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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
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Purity: >99.9% Palladium (II) di(4-oxopent-2-en-2-oate) based on 34.94% Pd content
Analytical monitoring:
yes
Remarks:
concentrations of the test item in the test medium were confirmed by measuring dissolved Pd concentrations by ICP-MS
Details on sampling:
- Concentrations: Control and all test concentrations
- Sampling method: Samples taken at test start and test end
- Sample storage conditions before analysis: The samples were filtered (0.2 μm polyether sulphone membrane syringe filter) and immediately stabilised
by adding 0.1 mL conc. HNO3 to 10 mL test media. Samples are typically stable for about 24 hours. Samples were analysed on the day of
sampling, and were immediately stored at 4 °C until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: To prepare a stock solution in growth medium, 3.504 mg of the test item were transferred into
a 1 L sterile glass flask. Sterile growth medium (1 L) and an acetone washed, sterilised star
shaped stirring bar were added to the bottle and the stock solution was stirred vigorously for
24 hours at room temperature (about 20°C). Another flask was filled with growth medium
only for the control replicates and stirred in the same way.
To separate insoluble test item from the test medium, the stock solution was filtered through
a 0.22 μm PES filter. The stock solution was then diluted with sterile growth medium to obtain the required test
concentrations. All work was conducted on a clean bench using sterile equipment.
- Controls: Test medium (without added test item)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None after filtering
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: unicellular green alga
- Strain: Raphidocelis subcapitata, Chlorophycea, Chlorophyta. (former names Pseudokirchneriella subcapitata, Selenastrum capricornutum)
- Source (laboratory, culture collection): Experimental Phycology and Culture Collection of Algae at the University of Goettingen
(EPSAG), Nikolausberger Weg 18, 37073 Goettingen, Germany. Catalog No 61.81 SAG.
- Age of inoculum (at test initiation): The culture duration of the pre-cultures was about 3 days.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: Not stated
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not stated
Test temperature:
21.5 - 22 °C
pH:
pH of the controls: mean pH 7.65 at test start, mean pH 8.12 at test end
pH in test solutions: pH 7.51 to 7.56 at test start, pH 7.80 to 8.04 at test end
Dissolved oxygen:
Not stated
Salinity:
Not stated
Conductivity:
Not stated
Nominal and measured concentrations:
range-finder test: Nominal concentration 3-30-300 µg test item/L
definitive test: Nominal concentrations: control, 9.0, 22.4, 56.0, 140 and 350 μg test item/L equivalent to: control, 3.13, 7.83, 19.6, 48.9 and 122 μg Pd/L.

Measured concentrations in control: 0.014 µg Pd/L at test start, 0.017 μg Pd/L at test end
Measured concentrations at test start: 1.15, 2.83, 7.07, 18.6 and 50.7 μg Pd/L
Measured concentration after 72 hours (test end): 0.502, 1.24, 3.04, 10.5 and 36.8 μg Pd/L
Geometric mean of the measured concentrations at test initiation and termination (mean measured): 0.760, 1.87, 4.64, 14.0 and 43.2 μg Pd/L (equivalent to 2.18, 5.35, 13.3, 40.1 and 124 μg test item/L).

The values measured in the test solution represent the soluble part of palladium. The test item concentrations were assessed by chemical analyses of dissolved palladium in the aqueous phase and were calculated based on the palladium content of 34.94%

