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Toxicity to aquatic algae and cyanobacteria

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:

May 05, 2022 to August 09, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

yes

Remarks:

See 'Any other information on materials and methods incl. tables' section

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

GC-MS

Details on sampling:

Frozen samples to be analyzed:
Control (0 and 72 hours)
Test solutions (0, 24, 48 and 72 hours)

To the frozen water samples 20% toluene (based on the sample volume; e.g. 4 mL to a 20 mL test sample) was added and left to stand until completely defrosted. The samples were then homogenised and liquid/liquid extracted for 1 min by means of a vortex mixer. Afterwards, the samples were left to stand for phase separation. An aliquot of the organic phase of approx. 1 mL was transferred into a GC vial and used for analysis by GC-MS.
If necessary, final sample extracts were diluted in toluene to achieve final concentrations falling within the linear range of the calibration curve.

Vehicle:

yes

Details on test solutions:

Based on the results of a preliminary non-GLP range finding test conducted at 0.116, 0.695, 4.17 and 25.0 mg test item/L (see section 24), the following concentration steps in a geometrical series (spacing factor: square-root of five) were tested in the definitive test:
0.48, 1.07, 2.40, 5.37 and 12.0 mg test item/L.

At day of start of the exposure period a stock solution (S1) at a nominal concentration of 12.0 mg test item/L was prepared by dissolving 0.01210 g test item in 1000 mL test medium.
This stock solution (S1 / C5) was stirred for 80 minutes at 500 rpm, interrupted by short periods of intensive stirring at 1000 rpm (<5 minutes), followed by further stirring for 25 minutes at 700 rpm at ambient temperature in dark using a magnetic stirrer.
The details for preparing the above stock solution are as follows. The test medium was adapted to test temperature, and ca. 600 mL of the test medium was filled into a 1 L glass measuring flask. The test item was weighted on a glass-weighing boat and was rinsed into the 1 L glass measuring flask using the test medium and filled-up to the mark.
After a stirring period of 0.5 hours the stock solution was visually examined for undissolved/particulate matter and test item was visible at the surface of the water column.
Therefore, it was decided to continue stirring, see above, until no test item was visible while left to settle for 15 minutes. After the prolonged stirring period the stock solution (S1 / C5) was visually examined for undissolved/particulate matter in the free water column and no test item was visible.
This stock solution was used to prepare a dilution series in test medium. The test solutions were prepared by addition of the appropriate amount of the stock solution (S1 / C5) to a glass volumetric flask and thereafter the flask was filled up to the mark with test medium. The final test solutions were stirred for 10 minutes on a magnetic stirrer at 500 rpm at room temperature.
The volume of the stock solution was large enough to prepare the test solutions and all analytical samples.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test system used in this study was the freshwater algae Pseudokirchneriella subcapitata (SAG 61.81), currently valid species name: Raphidocelis subcapitata.
The organisms were originally supplied by Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany.
The unicellular green alga Pseudokirchneriella subcapitata was chosen as a representative of freshwater algae. The selection of the test system is based on the test guideline.

To adapt the algae to the test conditions a pre-culture was inoculated by a liquid algal culture and incubated under test conditions.
Species: Pseudokirchneriella subcapitata (SAG 61.81), currently valid
species name: Raphidocelis subcapitata
Supplier: Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany
Date of receipt: February 24, 2016
Charge No. (ECT): A/240216/A
pH-value of the algal medium: 7.9
Culture medium: OECD medium (OECD 201, EN ISO 8692)
Volume of liquid stock culture per pre-culture vessel: 100±5 mL
Pre-culture vessels: 300 mL Erlenmeyer flasks
Number of replicates: 2
Light: Permanent illumination (24/0 h light/dark); Econlux LED Sunstrip “daylight”
Light intensity: 60–120 μE m-2s-1; (measured: 85.8–88.6 μE m-2s-1)
Shaker: 100±5 oscillations/min
Temperature in the test room: 21–24 C; (measured: 23.3–23.8 °C)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.8–24.0 °C, mean 23.0 °C

pH:

pH of test medium: 8.2
pH in test solutions: 8.0-9.1

Nominal and measured concentrations:

0.48, 1.07, 2.40, 5.37 and 12.0 mg test item/L

Details on test conditions:

Test vessels: 60 mL glass vials (nominal), air-tightly closed by a screw-cap with PTFE lining
Volume of test solution per test vessel: 60 mL (nominal), filled to the rim
Age of the pre-culture: 4 days
Number of cells per mL in the pre-culture before inoculating the test solutions: 408.5×10^4
Number of cells per mL test solution at the beginning of the test: 0.20×10^4
Number of replicates per test item concentration: 3
Number of replicates per test item concentration used for analytical purposes only: 2
Number of replicates in the control: 6
Test medium: OECD medium + additional NaHCO3 (modified)
pH of test medium: 8.2
pH in test solutions: 8.0–9.1
Light regime: 24 h light/0 h dark
Type of light: Econlux LED Sunstrip “daylight”
Light intensity: 80.2–89.2 μE m–2s–1;mean 84.4 μE m–2s–1
Temperature: 22.8–24.0 °C, mean 23.0 °C
Shaker: 100±5 oscillations/min; the test vessels were placed randomly on a horizontal shaker, so that each test vessel was rotated around its own axis
Test duration (exposure): 72 hours
Counting of algae: Daily

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.131 mg/L

95% CI:

>= 0.035 - <= 0.485

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.72 mg/L

95% CI:

>= 0.183 - <= 3.01

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.36 mg/L

95% CI:

>= 0.153 - <= 0.864

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.47 mg/L

95% CI:

>= 1.93 - <= 15

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.289 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Results with reference substance (positive control):

Two reference tests using potassium dichromate (K2Cr2O7) as reference item were performed in separate studies. One was performed using a standard test design (study number: IAO2202, February 2022) and the other using a closed-bottle system (study number: IAO2203-CB, March 2022). The latter was performed to confirm that that the use of a modified test system to reduce losses of test item through volatility (completely filled and sealed vessels) is expected to give results consistent with that obtained in a conventional system.

