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Diss Factsheets

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Aug 2021 - 21 Jan 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
version 13.04.2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
version 31.5.2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
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:
Pd content 48.13%
Pd purity 99.9%

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The test item concentrations in the aqueous solutions were measured by chemical analysis. Fresh samples (2 samples of 20 mL per concentration) were taken from all five test solutions and the control at the beginning of the test and at medium renewal at 24 hours prior to distribution to the test vessels. Samples of aged solutions (2 samples of 20 mL per concentration) were taken directly from one representative replicate at medium renewal and test end per test concentration and control. One set of samples was filtered through a 0.45 µm PES filter before measurement. The other set of samples was measured without filtration. In addition, for each set of samples a set of retain samples was taken.
All samples were stabilised by acidification and if needed stored at 4 ± 3 °C until further analysis. All samples will be discarded after finalization of the study.

Test solutions

Vehicle:
no
Remarks:
Purified, Cu-reduced drinking water is used as holding- and dilution water. The purification includes filtration with activated charcoal and aeration. To avoid copper contamination, plastic water pipes are used for the testing facilities.
Details on test solutions:
Introduction of the test item
An appropriate amount of the test item was transferred into a glass flask, which was filled up to the required volume with dilution water to obtain a stock solution of 100 mg test item/L (as done in the range finding test). The stock solution was stirred vigorously for about 72 hours at room temperature. After stirring, the stock solution was filtered using a 0.45 µm PES filter in order to remove undissolved test item. The test concentrations were then prepared by dilution of the filtered stock solution with dilution water. For the control, dilution water without test item was used.
The test was performed under semi-static test conditions. Test solutions were renewed after 24 hours. The test solutions were freshly prepared before test start and before medium renewal.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The test organisms were juvenile Daphnia magna, 4 – 24 hours old. Origin of the cladocerans is the German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene. Specimens used in the test are bred in the laboratory of the Fraunhofer IME.

Pre-Treatment
Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving. Batches of 30 to 50 animals were held at room temperature in ca. 1.8 L dilution water for one week. During this week the daphnids were fed daily with an algal suspension (Desmodesmus subspicatus) and ArtemioFluid (JBL). Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a few mL of medium. Five mL of this suspension was given to 1.8 L Daphnia medium. The water was changed three times per week. Newborn D. magna were separated by sieving, the first generation was discarded. Individuals applied in the test were transferred with a cropped Pasteur pipette a few hours after sieving to ensure applying only healthy specimens.

Test course and sampling
Daphnia magna, 4 – 24 h old were exposed to five concentrations of the test item and a control under semi-static conditions for a period of 48 hours. Test specimens were added to the test solutions with a bore Pasteur pipette. The daphnids were exposed without aeration in 50 mL of test solution in numbered glass beakers. The test organisms were transferred into new test vessels with freshly prepared test solutions after 24 hours.
The beakers were subjected to a light/dark cycle of 16/8 hours. The test temperature during the test was between 18 – 22°C. Temperature did not vary by more than ± 1°C within these limits. Light intensity did not exceed 1000 lux.
The numbers of immobile daphnids were visually determined daily and immobile daphnids were removed. Any abnormalities in appearance and behaviour were recorded. Following the OECD guideline 202, the daphnids were not fed during the test.

Study design

Test type:
semi-static
Water media type:
freshwater
Remarks:
Purified, Cu-reduced drinking water is used as holding- and dilution water.
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
The test was performed under semi-static conditions over 48 hours with one medium renewal after 24 hours.

Test conditions

Hardness:
Hardness of the Cu-reduced water is below the recommended range (>140 mg/L as CaCO3) for Daphnia magna indicated in the guideline. Therefore, the Cu-reduced dilution water is enriched with an appropriate amount of CaCl2 to increase the hardness, resulting in values of about 250 – 350 mg CaCO3/L.
Test temperature:
temperature between 19.5 and 20.0 °C, mean temperature of 19.8 °C (temperature did not vary by more than ± 1°C)
pH:
The pH values throughout the test were within a range of 7.95 – 8.40 at all treatment levels (pH did not vary more than 1.5 units within one treatment)
Dissolved oxygen:
The oxygen concentration was between 8.49 mg/L and 9.07 mg/L (97 – 102 % of air saturation)
Nominal and measured concentrations:
The concentration of the test item in the aqueous test solution was assessed by analysis of Palladium (Pd) at the start of the test, at medium renewal after 24 hours, and at test end in filtered (0.45 µm) and in unfiltered samples. An ICP-MS system was used for measurements of Pd in the test solutions. The LOQ was 0.002 µg/L

The concentrations to be tested in the definitive test were selected based on results from the range-finding tests and agreed upon with the study monitor. The following nominal loadings with a spacing factor of 2.0 were applied:
Control, 0.625, 1.25, 2.50, 5.00 and 10 mg test item/L, corresponding to 0.300, 0.602, 1.20, 2.41 and 4.81 mg Pd/L (Pd content of 48.13 %).

