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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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

yes

Remarks:

HPLC/MS/MS analysis

Details on sampling:

Sampling schedule:
- Control: at 72 hours
- Test concentration/s: at 0 and 72 hours

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Name: Desmodesmus subspicatus (formerly Scenedesmus subspicatus) Strain No. 86.81 SAG
- Source: Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Maintenance and Acclimatisation : Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21 - 24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 µE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977)) was renewed once a week. Cell density measurements were made using a Particle and Count Size Analyzer, Z2 (Beckman Coulter).
- Preparation of pre-cultures: Pre-cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium
- Test cultures: The algal inocula for a test were taken from an exponentially-growing pre-culture and were mixed with the nutrient medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

1.3 °dH (22.5 mg/L CaCO3)

Test temperature:

21-24 °C

pH:

7.8-8.1

Salinity:

n.a.

Conductivity:

No data

Nominal and measured concentrations:

- 100 mg/L test concentration equals 0.005945 (HPLC/MS values) mg/L after 72 hours
- 100 mg/L test concentration (without algae) equals 0.020058 (HPLC/MS values) mg/L after 72 hours

Details on test conditions:

EXPOSURE CONDITIONS:
- Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers, test volume: 100 mL
- Culturing apparatus: Shaking incubator in which a temperature in the range 21 to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm; Temperature was measured and recorded daily
- Light intensity: A light intensity ranging from 60 to 120 µE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured; The light intensity was checked before the start of the study
- Cell density measurements: Cell densities were measured in a Particle and Size Analyzer (Z2, Beckman Coulter) by taking aliquots from each test flask, which were not replaced
- Experimental design: 5 test concentrations plus 1 control; 3 replicates per concentration; 3 replicates per control; Initial cell density in the test cultures approximately 5000 cells per millilitre; additionally highest test concentration without algae
- Test item concentration/s: 6.3, 12.5, 25, 50 and 100 mg/L
- Method of administration: stock solution
- Criteria of effects: The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.019 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.019 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.019 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.019 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

No toxic effects against algae were observed at the limit of water solubility under exposure conditions.

The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 0.040 % to 0.075 % of nominal values at 0 hours, and from 0.006 % to 0.073 % of nominal values at 72 hours.

Validity criteria fulfilled:

yes

Remarks:

cell density in control increased by a factor of at least 16 in 72 h; mean coefficient of variation for section-by-section specific growth rates in control <= 35 %; CV of average specific growth rates in replicate control cultures <= 7 %

Conclusions:

After 72 hours an EC50 of higher than 0.019 mg/L (NOEC >=f 0.019 mg/L) concerning the reduction of growth rate were determined. No toxic effects against algae were observed at the limit of water solubility under exposure conditions. The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 0.040 % to 0.075 % of nominal values at 0 hours, and from 0.006 % to 0.073 % of nominal values at 72 hours.

Executive summary:

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test' (2006). A range finding test preceded the main test and provided information about the range of concentrations which were used in the main test. The following nominal concentrations of the test item were tested in the range finding test: 0.10, 1.0, 10 and 100 mg/L. In the main test, the algae were exposed to the test item added to dilution water at a range of concentrations for a period of 72 hours. Defined concentrations of the test item led to a certain inhibition of algal yield and growth rate at the end of the 72 hour study period. Cell densities were recorded at 24 hour intervals. All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10). For the calculations all algae counts were divided by a factor of 10000. During the test a temperature range of 21 -24 °C was maintained in the test vessels. The pH was measured at the beginning of the test and after 72 hours of exposure. The maintenance of the test item concentrations was proved by analytical measurements. In order to avoid an impairment of the test system, an additional replicate was used for analysis and pH measurement at the beginning of the test. Chemical analysis and pH measurement at the end of the test were performed using replicate I of the test concentrations and the control vessels. In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, a test flask at the highest test concentration without algae was run in parallel to the geometric series of test concentrations. The validity criteria of the test were met. After 72 hours an EC50 of higher than 0.019 mg/L (NOEC >=f 0.019 mg/L) concerning the reduction of growth rate were determined. No toxic effects against algae were observed at the limit of water solubility under exposure conditions. The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 0.040 % to 0.075 % of nominal values at 0 hours, and from 0.006 % to 0.073 % of nominal values at 72 hours. This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to algae.

Description of key information

After 72 hours an EC50 of higher than 0.019 mg/L (NOEC >=f 0.019 mg/L) concerning the reduction of growth rate were determined. No toxic effects against algae were observed at the limit of water solubility under exposure conditions (for details see also chapter 4.8). The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 0.040 % to 0.075 % of nominal values at 0 hour, and from 0.006 % to 0.073 % of nominal values at 72 hours (Spoo-Kloeppel, 2018).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.019 mg/L

EC10 or NOEC for freshwater algae:

0.019 mg/L

Additional information

"Should read: 72 h-EC 50 >0.019 mg/L and 72 h-NOEC >= 0.019 mg/L"