The evaluation was based on the geometric mean measured Pd concentrations, since the measured Pd concentrations were more than 20% below the nominal values.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical glass flasks, acid-washed (10% HNO3) and rinsed six times with purified water
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 250 mL conical glass flasks covered with air-permeable silicone-sponge caps (vessels and silicone-sponge caps sterilised prior to use by autoclaving).
- Aeration: Not stated
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: 10 000 cells /mL
- Control end cells density: 131.8 x 10(4) cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: Yes. The sterilised synthetic growth medium (OECD medium) according to OECD 201 with
reduced EDTA concentration was used as growth medium.
In a pre-test, it was demonstrated that the growth of R. subcapitata in the OECD 201 growth
medium with 50% EDTA concentration fulfils the validity criteria of the OECD 201 guideline
while it did not in an EDTA-free OECD 201 medium. All stock solutions and the medium were
prepared with purified water processed using an ELGA „PURELAB Ultra“. The pH of the
medium was obtained at equilibrium between the carbonate system of the medium and the
partial pressure of CO2 in atmospheric air.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: The pH of the medium was obtained at equilibrium between the carbonate system of the medium and the partial pressure of CO2 in atmospheric air.
- Photoperiod: Continuous light
- Light intensity and quality: light intensity (OSRAM Standard “cool white”) adjusted to 60 - 120 μE m-2 s-1
(4440 - 8880 lux) close to the surface of the liquid. The light intensity was measured daily
using a cosine (2 π) receptor (LI-250A, LI-COR) in μE m-2 s-1 at level of the test media
surface. measured light intensity 92.01 to 93.08 μE m-2 s-1
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (CASY TT, OMNI Life Science GmbH & Co. KG, Bremen, Germany)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Yes
- Test concentrations:
Nominal concentration 3-30-300 mg test item/L
- Results used to determine the conditions for the definitive study: The nominal concentrations to be tested in the definite test were selected on the basis of the results from the range-finding test
Reference substance (positive control):
yes
Remarks:
sensitivity of test organism routinely checked using 3,5-dichlorophenol; latest (April 2018) nominal growth rate ErC50=2.61 mg/L (95% CL 2.36-2.89 mg/L) in good agreement with results of international ring test (ErC50 3.38±1.3 mg/L ).
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
81 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 75.0-87.0 µg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
25.5 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 21.2-29.5 µg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
13.3 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
32.1 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL 25.1-39.2 µg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
14.9 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95%CL 6.8-20.3 µg/L
Details on results:
Microscopic examinations were performed to verify a normal and healthy appearance of the
inoculum culture and to detect any abnormal appearance of the algae (as may be caused by
the exposure to the test substance) at the end of the test - Normal appearance of algal cells in control and all treatments throughout test, concentration-dependent decrease of number of algal cells.
Validity criteria fulfilled:
yes
Remarks:
cell number in control cultures increased by factor of 132 within 72h; mean of replicate CV in section-by-section growth rate of controls was 8.8% during test period; CV of average specific growth rate at test end in replicate control cultures was 2.1%
Conclusions:
The 72-hour ErC10 and ErC50 for the inhibition of growth rate were estimated with 8.92 μg Pd/L (25.5 μg test item/L) and 28.3 μg Pd/L (81.0 μg test item/L), based on mean measured Pd concentrations.

For yield, EyC10 and EyC50 values of 5.19 and 11.2 μg Pd/L (14.9 and 32.1 μg test item/L) were determined, respectively, based on mean measured Pd concentrations.

The NOEC for the inhibition of both growth rate and yield was determined with 4.64 μg Pd/L (13.3 μg test item/L), based on mean measured Pd concentrations.
Executive summary:

A GLP compliant study was performed to determine the toxicity of Palladium (II) di(4-oxopent-2-en-2-oate) (CAS 14024-61-4) on the growth of the unicellular freshwater green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). Exponentially-growing cultures of the green alga were exposed to five concentrations of the test item plus a control over several generations under defined conditions for 72 hours according to the OECD guideline 201.

The nominal test concentrations were prepared with sterile growth medium under sterile conditions. For the examination of the inhibition of alga growth, a control (test medium only) and 5 test item treatments were tested (nominal concentrations of 9.0, 22.4, 56.0, 140 and 350 μg test item/L and a control (equivalent to 3.13, 7.83, 19.6, 48.9 and 122 μg Pd/L)).

