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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:
2018-01-09 - 2018-02-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
28th July 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA Ecological Effects Test Guidelines, OCSPP 850.5400, Algal Toxicity, Tiers I and II, EPA 712-C-96-164
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch/Lot Number: 7703200
Description: clear, pale yellow liquid
Purity: considered as 100%
Manufacture date: 04 February 2016
Expiry date: 28 February 2019
Storage condition: Controlled room temperature (15-25°C, below 70 RH%)
Analytical monitoring:
yes
Details on sampling:
Analytical measurement was performed at the control and at the applied test concentration levels at the beginning of the test and in 24-hour intervals thereafter during the experiment.
Determination of test substance concentrations was performed at each test concentration level and from the additional samples without algae in order to distinguish degradation and adsorption of the test item to algae.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Because the test item is very poorly soluble in water, test solutions were prepared using a saturated solution method (water accommodated fraction, WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
Saturated test item solutions (nominal loading rates of 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L) were prepared individually by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the study. These solutions were shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C.
The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give test solutions at the appropriate nominal loading rates.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd
- Initial cell number: The initial cell number in the test cultures was 104 cells/mL.
- Pre-culturing: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
not applicable
Hardness:
Not reported
Test temperature:
22.6 – 23.0 °C
pH:
7.29 – 8.60
Dissolved oxygen:
Not reported
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rates WAFs
The corresponding measured geometric mean test item concentrations were: 1.78; 2.02; 8.95; 18.33 and 25.38 mg/L in presence of algae, while the measured geometric mean test item concentrations were: 3.46; 6.15; 11.99; 18.18 and 24.79 mg/L in absence of algae
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: fitted with air-permeable stoppers
- Material, size, headspace, fill volume: glass
- Initial cells density: 10E04 algal cells per mL test medium
- Control end cells density: 69.33E04 cell/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 24 h light
- Light intensity and quality: Approx 7813 lux (equivalent to 106 μE/m2/s). Fluorescent lamps (with a spectral range of 400-700 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
- Other: Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x2
- Range finding study
- Test concentrations: Nominal concentrations (% saturated solution) - 0.1, 1, 10, 100
- Results used to determine the conditions for the definitive study:
Nominal concentration (% saturated solution)/ Average cell number at 72 hours (x10E04 cell/mL): untreated control/72.00; 0.1/69.50; 1/67.00; 10/45.00; 100/4.00
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 28.92 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15.29 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.53 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.71 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.71 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: none reported
- Colour differences: none reported
- Flocculation: none reported
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no (NOEC)
Results with reference substance (positive control):
The date of the last study (Study Code: 17/367-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 04 – 07 December 2017.
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EbC 50: 0.63 mg/L, (95 % confidence limits: 0.58 – 0.69 mg/L)
The 72h EyC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).
Reported statistics and error estimates:
Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.
The ErL50/ErC50 values of the test item could not be calculated (due to the slight effect observed) and therefore determined directly from the raw data. The EbL50/EbC50 and EyL50/EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.
With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates were significantly different from that of the control group in the
initial nominal loading rate range of 25.0 - 100.0 mg/L (WAFs). The overall NOELR was determined as 12.5 mg/L nominal loading rate (WAF); the overall LOELR was determined as 25.0 mg/L nominal loading rate (WAF). The 0-72 h areas and yield were significantly different from that of the control group in the examined nominal concentration range of 12.5 - 100.0 mg/L (nominal loading rates, WAFs). The overall NOELR was determined as 6.25 mg/L nominal loading rate WAF; the overall LOELR was determined as 12.5 mg/L nominal loading rate WAF.
Validity criteria fulfilled:
yes
Executive summary:

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours (OECD TG 201).

A significant toxic response was observed during the preliminary range-finding test, therefore five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment.

The concentrations of test item used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rates WAFs. Test concentrations were analytically determined at the start of the test and at 24 hour intervals thereafter in order to better define loss of the test substance during the exposure period.

The test design included three replicates at each test concentration and six replicates for the untreated controls. There was an additional replicate without algae at each test concentration level for further analytical measurements (0, 24, 48 and 72 hours) in order to distinguish degradation and adsorption of the test item to algae.

According to the relevant guideline (OECD 201) disappearance of the test substance from solution by absorption to the increasing algal biomass does not mean that it is lost from the test system. When the result of the test was analysed, it was checked whether a decrease in concentration of the test substance in the course of the test was accompanied by a decrease in growth inhibition.

The corresponding measured geometric mean test item concentrations were: 1.78; 2.02; 8.95; 18.33 and 25.38 mg/L in presence of algae (more details are given in 4.2.). In addition, results of the analytical measurements of samples in absence of algae showed measured geometric mean test item concentrations of 3.46; 6.15; 11.99; 18.18 and 24.79 mg/L for the nominal concentrations of 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rate WAFs respectively (more details are given in 4.2.).