Result:
Standard: Growth rate ErC50 (0–72 h): 1.001 mg/L (0.854 – 1.171 mg/L; 95%-CL)
Closed-Bottle: Growth rate ErC50 (0–72 h): 0.773 mg/L (0.687 – 0.871 mg/L; 95%-CL)

Based on an international ring test (ISO (2004). Water Quality – Freshwater algal growth inhibition test with unicellular green algae. European Standard EN ISO 8692, October 2004.) mentioned in OECD guideline 201, the ErC50 (72h)-values for potassium dichromate obtained from different laboratories was 1.19 mg/L with a standard deviation of 0.27 mg/L.
The ErC50 value for the toxic reference item, potassium dichromate, was determined as 1.001 mg/L (standard system) and 0.773 mg/L (closed system). The value for the standard system is within the range of the international ring test (ISO (2004)). Although the value for the closed system reference test is just outside the range of the aforementioned ring test, it is within the historical range of the general reference test results (open test system) of our laboratory (mean ErC50 value = 1.041 ± 0.328 mg/L) and also within the range of the narrow upper (1.361 mg/L) and lower (0.724 mg/L) warning limit. The ErC50 value is also within the historical range of the reference test results (closed test system) of our laboratory (mean ErC50 value = 0.769 ± 0.082 mg/L) and also within the range of the upper (0.854mg/L) and lower (0.685 mg/L) warning limit.
The narrow warning limits were calculated as one standard deviations from the historical mean of the ErC50 values. The calculation of the warning limits was not conducted in compliance with Good Laboratory Practice Principles (all calculated ErC50 using the log concentration; according to Environment Canada (2005), Guidance Document on Statistical Methods for Environmental Toxicity Tests, Method Development and Applications Section, Environment Canada, Ottawa, ON, Report EPS 1/RM/46).
Additionally, the ErC50 values for the toxic reference item (standard and close system) are within the refined range of 1.10 ± 0.48 mg/L, recommended based on an international ring test (Pattard, M.; Römbke, J.; Moser, T. (2009). Range of Reference Tests in Aquatic Tests. [in] Moser, H.; Römbke, J. “Ecotoxicological Characterization of Waste. Results and Experiences of an International Ring Test”, chapter 5, pp. 61-70.).

Therefore, the results of these reference tests (standard and closed bottle) are acceptable and the test conditions are reliable.

Validity criteria fulfilled:

yes

Conclusions:

The EyC50 of Pseudokirchneriella subcapitata was determined to be 1.06 mg test item/L based on nominal concentrations, equivalent to 0.720 mg test substance/L based on measured concentrations.
The NOEC based on nominal concentration was 0.48 mg test item/L, equivalent to 0.289 mg test substance/L based on geometric mean measured concentrations.

The ErC50 of Pseudokirchneriella subcapitata was determined to be 6.75 mg test item/L based on nominal concentrations, equivalent to 5.47 mg test substance/L based on measured concentrations.
The NOEC based on nominal concentration was 0.48 mg test item/L, equivalent to 0.289 mg test substance/L based on measured concentrations.

Executive summary:

An algal growth inhibition test with Desmodesmus subspicatus was performed with the test substance according to OECD guideline No 201. The corresponding end-points at 72 hours of incubation are :
72h ErC10 = 0.36 mg/L
72h ErC20 = 0.92 mg/L
72h ErC50 = 5.47 mg/L
72h NOErC = 0.289 mg/L
72h EyC50 = 0.72 mg/L
72h EyC10 = 0.13 mg/L
72h NOEyC = 0.289 mg/L

Description of key information

An algal growth inhibition test with Desmodesmus subspicatus was performed with the test susbtance according to OECD guideline No 201. The corresponding end-points based on geometric mean measured concentrations at 72 hours of incubation are:
72h ErC10 = 0.36 mg/L
72h ErC20 = 0.92 mg/L
72h ErC50 = 5.47 mg/L
72h NOErC = 0.289 mg/L
72h EyC50 = 0.72 mg/L
72h EyC10 = 0.13 mg/L
72h NOEyC = 0.289 mg/L

The preferred observational endpoint in th​e algal test is algal growth rate inhibition because it is not dependent on the test design, whereas biomass depends both on growth rate of the test species as well as test duration and other elements of test design​ ​(ECHA guidance Chapter R.7b v​4.0, OECD 201 Guideline). The EU CLP regulation (No 1272/2008 and its adaptation 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EC50 and EC10 based on growth rate are selected as key values and used for classification purposes, which were determined in this study to be ​5.47 mg/L and ​0.36 mg/L respectively.​

Key value for chemical safety assessment

EC50 for freshwater algae:

5.47 mg/L

EC10 or NOEC for freshwater algae:

0.36 mg/L