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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
For determination of the test item concentrations five replicate samples (5 mL per replicate) were taken from each test concentration and from the control group at the start of the test and three replicate samples (5 mL per replicate) on subsequent days thereafter during the experiment. For these daily measurements, one extra replicate was used in each test group which was treated same as the replicates used for determination of the algal cells.
The analysis was performed by the analytical laboratory of TOXI-COOP ZRT according to the results of the analytical method validation (Study number: 880-100-1595. See Section 8, Nemeth 2017) using HPLC method with UV detection.
Vehicle:
no
Details on test solutions:
The test solutions used in the test were prepared by mechanical dispersion. A stock solution was first prepared by dissolving an amount of 0.1 g test item in 1000 mL dilution water (OECD medium) resulting a nominal concentration of 100 mg/L. This stock solution was ultrasonicated for approx. 15 min and then agitated by orbital shaker for about 1 hour to obtain clear solution. Subsequent lower concentrations were prepared by appropriate dilution of this stock solution. The test solutions were prepared just before introduction of algae (start of the experiment).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Origin: The algae were supplied by the SAG: Collection of Algal Cultures, D-37073 Göttingen, Germany
Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.
Pre-culturing: The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 2-4 days. (The pre-culture was incubated for four days at this test.) The algal cultures used in this study did not contain deformed or abnormal cells.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
According to the guideline.
Post exposure observation period:
No.
Hardness:
Not reported.
Test temperature:
Culture temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was between 22.1 and 22.7°C measured in the flask and in the range of 21.9 – 23.9°C measured within the climate chamber.
pH:
pH: The pH was measured in each test group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of the test. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.26 – 8.55 during the experiment.
Dissolved oxygen:
Not reported.
Salinity:
No.
Conductivity:
Not reported.
Nominal and measured concentrations:
0 - 6.25 - 12.5 - 25 - 50 - 100 mg/L nominal.
0 - 3.5 - 6.9 - 13.3 - 29.5 - 62.1 mg/L calculated geometric mean concentrations, based on measured concentrations.
Details on test conditions:
The measured concentrations deviated more than 20 % from the nominal during the experiment therefore the geometric mean of the measured concentrations were calculated to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings: 3.5, 6.9, 13.3, 29.5 and 62.1 mg/L.
Biological results and endpoints are based on the measured geometric mean concentrations.

Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment.

Light Intensity: The light intensity was checked and recorded at the start of the test. The algal culture flasks were continuously illuminated. The average light intensity measured at the position occupied by algal culture flasks during the test was 7998 lux, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

In order to select appropriate test concentrations for use in the definitive test, a non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item. Based on the results of the preliminary range-finding test five test concentrations in a geometric series with a spacing factor of 2 and a concurrent untreated control were used in the main test.

Performance of the Test
The test was performed with three replicates per test concentration and six replicates in the control group.
The test was started (0 hours) by inoculation of 0.1 mL algal biomass (107 algal cells per mL) to 100 mL test item solution. The initial cell density was about 104 cells/mL, in each test flask.
Volumes of 100 mL algal suspension per replicate were continuously shaken by a laboratory orbital shaker in 250 mL Erlenmeyer flasks. The test flasks were covered with air-permeable stoppers.
The cell concentration was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. The cell morphology was examined in parallel.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
19.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
35.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Analytical Results
Five different concentrations arranged in a geometric series with a spacing factor of 2 and a concurrent control was tested in the main experiment.
The measured concentrations deviated more than 20 % from the nominal therefore the exposure concentrations were calculated as the geometric mean of the concentrations measured at each day of the test .
In the untreated control group the test item was not detected.

Biological Results:
Average Specific Growth Rate:
The results of the statistical evaluation show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value in the concentrations of 6.9, 13.3, 29.5 and 62.1 mg/L (measured). Accordingly the 72-h NOEC based on growth rate was determined to be 3.5 mg/L. The 72 h ErC50 value was determined to be >100 mg/L (calculated as 242.8 mg/L; 95% confidence limits: 159.2 – 450.7 mg/L).
Yield:
The results of the statistical evaluation show that the 0-72 h yield was statistically significantly different from the untreated control value in the concentrations of 6.9, 13.3, 29.5 and 62.1 mg/L (measured). Accordingly the 72-h NOEC based on yield was determined to be 3.5 mg/L. The 72 h EyC50 value was determined to be 35.7 mg/L (95 % confidence limits: 27.4 – 51.3 mg/L).
Morphological Changes of Algal Cells:
No abnormal shape of algal cells was noticed in the control and in the concentrations of 3.5, 6.9 and 13.3 mg/L at the 24-h and 48-h observation periods and in the concentrations of 3.5 and 6.9 mg/L at the end of the test. Swollen cells were observed together with the normal cells in the concentrations of 29.5 and 62.1 mg/L at the 24-h and 48-h observation periods and in the concentrations of 13.3, 29.5 and 62.1 mg/L at the end of the test.
Results with reference substance (positive control):
The date of the last study (Study number: 392-201-1921) with the reference item Potassium dichromate was: 07 – 10 March 2017.
The 72h ErC50: 0.99 mg/L, (95 % confidence limits: 0.68 – 1.49 mg/L)
The 72h EyC50: 0.59 mg/L, (95 % confidence limits: 0.47 – 0.75 mg/L)

The cell density in the control cultures increased by a factor of 55.33 within 72 hours. This corresponds to a specific growth rate of 1.34 day-1.

 The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %

- CV for section-by-section growth rate day 0-1: 21.73 %

- CV for section-by-section growth rate day 1-2: 14.32 %

- CV for section-by-section growth rate day 2-3: 4.82 %

The mean coefficient of variation for section-by-section specific growth rates: 13.62 %.

 The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 2.10 %.

All validity criteria were met, therefore the study can be considered as valid.

Validity criteria fulfilled:
yes
Conclusions:
The 72-h ErC50 is >100 mg/L (calculated: 242.8 mg/L).
The NOEC growth rate is 3.5 mg/L.
Executive summary:

A 72-h algal growth inhibition test with Raphidocelis subcapitata, according to OECD and EC-guidelines was performed. The 72-h ErC50 is >100 mg/L (calculated: 242.8 mg/L). The NOEC growth rate is 3.5 mg/L. The ErC10 = 19.2 mg/L.

All validity criteria were met.

Description of key information

A 72-h algal growth inhibition test with Raphidocelis subcapitata, according to OECD and EC-guidelines was performed. The 72-h ErC50 is >100 mg/L (calculated: 242.8 mg/L). The NOEC growth rate is 3.5 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
242.8 mg/L
EC10 or NOEC for freshwater algae:
19.2 mg/L

Additional information