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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:
2019-04-15 to 2019-07-11
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:
2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals", 2nd Ed., February 08, 2019.
Version / remarks:
2019
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (partA; Section 5) of directive 91/414
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and 100 mg/L
- Sampling method: Duplicate samples from the freshly prepared test media (containing algae) of the only test concentration and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of the only test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers (replicate 1-6) of each treatment. In addition, from the six additionally prepared biological replicates (replicates 7-12) separate duplicate samples were taken by pouring together the contents of these test beakers. All samples were adjusted to pH 3 with HCl, and remained undiluted until analysis.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ -20 °C)
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
The test medium of the only test item concentration of nominal 100 mg/L was prepared by dissolving 101.6 mg test item into 1016 mL test water by intense stirring for 10 minutes. The pH was adjusted from pH 5.6 to pH 8.7 with 1 M NaOH. The solution was sterilized by membrane filtration (cellulose acetate, 0.2 µm). The test medium was freshly prepared just before the start of the test (=introduction of algae). All glass vessels were sterilized by either dry heating (180 °C, 3 h) or by autoclaving (121 °C, 20 min) prior to use.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (KORSHIKOV)
- Strain: 61.81 SAG
- Source: Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): The algae cells were taken from an exponentially growing pre-culture, which was set up 4 days prior to the test start under the same conditions as in the test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (=24 mg/L) as CaCO3
Test temperature:
22.3 to 23.3 °C
pH:
pH value in the control at test start: 8.2;
pH value in the control at test end: 8.7;
pH value in the test item treatments at test start: 8.7;
pH value in the test item treatments at test end: 8.9
Salinity:
Not applicable
Conductivity:
no data
Nominal and measured concentrations:
Nominal: 100 mg test item/L and a control
Measured concentrations: The concentrations of the test item were within ±20% of the nominal concentrations during the test. Thus, all reported results refer to nominal values.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 mL volume with approx. 50 mL of test medium
- Type: closed (covered with sterile caps)
- Initial cells density: 5000 algae cells per mL test medium
- No. of vessels per concentration (replicates): 12
- No. of vessels per control (replicates): 6
- No. of vessels per blank control (replicates): Additionally, two replicated of the only test concentration and one replicate of the control were prepared without algae to provide a "blank" for the spectrophotometric measurements. These additional replicates were incubated under the same conditions as described above. These blank values were subtracted from the absorptions measured in the samples containing algae in order to eliminate absorption caused by the test item.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to OECD guideline

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity (mean): 5007 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: spectrophotometer

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield and Growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield and Growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield and Growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield and Growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield and Growth rate
Details on results:
Microscopical Observation: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing at the nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected at the only concentration tested.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 72-hour EC50:
Yield: 0.445 mg/L
Growth rate: 0.941 mg/L
Biomass: 0.494 mg/L
Reported statistics and error estimates:
Based on the calculated cell densities, the 72 hours ErC50 and the 72 hour EyC50 could not be calculated by a statistical analysis due to the lack of effects and were therefore determined directly from the raw data.
For the determination of the 72 hour LOEC and the 72 hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by two-sample t-test.
The six replicates of the only test item concentration which were treated like the control (i.e. daily determination of cell density) were used for statistical analysis.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat® Solutions GmbH.

Analytical Results:
The quantification of the test item in the test samples was performed using liquid chromatography with UV detection. At the start of the test 97% of the nominal test concentrations were found. After 72 hours test duration, 100% of the nominal value was determined. Analysis of the additionally prepared replicates which were kept closed throughout the test to ensure sterility and sampled only at test end showed 100% of the nominal value. Bacterial degradation of the test item could therefore be rules out.

Table 1: Summary of Analytical Results

Nominal

[mg test item/L]

% of nominal1

RSD

[%]

n

Control

n.a.

n.a.

4

100

99

2

4

1mean value of all measured samples per treatment group

RSD: relative standard deviation per treatment group

N: number of analysed samples (only replicates 1-6 were evaluated)

N.a.: not applicable


Validity criteria of the study:
Cell density
Increase in Control Cultures: 147.7-fold increase within 72 hours and thus, the validity criterion was met.
Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 18.5% and thus, the validity criterion was met.
Coefficient of Variation of Average Growth between Control Replicates: 1.6% and thus, the validity criterion was met.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this algae growth inhibition test, no toxicity of the test item up to nominal 100 mg/L was determined. Thus, the 72-hour LOEC and the 72-hour EC50 were clearly higher than 100 mg test item/L.
Executive summary:

The inhibitory effect of the test item on the growth of the freshwater algae Pseudokirchneriella subcapitata was investigated in a static limit test according to OECD TG 201. Exponentially growing cultures of this green algal species were exposed to the only test item concentration of nominal 100 mg/L under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The 72-hour EC50 and the 72-hour EC10 for the parameters growth rate and yield could not be calculated due to the low effects of the test item. Therefore the 72-hour EC50 and the 72-hour EC10 was determined to be higher than nominal 100 mg test item/L, the highest concentration tested. The 72-hour NOEC was determined to be at least nominal 100 mg test item/L for the parameters growth rate and yield and the associated 72-hour LOEC was determined to be higher than nominal 100 mg test item/L for both parameters. All reported results refer to nominal values since the concentrations of the test item were within ±20% of the nominal concentrations during the test

Description of key information

In an Algal Growth Inhibition test according to OECD TG 201, no toxicity of the test item was determined up to a nominal concentration of 100 mg/L. The 72-hour NOEC was determined to be at least nominal 100 mg test item/L for the parameters growth rate and yield. The 72-hour EC50 and the 72-hour EC10 was determined to be higher than nominal 100 mg test item/L (reference 6.1.5-1).

Key value for chemical safety assessment

Additional information

The inhibitory effect of the test item on the growth of the freshwater algae Pseudokirchneriella subcapitata was investigated in a static limit test according to OECD TG 201. Exponentially growing cultures of this green algal species were exposed to the only test item concentration of nominal 100 mg/L under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The 72-hour EC50 and the 72-hour EC10 for the parameters growth rate and yield could not be calculated due to the low effects of the test item. Therefore the 72-hour EC50 and the 72-hour EC10 was determined to be higher than nominal 100 mg test item/L, the highest concentration tested. The 72-hour NOEC was determined to be at least nominal 100 mg test item/L for the parameters growth rate and yield and the associated 72-hour LOEC was determined to be higher than nominal 100 mg test item/L for both parameters. All reported results refer to nominal values since the concentrations of the test item were within ±20% of the nominal concentrations during the test (reference 6.1.5-1).