Palladium(II) acetate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 Feb - 11 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Palladium(II) acetate

EC Number:

222-164-4

EC Name:

Palladium(II) acetate

Cas Number:

3375-31-3

Molecular formula:

C2H4O2.1/2Pd

IUPAC Name:

palladium(II) acetate

Details on test material:

Orange powder, Lot number: 11310, stored at room temperature in the dark

Specific details on test material used for the study:

Palladium content: 48.13%
Palladium purity: 99.9%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Two samples of 20 mL were taken from the stock solution, all test solutions and controls at the beginning of the test prior to addition of the algae. At the end of the growth test, samples (2 samples of 20 mL) were taken directly from representative replicates per test concentration and controls. Aged samples were filtered using a 0.2 μm PES filter. In addition, unfiltered samples were taken at the beginning of the test and at the end of the test.
Therefore, at test initiation there were two sets of samples (analytical and retain) for the stock solution, each test concentration and the control. At test termination there were four sets of samples (filtered: analytical and retain; unfiltered: analytical and retain) for each test concentration and the control.
All samples were stabilised by acidification (2.5 mL of concentrated HNO3 on 20 mL sample) with concentrated nitric acid and stored at 4 ± 3 °C until further analysis.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the test concentrations
An amount of 5.01 mg of the test item was transferred into a sterile glass flask filled up to 1 L with growth medium (sterile) to obtain an appropriate stock solution (as used in the range finding test). The test medium was stirred for 72 hours at room temperature. All test concentrations were prepared by dilution of the stock solution with sterile growth medium.
For the blank control, growth medium without test item was used. All work was conducted on a clean bench using sterile equipment.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Raphidocelis subcapitata, Chlorophycea, Chlorophyta.
Origin: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, 37073 Göttingen.
Strain number: Catalog No 61.81 SAG.
Cultivation: The stock cultures were maintained fulfilling the criteria of the OECD guide-line (culture medium recommended by Bringmann und Kühn (1980) [6]). Prior to testing a pre-culture was established in OECD growth medium (as described below) to obtain exponentially-growing algae for the test. The culture duration of the pre-cultures was 3 days.

Study design

Test type:

static

Water media type:

freshwater

Remarks:

The sterilised synthetic growth medium (OECD medium) according to OECD 201 was used as growth medium.

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

During the test, all test vessels were kept at a temperature within the range of 21 to 24 °C, controlled at ± 2 °C. Measured temperature remained at 22.0 °C during the test.

pH:

The pH of the controls slightly decreased during the test from 8.06 at test start to a mean value (n = 8) of 8.03 at test end. In the test concentrations, the initial pH in the treatments ranged from 8.01 to 8.09 and the mean pH value was between 8.04 and 8.09 at the end of the test.

Nominal and measured concentrations:

Test concentrations
Prior to the definitive test, a non-GLP range-finder with nominal concentrations of 0.005, 0.025 and 0.125 mg Pd/L, equivalent to 0.010, 0.052 and 0.260 mg test item/L (considering the Pd content of 48.13%) was conducted.
Based on the results of the range-finder, the following nominal concentrations with a spacing factor of approx. 2.5 were selected for the main test:
1.33, 3.32, 8.31, 20.8 and 51.9 µg test item/L,
equivalent to 0.64, 1.60, 4.00, 10.0 and 25.0 µg Pd/L.

Based on the measured Pd concentration in the stock solution of 126% (measurement sample) and 131% (retain sample) at test initiation, the nominal concentrations were re-calculated at 1.70, 4.26, 10.7, 26.7 and 66.5 µg test item/L equivalent to 0.820, 2.05, 5.13, 12.8 and 32.0 µg Pd/L.

At test initiation, the analytical sample and the retain sample were measured. The mean concentration was determined and ranged from 52.8% to 86.0% of the nominal concentrations in the different treatments. Therefore the measured concentrations were not within a range of 80 to 120% of the nominal concentrations.
Thereafter, the measured concentrations did not remain within ± 20% throughout the test period of 72 hours (% of initial: 27.1% to 78.8%; % of nominal: 14.3% to 66.3%). Therefore, the evaluation of biological results was based on the geometric mean measured concentrations of 0.226, 1.01, 2.77, 7.91 and 23.9 µg Pd/L (equivalent to 0.469, 2.10, 5.75, 16.4 and 49.7 µg test item/L; re-calculated based on a Pd content of the test item of 48.13%).
The results of the unfiltered samples at test termination showed that there is an influence of the algae on the recovery of the test item. Since filtration (0.2 µm) is the standard procedure at test termination to obtain results of the dissolved test item concentration, the unfiltered samples were not considered for the evaluation and only used as additional information.

Details on test conditions:

Growth medium
The sterilised synthetic growth medium (OECD medium) according to OECD 201 was used as growth 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.

Test vessels
Test vessels were 250 mL conical glass flasks covered with air-permeable silicone-sponge caps. Test vessels were acid-washed (10% HNO3) and rinsed six times with purified water. The vessels and caps were sterilized prior to use (autoclaving).
Test conditions
During the test, all test vessels were kept at a temperature within the range of 21 to 24 °C, controlled at ± 2 °C, with a light intensity (OSRAM Standard “cool white”) of
60 - 120 µE m-2 s-1 (4440 - 8880 lux). 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 and at different positions in the incubation chamber.
During the course of the test all test vessels were kept on a laboratory shaker (Incubation Shaker Multitron®, INFORS Typ HT, Switzerland) shaking continuously at 150 rpm.
The solution pH was measured in one test vessel per test concentration including the control. At the end of the test, the solution pH values were measured directly in the test vessels.
The test culture temperature was measured in one additional test vessel.
At test start all the test vessels were randomly placed in the incubator and randomly repositioned daily afterwards.

