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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Storage: Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light.
Vehicle:
no
Details on test solutions:
PRE-TREATMENT OF TEST ITEM AND PREPARATION OF TEST ITEM CONCENTRATIONS
A stock solution was prepared to give the desired series of test concentrations. 200.2 mg of the test item were added to 2 litres of dilution water and treated for 1 h in an ultrasonic. The pH was measured to be 7.8.
To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL and 0.690 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 3 replicates were prepared. All flasks were sealed with cotton stoppers.

STANDARD SOLUTIONS USED FOR CALIBRATION OF THE TEST ITEM
20.9 mg of the test item were dissolved and made up with Millipore water/Acetonitrile (1:1) in a 100 mL volumetric flask to prepare a stock solution of 209 mg/L. Defined volumes of this stock solution were diluted with Millipore water to obtain standard solutions in the range of 0.0523 to 2.09 mg/L.
The concentration of the lowest used calibration solution was employed as the limit of quantification.
These solutions were used to calibrate the HPLC-system.

STANDRAD SOLUTIONS USED FOR VERIFICATION OF THE CALIBRATION OF THE TEST ITEM AND BLANK TEST
22.8 mg of the test item were dissolved and made up with Millipore water/Acetonitrile (1:1) in a 100 mL volumetric flask to prepare a verification
solution with a nominal concentration of 228 mg/L. This solution was diluted with Millipore water to obtain a concentration of 0.912 mg/L. The daily
verification of the calibration was performed with this diluted Standard Solution of Verification. The mean recovery rate was calculated.
Millipore water without the test item was analysed (analytical blank).
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
-Name: Desmodesmus subspicatus (formerly Scene-desmus 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 cell counter, 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 the test were taken from an exponentially growing pre culture and were mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
1.3 °dH corresponding to 22.5 mg/L CaCO3
Test temperature:
21 - 24 °C
pH:
7.8 - 7.9
Nominal and measured concentrations:
3.1, 6.3, 12.5, 25, 50 and 100 mg/L (nominal)
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 °C 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 cell counter (Z2, Beckman Coulter) by taking aliquots from each test flask, which were not replaced.
-Experimental design:
6 test concentrations plus 1 control
3 replicates per concentration, 3 replicates per control
Initial cell density in the test cultures appro-ximately 5000 cells per millilitre.
Additionally highest test concentration without algae.
-Test item concentration/s: 3.1, 6.3, 12.5, 25, 50 and 100 mg/L
-Method of administration: stock solution
-Duration of exposure: 72 hours
-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.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence limits [mg/L]: 18-32
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 6.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
39 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Yield was calculated as the biomass; 95 % confidence limits [mg/L]: 30-53
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Yield was calculated as the biomass; 95 % confidence limits [mg/L]: 1.5-5.6
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 3.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Yield was calculated as the biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
6.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Yield was calculated as the biomass
Details on results:
After 24 hours of exposure, the test media coloured to beige brown in the test item concentrations 50 mg/L and 100 mg/L (nominal).
No beige brown colour was visible over the test period at the test item concentrations of 3.1-25 mg/L (nominal).
NOEC/EC 10 for Yield and Growth Rate were determined at test concentrations which showed no colouring, while the ErC 50 was determined at concentrations which showed colouring and for which an influence on the result may not be excluded.
The results are expressed in terms of nominal concentrations. Effective concentrations ranged from 100.7 % to 102.1 % of nominal values at 0 hours, and from 89.0 % to 93.1 % of nominal values at 72 hours.
Reported statistics and error estimates:
All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10).
Validity criteria fulfilled:
yes
Conclusions:
Toxic effects of Anthraquinone-2-sulfonic acid sodium salt monohydrate on the growth rate of the algal species Desmodesmus subspicatus over several generations was determined and showed an ErC 50 > 100 mg/L, ErC 10 = 25 mg/L and a NOEC[r] ≥ 6.3 mg/L.
Executive summary:

A study was performed to assess the adverse effects of Anthraquinone-2-sulfonic acid sodium salt monohydrate on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.

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 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 3.1, 6.3, 12.5, 25, 50 and 100 mg/L of Anthraquinone-2-sulfonic acid sodium salt monohydrate dissolved in dilution water. Auxiliary used to prepare the test media was an ultrasonic bath.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to Williams Multiple Sequential t-test Procedure. The following values were determined: 72hr-ErC50 >100 mg/L, 72hr-ErC10=25 mg/L, NOEC≥6.3 mg/L, LOEC≤ 12.5 mg/L.

After 24 hours of exposure, the test media coloured to beige brown in the test item concentrations 50 mg/L and 100 mg/L (nominal).

No beige brown colour was visible over the test period at the test item concentrations of 3.1-25 mg/L (nominal).

NOEC/EC 10 for Growth Rate were determined at test concentrations which showed no colouring, while the EC 50 for Growth Rate was determined at concentrations which showed colouring and for which an influence on the result may not be excluded.

The results are expressed in terms of nominal concentrations. Effective concentrations ranged from 100.7 % to 102.1 % of nominal values at 0 hours, and from 89.0 % to 93.1 % of nominal values at 72 hours.

All validity criteria are met.This toxicity study is classified as acceptable and satisfies the guideline requirements for toxicity to aquatic algae.

Description of key information

Toxic effects of Anthraquinone-2-sulfonic acid sodium salt monohydrate on the growth rate of the algal species Desmodesmus subspicatus over several generations was determined and showed an ErC 50 > 100 mg/L, ErC 10 = 25 mg/L, NOEC[r] ≥ 6.3 mg/L and LOEC[r] ≤ 12.5 mg/L.

Key value for chemical safety assessment

Additional information