The concentrations of dissolved palladium (Pd) in the media were measured at test initiation and test termination by ICP-MS. Measured palladium concentrations were within a range of 36.1 – 41.5% of the nominal concentrations at test start and between 15.5 – 30.2% of nominal at test termination. The evaluation of the test results was based on geometric mean measured Pd concentrations of 0.760, 1.87, 4.64, 14.0 and 43.2 μg Pd/L (equivalent to 2.18, 5.35, 13.3, 40.1 and 124 μg test item/L).

Concentration-dependent inhibiting effects on the growth of the freshwater green algae were observed over the range of the tested concentrations. The 72-hour ErC10 and ErC50 for the inhibition of growth rate were estimated with 8.92 μg Pd/L (25.5 μg test item/L) and 28.3 μg Pd/L (81.0 μg test item/L), based on mean measured Pd concentrations.

For yield, EyC10 and EyC50 values of 5.19 and 11.2 μg Pd/L (14.9 and 32.1 μg test item/L) were determined, respectively, based on mean measured Pd concentrations.

The NOEC for the inhibition of both growth rate and yield was determined with 4.64 μg Pd/L (13.3 μg test item/L), based on mean measured Pd concentrations.

Description of key information

A GLP compliant study was performed to determine the toxicity of Palladium (II) di(4-oxopent-2-en-2-oate) (CAS 14024-61-4) on the growth of the unicellular freshwater green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). Exponentially-growing cultures of the green alga were exposed to five concentrations of the test item plus a control over several generations under defined conditions for 72 hours according to the OECD guideline 201.

The nominal test concentrations were prepared with sterile growth medium under sterile conditions. For the examination of the inhibition of alga growth, a control (test medium only) and 5 test item treatments were tested (nominal concentrations of 9.0, 22.4, 56.0, 140 and 350 μg test item/L and a control (equivalent to 3.13, 7.83, 19.6, 48.9 and 122 μg Pd/L)).

The concentrations of dissolved palladium (Pd) in the media were measured at test initiation and test termination by ICP-MS. Measured palladium concentrations were within a range of 36.1 – 41.5% of the nominal concentrations at test start and between 15.5 – 30.2% of nominal at test termination. The evaluation of the test results was based on geometric mean measured Pd concentrations of 0.760, 1.87, 4.64, 14.0 and 43.2 μg Pd/L (equivalent to 2.18, 5.35, 13.3, 40.1 and 124 μg test item/L).

Concentration-dependent inhibiting effects on the growth of the freshwater green algae were observed over the range of the tested concentrations. The 72-hour ErC10 and ErC50 for the inhibition of growth rate were estimated with 8.92 μg Pd/L (25.5 μg test item/L) and 28.3 μg Pd/L (81.0 μg test item/L), based on mean measured Pd concentrations.

For yield, EyC10 and EyC50 values of 5.19 and 11.2 μg Pd/L (14.9 and 32.1 μg test item/L) were determined, respectively, based on mean measured Pd concentrations.

The NOEC for the inhibition of both growth rate and yield was determined with 4.64 μg Pd/L (13.3 μg test item/L), based on mean measured Pd concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:
81.1 µg/L
EC10 or NOEC for freshwater algae:
25.5 µg/L

Additional information

A GLP compliant study was performed to determine the toxicity of Palladium (II) di(4-oxopent-2-en-2-oate) (CAS 14024-61-4) on the growth of the unicellular freshwater green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) according to the OECD guideline 201. The geometric mean measured Pd concentrations were control, 0.760, 1.87, 4.64, 14.0 and 43.2 μg Pd/L (equivalent to control, 2.18, 5.35, 13.3, 40.1 and 124 μg test item/L). The 72-hour ErC10 and ErC50 for the inhibition of growth rate were estimated with 8.92 μg Pd/L (25.5 μg test item/L) and 28.3 μg Pd/L (81.0 μg test item/L), based on mean measured Pd concentrations.