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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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
For determination of the test item content, three replicate samples were taken from each test concentration every day. One control sample was directly analysed at the start and six control samples were directly analysed at the end.
Samples were diluted with acetonitrile / 25 mM tetrabutylammonium hydroxide (4/6) mixture. The control samples were directly analysed.
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.
After the formulation procedure algal cells were immediately introduced into the test solution (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 three 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 22.0 and 22.5°C measured in the flask and in the range of 21.2 – 23.0°C measured within the climate chamber.
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 5.48 – 9.05 during the experiment.
Dissolved oxygen:
Not reported.
Salinity:
No.
Conductivity:
Not reported.
Nominal and measured concentrations:
6.25, 12.5, 25, 50 and 100 mg/L nominal.
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: 5.9, 12.0, 24.0, 47.3 and 66.3 mg/L.
Details on test conditions:
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 7984 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.

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 (10E7 algal cells per mL) to 100 mL test item solution. The initial cell density was about 10E4 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.

Mean values and standard deviations were calculated for each concentration at each observation period using Excel for Windows software.
For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values using analysis of variance (ANOVA) and Dunnett’s test (α = 0.05; 2-sided) by SPSS software.
For determination of the ECx values analysis Probit analysis by SPSS software was performed.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
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.:
51 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.:
47.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Average Specific Growth Rate:
The results of the statistical evaluation show that the 0-72 h average specific growth rate was statistically not significantly different from the untreated control value in the concentrations of 5.9, 12.0, 24.0 and 47.3 mg/L (measured) and significantly different in the 66.3 mg/L concentration (measured).
Accordingly the 72-h NOEC based on growth rate was determined to be 47.3 mg/L.
The 72 h ErC50 value was determined to be >100 mg/L.

Yield:
The results of the statistical evaluation show that the 0-72 h yield was statistically not significantly different from the untreated control value in the concentrations of 5.9, 12.0, 24.0 and 47.3 mg/L (measured) and significantly different in the 66.3 mg/L concentration (measured).
Accordingly the 72-h NOEC based on yield was determined to be 47.3 mg/L.
The 72 h EyC50 value was determined to be value was determined to be >100 mg/L.

Morphological Changes of Algal Cells:
No abnormal shape of algal cells was noticed in the control and in the lowest concentration of 5.9 mg/L (measured) during the test.
Thin cells, swollen cells and pinches on cells were noticed in the higher concentrations together with the normal cells during the test.
Results with reference substance (positive control):
The date of the last study (Study number: 392-201-2532) 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)
Reported statistics and error estimates:
See above.

Analytical results:

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. The calculated geometric mean of the measured concentrations were: 5.9, 12.0, 24.0, 47.3 and 66.3 mg/L. All biological results are based on the measured geometric mean concentrations.

Validity of the Test:

 The cell density in the control cultures increased by a factor of 112.83 within 72 hours. This corresponds to a specific growth rate of 1.57 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: 56.16 %

- CV for section-by-section growth rate day 1-2: 31.42 %

- CV for section-by-section growth rate day 2-3: 7.06 %

The mean coefficient of variation for section-by-section specific growth rates: 31.55 %.

 The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 3.70 %.

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

Validity criteria fulfilled:
yes
Conclusions:
NOECr = 47.3 mg/L; NOECy = 47.3 mg/L
ECr10 = 51 mg/L; ECy10 = 4.2 mg/L
ECr50 = >100 mg/L; ECy50 = >100 mg/L
Executive summary:

A 72-h algal growth inhibition test with Raphidocelis subcapitata according to the OECD guideline 201 was performed. Results:

NOECr = 47.3 mg/L; NOECy = 47.3 mg/L

ECr10 = 51 mg/L; ECy10 = 4.2 mg/L

ECr50 = >100 mg/L; ECy50 = >100 mg/L

Description of key information

NOECr = 47.3 mg/L; NOECy = 47.3 mg/L

ECr10 = 51 mg/L; ECy10 = 4.2 mg/L

ECr50 = >100 mg/L; ECy50 = >100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
51 mg/L

Additional information

The EC50 is not 100 mg/L, as one is forced to enter in the field above, but the EC50 is >100 mg/L.