Determination of cell numbers
The cell concentrations were determined in the inoculum culture prior to the addition to the test vessels at test start and after 24, 48 and 72 h in the test cultures.
The cell density was measured using an electronic particle counter (CASY TT, OMNI Life Science GmbH & Co. KG, Bremen, Germany)

Test performance
For the definitive growth test, four replicates of each test concentration and eight replicates of the control were prepared. The test vessels were filled with 100 mL of the respective test solution containing the test item. The blank control consisted of fresh growth medium only.
Cell concentrations were determined in pre-cultures (0 hours) and after 24, 48 and 72 hours of exposure in the test concentrations.
At the beginning of the test, the initial cell concentration was calculated based on the cell number of the pre-culture. According to a cell density of 1 827 000 cells/mL 547 µL of pre-culture was added to each test vessel to achieve an initial cell concentration of 10 000 cells/mL.
All test preparations were performed under sterile conditions.

Reference substance (positive control):

yes

Remarks:

sensitivity of test organism routinely checked using 3,5-dichlorophenol as primary standard following internal SOPs in non-GLP test

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.47 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.73 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.01 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.32 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.64 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

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 at the end of the test:
control,0.226 µgPd/L: normal appearance of algal cells
1.01 µgPd/L: reduced number of algae cells
2.77, 7.91, 23.9 µg Pd/L: significantly reduced number of algae cells

Results with reference substance (positive control):

The sensitivity of the test organism is routinely checked using 3,5-dichlorophenol as primary standard following internal SOPs in a non-GLP test twice a year. The latest (February 2021) ErC50 value of 2.47 mg/L (nominal; 95% confidence limits: 2.22 – 2.78 mg/L) is in good agreement with the results of an international ring test with an ErC50 of 3.38 ± 1.30 mg/L.

Reported statistics and error estimates:

Biological data were statistically analysed to determine EC50 and EC10 values together with 95% confidence intervals, where possible. The ErC50/10 and EyC50/10 were determined using the 3-parametric non-linear regression procedures.

The NOEC for growth rate and yield were determined by Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm (significance level of 0.05, one-sided smaller).

The computer program ToxRat® was used for statistical evaluations.

Any other information on results incl. tables

Light intensity ranged from 94.89 to 97.04 µE m^-2 s^-1 and the temperature remained at 22.0 °C during the test.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

*cell number in control cultures increased >16 fold within the 72h test period *mean of the replicate CV % in the section-by-section growth rate of controls was <=35% *CV% of average specific growth rate at test end in replicate control cultures <=7%

Conclusions:

The 72-hour toxicity of the test item to the unicellular green alga Raphidocelis subcapitata was determined in a static system (OECD 201) exposed to nominal concentrations of 1.33 - 51.9 µg test item/L (factor: approx. 2.5) and a control. Based on the measure geometric mean test item concentrations, the 72-hour ErC50 for the inhibition of growth rate was 2.64 µg test item/L and the ErC10 was 1.32 µg test item/L. The NOEC for growth rate was2.10 µg test item/L. For yield, an EyC50 of 2.01 µg test item/L was determined. The EC10 value for yield was 1.73 µg test item/L and the NOEC was 0.470 µg test item/L.

Executive summary:

A study was performed to determine the toxicity of Palladium(II) acetate on the growth of the unicellular freshwater green alga Raphidocelis 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, eight replicates of the control (test medium only) and four replicates of each treatment were tested at the following nominal concentrations of 1.33 - 51.9 µg test item/L equivalent to 0.64 - 25.0 µg Pd/L (factor: approx. 2.5).

The concentrations of the test item in the media were confirmed by ICP-MS and ICP-OES measurements of dissolved palladium at test initiation and test termination. The LOQ (ICP-MS) was at 0.002 µg Pd/L at test initiation and test termination. In addition,  one retain sample from test initiation was measured via ICP-OES with a LOQ of 0.461 µg Pd/L (additional confirmatory measurement).

The measured Pd concentration in the stock solution was 126% (measurement sample) and 131% (retain sample) at test initiation. Based on this measured concentration, the nominal concentrations were re-calculated at 1.70 - 66.5 µg test item/L equivalent to 0.820 - 32.0 µg Pd/L.

At test initiation, the analytical sample and the retain sample of each dosing level were measured. The mean concentration was determined and ranged from 52.8% to 86.0% of the nominal concentrations in the different treatments. Therefore the measured concentrations were not within a range of 80 to 120% of the nominal concentrations.

Thereafter, the measured concentrations did not remain within ± 20% throughout the test period of 72 hours (% of initial: 27.1% to 78.8%; % of nominal: 14.3% to 66.3%). Therefore, the evaluation of biological results was based on the geometric mean measured concentrations of 0.226 - 23.9 µg Pd/L (equivalent to 0.469 - 49.7 µg test item/L; re-calculated based on a Pd content of the test item of 48.13%).

A concentration-dependent inhibiting effect on the growth of the freshwater green algae was observed over the range of the tested concentrations.

The respective 72-hour E_rC₅₀ for the inhibition of growth rate was 1.27 µg Pd/L (equivalent to 2.64 µg test item/L) and the E_rC₁₀ was 0.636 µg Pd/L (equivalent to 1.32 µg test item/L). The NOEC for growth rate was 1.01 µg Pd/L (equivalent to 2.10 µg test item/L).

For yield, an E_yC₅₀ of 0.967 µg Pd/L (equivalent to 2.01 µg test item/L) was determined. The EC₁₀ value for yield was at 0.834 mg test item/L (equivalent to 1.73 µg test item/L) and the NOEC was at 0.226 µg Pd/L (equivalent to 0.470 µg test item/L).