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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:
2012-07-04 to 2012-07-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted 2011-07-28
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Series on testing and assessment, Number 23. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. ENV/JM/MONO(2000)6, 15-Dec-2000.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2011-02-07
Analytical monitoring:
yes
Details on sampling:
Zinc concentrations of filtered samples (0.2 μm, Polyethersulfone membrane) were determined by ICP-OES (LOD 1.65 µg/L; LOQ 5.49 μg/L).
To assess the concentration of dissolved zinc, all test solutions were sampled at the beginning of the exposure periods prior to addition of the algae. At the end of each exposure period, samples were taken from each replicate of each treatment, pooled for chemical analysis and solutions and algae were separated using a 0.45 μm PET filter.

- Preparation, treatment and storage of samples for chemical analysis:
Two replicates of each treatment and of the controls were sampled at the beginning and at the end of each exposure period, respectively. Aqueous samples for zinc analysis were filtered (0.2 μm, Polyethersulfone membrane) immediately after sampling, transferred into disposable polyethylene vials (Scintillation vials, Sarstedt, Nuembrecht, Germany), acidified (target conc. 3% nitric acid) and stored at approx. 4°C until analysis. Zinc concentrations of test solutions were determined by ICP-OES. Zn measurements were performed within 20-26 days after sampling. Under the described storage conditions, samples are typically stable for at least three months. These are the same conditions as required for storage of certified reference materials. The analyzed CRM TMDA-70 is stable for at least one year under these conditions.
Acidification is the stabilization method that is recommended in standard methods for metal analysis (e.g. ISO 11885).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: since the test item is a complex mixture of individual substances with different solubility and physical-chemical properties, a UVCB according to Regulation EC No 1907/2006, and of low water solubility, toxicity was determined using dissolved fractions. Dissolved fractions for the various test concentrations were prepared individually according to the OECD guidance document No. 23*.
Amounts of 20, 200, 2 000 and 20 000 μg test item, respectively, were transferred into 2 L sterile glass flasks. The flasks were brought to the volume of 2 L with growth medium (sterile) to obtain nominal loadings of 10, 100, 1 000 and 10 000 μg/L. One flask was filled with growth medium only for the controls.
Flasks were stirred slowly to avoid bubble and foam formation using a star shape magnetic stirring bar for 48 h at room temperature (around 20°C).
To separate insoluble parts from the aqueous phase and to sterilise the dissolved fractions by filtration, the test preparations were filtered into a sterile glass beaker by using a 0.22 μm filter (Syringe Filters DIAFIL PS (Polyether sulfone, PES) DIA-Nielsen GmbH & Co. KG, Düren, Germany) under sterile conditions. A volume of 100 mL of the filtrated dissolved fractions was directly used for the algal growth test. The volume was measured using a graduated glass cylinder.

* Reference:
- OECD Series on testing and assessment, Number 23. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. ENV/JM/MONO(2000)6, 15-Dec-2000.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Pseudokirchneriella subcapitata, Chlorophycea, Chlorophyta.
- Source: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Catalog No 61.81 SAG.
- Method of cultivation: the stock cultures are maintained fulfilling the criteria of the OECD guideline 201. Prior to testing, a pre-culture was established in standard OECD growth medium to obtain exponentially-growing algae for the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
not applicable
Hardness:
no data
Test temperature:
22.5 - 23.0°C
pH:
The pH of control and test culture solutions did not increase by more than 1.5 units during the test periods (pH of control solutions at test start and end of the exposure periods: 8.23 and 7.89).
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
Test concentrations (nominal loadings): 10, 100, 1000 and 10000 μg test item/L
Details on test conditions:
TEST SYSTEM
- Test vessel: the test vessels used were 250 mL conical glass flasks covered with air-permeable siliconesponge caps. The test vessels were acid-washed with 10% HNO3 and rinsed six times with purified water. The vessels and caps were sterilised prior to use by autoclaving.
- Initial cells density: the algal pre-culture (221 μL at a cell density of 4.519 x 10^6 cells/mL) was added to the test vessels to achieve the initial cell concentration of 10,000 cells/mL. At test start, the initial cell concentration was calculated based on the cell number of the pre-culture.
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

All test preparations were performed under sterile conditions.

