at least two weeks prior to test initiation. Algal cells for this study were taken from a culture that had been transferred to fresh medium four days prior to test initiation. The negative and solvent control organisms were expected to exhibit exponential growth over the 72-hour exposure period. Exponential growth phase, defined as the period of growth where the algal cells are dividing at a constant rate, is indicated by the linear section of the growth curve on a logarithmic scale.

Culture medium: The algal cells were cultured and tested in freshwater algal medium. Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified well water. The test medium was then prepared by adding appropriate volumes of the stock nutrient solutions to purified well water. In addition to the medium nutrient components, the medium also contained supplemental sodium bicarbonate (35 mg/L supplemental was added, resulting in a tinal concentration of 50 mg/L). The pH was adjusted to 7.5 ± 0.1 using 10% hydrochloric acid and was sterilized by filtration (0 .22 μm) prior to use.

Inoculation of test vessels: Prior to test initiation, an inoculum of the algal cells was prepared in freshwater algal medium at a concentration of approximately 0.98 X 106 cells/mL. The concentration of algal cells was verified using a hemacytometer and microscope, and 1.5 mL of the inoculum was added to each test chamber to achieve a nominal concentration of approximately 5,000 cells/mL at test initiation.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
23.8 to 24.8°C
pH:
7.8 to 7.9 (day 0), 9.8-10.1 (day 3)
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (Control), 0 (Solvent control), 4.6, 9.1 , 18, 37 and 73 ng/L

Mean measured concentrations in two highest treatments levels (37 and 73 ng/L):
Details on test conditions:
Test apparatus: Test chambers were sterile, 300-mL BOD bottles plugged with glass stoppers. The bottles were completely filled with test solution to avoid headspace. The test chambers were labelled with the project number, concentration and replicate and were indiscriminately positioned daily on mechanical shakers in an environmental chamber designed to maintain the desired test temperature throughout the test. The test flasks were shaken continuously at 100 rpm.

Test conditions: Test chambers were held in an environmental chamber at a temperature of 23 ± 2°C. The temperature of a container of water adjacent to the test chambers in the environmental chamber was recorded twice daily during the test. In addition, temperature in the environmental chamber was measured continuously. The algae were held under continuous cool-white fluorescent lighting throughout the test. The target light intensity was 5,000 lux ± 20%. Light intensity was measured at five locations surrounding the test flasks on the shaker table at test initiation. The pH of the medium in each treatment and control group was measured at test initiation and at test termination. At test initiation, pH was measured in the individual batches of test solution prepared for each treatment and the control group. At test initiation, pH was measured in pooled samples of test solution collected from each of the remaining replicates in each treatment and control group, and the single abiotic replicate.

Observations: Test medium samples were collected at approximately 24-hour intervals during the 72-hour exposure from three replicates of the treatment and control groups, after sampling, these replicates were removed from the study. These samples were collected for the determination of algal cell densities and were held for a maximum of three days under refrigerated conditions sufficient to inhibit growth until cell counts could be performed. Cell counts were performed using an electronic particle counter (Coulter Electronics, Inc.). Prior to conducting cell counts, the linearity of the instrument response was determined at settings previously established for P. subcapitata. Microscopic examination of the inoculum culture prior to initiation of the test and of a pooled sample from each test concentration and the control at the end of the test was performed to assess whether the algae were normal in appearance.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 15 ng/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 15 ng/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 15 ng/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 15 ng/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
Reported statistics and error estimates:
The calculation of cell densities, yields, growth rates, and percent inhibition values, as well as all statistical analyses, were conducted using "The SAS System for Windows," Version 8.2. There were no statistical differences in calculated growth parameters between the treated cultures and the controls.

Table 1. Results of analysis of test media

 

Nominal concentration (ng/l)

Geometric mean measured concentration (ng/l)

Measured concentration as % of nominal

0 (Control)

<LOQ

-

0 (Solvent control)

<LOQ

-

37

<LOQ

9.5

73

15

21

 

Table 2. Test results

 

Nominal concentration (ng/l)

Mean yield after 72 hours (cells/mL)

Percentage inhibition of yield after 72 hours

Mean Growth rate after 72 hours

Percentage inhibition of growth rate after 72 hours

0 (Control)

283462

-

0.0557

-

0 (Solvent control)

211516

-

0.0520

-

4.6

323788

-14

0.0580

-4

9.1

239125

16

0.0540

3

18

368297

-30

0.0599

-8

37

310581

-10

0.0574

-3

73

377541

-33

0.0602

-8

 

Validity criteria fulfilled:
yes
Conclusions:
A 72-hour EC50 value of >15 ng/l and NOEC of ≥15 ng/l have been determined for the effects of the test substance on growth rate of Pseudokirchneriella subcapitata. The test substance has a water solubility of approximately 7.3 ng/l and therefore the results indicate that it is not toxic at its solubility limit.

Description of key information

EC50 and NOEC (72-h): EC50 >15 ng/l and NOEC: ≥15 ng/l, growth rate of Pseudokirchneriella subcapitata, reliability 1 (Wildlife International Ltd., 2012)

Key value for chemical safety assessment

Additional information

A 72 hour EC50 value of >15 ng/l and NOEC of ≥15 ng/l have been determined for the effects of the test substance on growth rate of Pseudokirchneriella subcapitata in accordance with OECD TG 201 and in compliance with GLP (Wildlife International, 2012).

Due to the slow hydrolysis of the test substance and the test media preparation and exposure method, it is likely that the test organisms were predominantly exposed to the parent substance.

The test substance has a water solubility of <10 ng/l and therefore, the results indicate that it is not toxic at its solubility limit.