At test initiation and medium renewal, the mean measured concentrations of Pd in filtered samples of the fresh test solutions ranged from 3.92 to 4.48 % of nominal. The concentrations measured in filtered samples of aged test solutions were between 1.54 and 4.17 % of nominal. During the 24h incubation until medium renewal, the four lower concentrations remained between 75.5 to 98.5 % of the initial concentrations, while the highest concentration decreased to 38.4 % of the initial concentration.
The evaluation of biological results was based on the geometric mean measured test concentrations. Geometric mean measured Pd concentrations of 0.011, 0.024, 0.050, 0.086 and 0.110 mg Pd/L were calculated. Considering the Pd content of 48.13 %, geometric mean measured concentrations of 0.023, 0.050, 0.104, 0179 and 0.229 mg test item/L were used for evaluations. The geometric mean measured concentrations showed recovery rates of 2.29 – 4.17 % of nominal concentrations.

The control consists of dilution water only.
Details on test conditions:
Light intensity was measured using an illuminance meter (MINOLTA) with a photometric sensor in Lux. The light intensity was between 872 – 969 lx (corresponding to 11.6 – 12.9 µE/(m² * s)) throughout the test
Reference substance (positive control):
yes
Remarks:
K2Cr2O7 is used as reference substance.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
75 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: immobility
Remarks on result:
other: 95%CI: 64-88 µg TI/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
50 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: immobility
Details on results:
The test item had a statistically significant effect on the immobilization of the daphnids in the geometric mean measured test concentrations of 0.104, 0.179 and 0.229 mg test item/L (0.050, 0.086 and 0.110 mg Pd/L).
Results with reference substance (positive control):
The latest 24h-EC50 with K2CrO4 (September 2021) was 0.937 mg/L (95 % confidence limits: 0.565 – 1.56 mg/L).
Reported statistics and error estimates:
The evaluation of the concentration-effect-relationships and the calculations of effect concentrations is based on the geometric mean measured test item concentrations since measured concentrations varied by more than 20 % from the nominal concentrations throughout the test.

The results were statistically analyzed to determine the EC50 and EC10 value together with 95 % confidence intervals using Probit-analysis assuming log-normal distribution of the values. However, if results showed inhibition of less than 50%, no EC50 value can be calculated. Therefore, in that case, the EC50 will be reported as > highest geometric mean measured concentration tested.
The NOEC and LOEC values were determined using the Step-down Cochran-Armitage Test procedure.

The computer program ToxRat was used for statistical evaluations.

Any other information on results incl. tables

No significant signs of disease or stress like discolouration or abnormal behaviour were observed in any replicate.


 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
With 0 %, the percent mortality observed in the controls over the test period is ≤ 10 %. The dissolved oxygen concentration at the end of the aging period (48 h) was > 3 mg/L in control and test vessels.
Conclusions:
The test item had a statistically significant effect on the immobilization of the daphnids at test concentrations of 0.104 mg test item/L and above. Based on geometric mean measured concentrations, the EC50 for immobility is calculated as 0.075 mg test item/L and the NOEC is determined to be 0.050 mg test item/L. Considering the palladium content of 48.13 %, the corresponding EC50 and NOEC were calculated at 0.036 mg Pd/L and 0.024 mg Pd/L, respectively.
Executive summary:

A study was performed to investigate the influence of the test item Palladium(II) acetate on the mobility of Daphnia magna. The test was conducted under semi-static conditions over a period of 48 hours with one medium removal after 24 hours. Effects on immobilization were determined daily.


The daphnids were placed in water containing the test item in nominal concentrations of 0.625 - 10.0 mg test item/L. Considering the palladium (Pd) content of 48.13 %, 0.301 - 4.81 mg Pd/L were introduced. Cu-reduced dilution water enriched with CaCl2 was used as test medium. A control with test medium only was run in parallel.


The concentrations of the test item in the media were confirmed by measurements of Pd concentrations at beginning of the test, before and after media renewal at 24 hours and at test end using an ICP-MS system.


Concentrations of freshly prepared test media showed recovery rates between 3.92 and 4.48 % of nominal concentrations. Concentrations in aged test media showed recovery rates between 1.54 and 4.17 % of nominal concentrations. During the 24 h incubation until medium renewal, the four lower concentrations remained between 75.5 to 98.5 % of the initial concentrations, while the highest concentration decreased to 38.4 % of initial.


The measured concentrations were not within a range of 80 – 120 % of nominal concentrations. Thus, the evaluation of effects was based on the geometric mean measured concentrations of 0.023 - 0.229 mg test item/L (corresponding to 0.011 - 0.110 mg Pd/L).


The test was considered valid according to OECD 202.


The test item had a statistically significant effect on the immobilization of the daphnids in the three highest test concentrations of 0.104 mg test item/L (geometric mean measured concentration) and above. The EC50 was calculated at 0.075 mg test item/L (corresponding to 0.036 mg Pd/L), the NOEC is calculated at 0.05 mg test item/L (corresponding to 0.024 mg Pd/L).