GROWTH MEDIUM
The sterilised synthetic OECD medium according to OECD 201 was used as growth medium. As stated in the guideline OECD 201, the molar ratio of EDTA to iron slightly exceeds unity in this medium. This prevents iron precipitation and at the same time, chelation of heavy metal ions is minimised (molar ratio EDTA / Fe(III+) = 1.135). Considering the molar ratio and the stability constants of EDTA with Fe(III+) and Zn (II+) ions (log Ks are 25.0 and 16.44, respectively), the chelation of zinc remains negligible in comparison with nominal test concentrations. All stock solutions and the medium were prepared with purified water processed using an ELGA „PURELAB Ultra“. Please also refer to table 1 in the field "Any other information on materials and methods incl. tables" below.

OTHER TEST CONDITIONS
- Light intensity: the culture vessels were incubated with a light intensity (OSRAM “day light”) adjusted to approximately 4440 - 8880 lux (60 - 120 μE m-2 s-1) close to the surface of the liquid. The light intensity was measured daily using an illuminance meter LI-189 (LI-COR, Lincoln, USA with radiation sensor) with a cosine (2π) receptor in lux on a level with the surface of the test media.
- The cultures were oscillated by continuously stirring on a laboratory shaker with 100 rpm (Incubation Shaker Multitron ®, INFORS, Switzerland).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: cell concentrations were determined using an electronic particle counter (CASY 1 Model TT, Roche Diagnostics GmbH, Germany). The correctness of the electronic counts was checked by microscope counts following internal standard operation procedures. During the test, the cell concentrations were determined after 24, 48 and 72 h in samples taken directly from the test vessels.
- During the exposure period, the incubation temperature was measured daily with a calibrated thermometer in an additionally prepared control vessel kept under the same conditions.
- The pH values were measured in the additionally prepared replicate at test start and directly in the test vessels at the end of the test.

TEST CONCENTRATIONS
- Test concentrations: nominal loadings of 10, 100, 1000 and 10000 μg test item/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
198.4 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 %-Cl lower: 165.9 µg/L; 95 %-Cl upper: 239.2 µg/L
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
32.9 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 %-Cl lower: 22.1 µg/L; 95 %-Cl upper: 44.2 µg/L
Duration:
72 h
Dose descriptor:
other: NOEL
Effect conc.:
10 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
39.6 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 %-Cl lower: 34.1 µg/L; 95 %-Cl upper: 45.8 µg/L
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
6.63 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 %-Cl lower: 4.83 µg/L; 95 %-Cl upper: 8.55 µg/L
Duration:
72 h
Dose descriptor:
other: NOEL
Effect conc.:
< 10 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- There were concentration-dependent effects on the growth of P. subcapitata at loadings higher than 10 μg/L.
- Corresponding microscopy revealed normal appearances of the algae cells despite increasing cell debris with increasing growth inhibition.
- The NOEL for growth rate was 10 μg/L.
- At test end, the measured test item concentrations were between 92.8 and 113 % of the initial measured levels.
Please also refer to "Any other information on results incl. tables" and "Attached background material" below.
Results with reference substance (positive control):
The nominal ErC50 value of 2.91 mg/L (95% confidence limits 2.35 - 3.63 mg/L) of the last test was in good agreement with the results of an international ring test with ErC50 of 3.38 ± 1.30 mg/L (International Organisation for Standardisation, 1993)*.

*Reference:
- International Organisation for Standardisation, 1993. ISO 8692 Water quality - Algal growth inhibition test
Reported statistics and error estimates:
-The evaluation of the concentration-effect-relationships and the calculations of effect concentrations were based on the nominal loadings of the test item.
- The mean value of the cell counts for each concentration was used for plotting growth curves.
- The EL (effective level) values for growth rate and yield after 72 h (growth rate) were determined since there was an inhibition > 50 % at the highest loading.
- Calculation of the percent inhibition of growth rate [r] and yield [y] was performed according to the OECD 201 guideline and listed in a table.
The test results were statistically analysed to determine an EL50, EL20 and EL10, and the respective 95 % confidence intervals, using Probit-analysis (Finney, 1984)* assuming log-normal distribution of the values. Individual replicate responses were used for the regression analysis. The NOEL and LOEL values for growth rate and yield were determined by the Welch t-test for inhomogeneous variances (Welch, 1937)* or Williams Multiple Sequential t-test (Williams, 1971)* (Williams, 1972)* using the computer program ToxRat Solutions (ToxRat® Professional 2.10.)*.