However based on the section-by-section average specific growth rates, it is indicating that a decrease of concentration of the test item was not clearly accompanied by a decrease in growth inhibition. Based on these results most probably the test item is indeed absorbed to the increasing algal biomass, and should not be considered as lost from the test system.

Therefore it is recommended to express the EL50/EC50 and NOELR/NOEC//LOELR/LOEC values in the initial test item concentrations (nominal or measured) and not in the mean measured concentrations from the algae containing samples, thus biological results are related to the initial nominal loading rates (WAFs) and initial measured test item concentrations.

Results based on initial nominal loading rates (WAFs):

72 -h ErL50 > 100 mg/L; 72 -h EγL50 = 37.8 mg/L; 72 -h EbL50 = 36.51 mg/L

72 -h NOErLR = 12.5 mg/L; 72 -h NOEγLR = 6.25 mg/L; 72 -h NOEbLR = 6.25 mg/L

Results based on initial measured concentrations:

72 -h ErC50 > 28.92 mg/L; 72 -h EγC50 = 15.70 mg/L; 72 -h EbC50 = 15.29 mg/L

72 -h NOErC = 6.53 mg/L; 72 -h NOEγC = 3.71 mg/L; 72 -h NOEbC = 3.71 mg/L

Description of key information

Results based on initial nominal loading rates (WAFs):

72 -h ErL50 > 100 mg/L; 72 -h EγL50 = 37.8 mg/L; 72 -h EbL50 = 36.51 mg/L

72 -h NOErLR = 12.5 mg/L; 72 -h NOEγLR = 6.25 mg/L; 72 -h NOEbLR = 6.25 mg/L

Results based on initial measured concentrations:

72 -h ErC50 > 28.92 mg/L; 72 -h EγC50 = 15.70 mg/L; 72 -h EbC50 = 15.29 mg/L

72 -h NOErC = 6.53 mg/L; 72 -h NOEγC = 3.71 mg/L; 72 -h NOEbC = 3.71 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
28.92 mg/L
EC10 or NOEC for freshwater algae:
6.53 mg/L

Additional information

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours (OECD TG 201).

A significant toxic response was observed during the preliminary range-finding test, therefore five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment.

The concentrations of test item used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rates WAFs. Test concentrations were analytically determined at the start of the test and at 24 hour intervals thereafter in order to better define loss of the test substance during the exposure period.

The test design included three replicates at each test concentration and six replicates for the untreated controls. There was an additional replicate without algae at each test concentration level for further analytical measurements (0, 24, 48 and 72 hours) in order to distinguish degradation and adsorption of the test item to algae.

According to the relevant guideline (OECD 201) disappearance of the test substance from solution by absorption to the increasing algal biomass does not mean that it is lost from the test system. When the result of the test was analysed, it was checked whether a decrease in concentration of the test substance in the course of the test was accompanied by a decrease in growth inhibition.

The corresponding measured geometric mean test item concentrations were: 1.78; 2.02; 8.95; 18.33 and 25.38 mg/L in presence of algae (more details are given in 4.2.). In addition, results of the analytical measurements of samples in absence of algae showed measured geometric mean test item concentrations of 3.46; 6.15; 11.99; 18.18 and 24.79 mg/L for the nominal concentrations of 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rate WAFs respectively (more details are given in 4.2.).

However based on the section-by-section average specific growth rates, it is indicating that a decrease of concentration of the test item was not clearly accompanied by a decrease in growth inhibition. Based on these results most probably the test item is indeed absorbed to the increasing algal biomass, and should not be considered as lost from the test system.

Therefore it is recommended to express the EL50/EC50 and NOELR/NOEC//LOELR/LOEC values in the initial test item concentrations (nominal or measured) and not in the mean measured concentrations from the algae containing samples, thus biological results are related to the initial nominal loading rates (WAFs) and initial measured test item concentrations.

Results based on initial nominal loading rates (WAFs):

72 -h ErL50 > 100 mg/L; 72 -h EγL50 = 37.8 mg/L; 72 -h EbL50 = 36.51 mg/L

72 -h NOErLR = 12.5 mg/L; 72 -h NOEγLR = 6.25 mg/L; 72 -h NOEbLR = 6.25 mg/L

Results based on initial measured concentrations:

72 -h ErC50 > 28.92 mg/L; 72 -h EγC50 = 15.70 mg/L; 72 -h EbC50 = 15.29 mg/L

72 -h NOErC = 6.53 mg/L; 72 -h NOEγC = 3.71 mg/L; 72 -h NOEbC = 3.71 mg/L