* References
- Finney. D.J.: Statistical Method in Biological Assay. 2nd ed., London. 1984.
- Welch, B.L. (1937). The significance of the difference between two means when the population variances are unequal. Biometrika 29, 350-362.
- Williams, D.A. (1971) A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics, 27, 103-117.
- Williams, D.A. (1972): The comparison of several dose levels with a zero dose control. Biometrics 28, 519-531.
- ToxRat® Professional 2.10. ToxRat® Solutions GmbH, Naheweg 15, D-52477 Alsdorf (http://www.toxrat-solutions.de).

Table: Percent inhibition of growth rate and yield by the test item compared to controls after 72 h.

Test item Loading [μg/L]

  

% Inhibition of growth rate


 

% Inhibition of yield

 

 

72 h exposure

72 h exposure

Control

0.0

0.0

10

3.8

16.2

100

30.6

74.7

1000

88.6

99.1

10000

92.5

99.5

Validity criteria fulfilled:
yes
Conclusions:
In a growth inhibition test with Pseudokirchneriella subcapitata exposed to dissolved fractions of Fatty acids, C8-10, zinc salts at loadings of 0.01, 0.1, 1 and 10 mg/L in standard test medium over a test period of 72 hours, the calculated EL50 value for growth rate was 198.4 µg/L based on dissolved fractions. The NOEL for growth rate was 10 µg/L while the EL10 was 32.9 µg/L, all based on dissolved fractions.

The effect concentrations are based on the loadings used to prepare the dissolved fractions in accordance with OECD Series on testing and assessment No. 23 (OECD 2000)*. Since the applied test medium is the standard OECD 201 test medium with regard to EDTA concentrations, these endpoints are considered for classification and labelling.

*Reference:
- OECD Series on testing and assessment, Number 23. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. ENV/JM/MONO (2000)6, 15-Dec-2000.


Description of key information

Data available for fatty acids, C8-10, zinc salts:

Acute toxicity to freshwater algae (standard test): EL50 = 0.20 mg/L (dissolved fraction after 7 days in accordance with OECD Series # 29) in standard test medium (Pseudokirchneriella subcapitata)

Chronic toxictiy to freshwater algae (standard test): EL10 = 0.03 mg/L (dissolved fraction after 7 days in accordance with OECD Series # 29) in standard test medium (Pseudokirchneriella subcapitata)

In the assessment of environmental toxicity of fatty acids, C8-10, zinc salts, read-across to the assessment entities zinc and C8 -10 fatty acids is applied since zinc cations and fatty acid anions determine its fate and toxicity in the environment. Please refer to the respective assessment entity for further details.

 

C8-10 fatty acids:

The lowest ErC50 and NOEC of 15 and 1.9 mg/L, respectively, for the growth inhibition of freshwater algae were observed for the effect of decanoic acid (C10) in a reliable, GLP-conform growth inhibition test with Pseudokirchneriella subcapitata conducted according to OECD 201.

 

Zinc:

Acute toxicity to freshwater algae: lowest IC50: 0.136 mg Zn/l (Selenastrum capricornutum; single value) (neutral/high pH)

Chronic toxicity to freshwater algae: lowest NOEC: 0.019 mg Zn/l (Pseudokirchneriella subcapitata =Selenastrum capricornutum; geomean of 27 data points)

Chronic toxicity to marine algae: 12 species available which NOECs range between 0.0078 and 0.67 mg/l (dissolved concentrations)

Respective NOEC/EC10 values were applied in the species sensitivity distribution.

Key value for chemical safety assessment

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