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Long-term toxicity to aquatic invertebrates

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long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as an unpublished report, no restrictions, fully adequate for assessment

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Diamminedichloropalladium (II)
Diamminedichloropalladium (II)
Constituent 2
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Diamminedichloropalladium (II)
- Molecular formula (if other than submission substance): Cl2H6N2Pd
- Molecular weight (if other than submission substance): 211.39 g/mole
- Physical state: yellow powder
- Analytical purity: >99.5%
- Composition of test material, percentage of components: 50.35% Palladium
- Lot/batch No.: 10912
- Expiration date of the lot/batch: 28 August 2014
- Storage condition of test material: Room temperature (15 - 30°C)

Sampling and analysis

Analytical monitoring:
Details on sampling:
At test start and at each renewal samples of all treatments were taken from the test solution preparation just before distributing it to the 10 replicates.At the renewals samples of the aged test solution were taken from each vessel, pooled per concentration and measured. Since analysis could not be applied immediately after sampling, samples were stored stabilized 1:1 (v/v) with ammonia solution 10 % in a refrigerator at 4 – 8 °C until analysis.

Test solutions

Details on test solutions:
Stock solutions were prepared by dissolving adequate amounts of test item in dilution water and these were used to prepare test solutions. The stock solutions were freshly prepared before each renewal.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: water flea
- Strain: Clone V
- Source: German Federal Environment Agency, Location Berlin-Marienfelde, Ecotoxicology Lab, 12307 Berlin, Germany
- Age at study initiation: 4-24 hours old

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
21 d

Test conditions

1 m mol/L as CaCO3
Test temperature:
Temperature: 19.5 – 20.9 °C throughout the test, with the permitted range of 18 – 22 °C (with a variance of less than 2 °C)
pH: 7.9 – 9.4 throughout the test. Usually the pH should be between 6 and 9. In weeks 1 and 2 the pH in aged solutions was > 9 due to algae. This is because the daphnids were fed at a constant rate but the feeding rate was lower for the juveniles. The pH did not vary by more than 1.5 units during the study and all other validity criteria were met so this was not considered to effect the validity of the test.
Dissolved oxygen:
Oxygen: 8.4 mg/L and 11.8 mg/L throughout the test which is above the required 3 mg/L
Nominal and measured concentrations:
Definitive test: nominal concentrations: Control, 0.051, 0.163, 0.522, 1.67, 5.35, and 17.1 µg Pd/L,
corresponding to Control, 0.101, 0.324, 1.04, 3.32, 10.63, and 34 µg DDP/L.
Mean measured (time weighted average) concentrations: control, 0.025, 0.062, 0.275, 1.16, 4.05, and 14.3 µg Pd/L,
corresponding to 0.049, 0.124,0.546, 2.31, 8.04 and 28.4 µg DDP/L
Details on test conditions:
- Test vessel: beaker
- Type: closed, covered with glass pane
- Material, size, headspace, fill volume: glass beakers filled with 50 mL test solution
- Dilution water: Purified drinking water. The purification included filtration with activated charcoal, passage through a lime-stone column and aeration
- Food: The unicellular alga Desmodesmus subspicatus at each test medium renewal, corresponding with 0.2 mg C/Daphnia/day
- Light intensity: 680 - 829 Lux
- Aeration: No
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

- Source/preparation of dilution water: Purified drinking water: filtered through activated charcoal, passed through a limestone column and aerated
- Conductivity: 171.6 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: 0 and 48 hours

- Adjustment of pH: No
- Photoperiod: 16 hours light: 8 hours dark

Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
21 d
Dose descriptor:
Effect conc.:
>= 28.4 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth, reproduction and time to first brood
Key result
21 d
Dose descriptor:
Effect conc.:
>= 14.3 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
Basis for effect:
growth, reproduction and time to first brood
Details on results:
The EC50 and EC10 could not be calculated but were considered to be > 28.4 µg DDP/L (14.3 µg Pd/L based on mortality, growth and reproduction and time to first brood.
Reported statistics and error estimates:
Regression analysis was used to determine EC values including the 95 % confidence interval using Probit-analysis assuming log-normal distribution of the values.
The LOEC was calculated by using ANOVA followed by Multiple comparisons (Dunnett's test or Mann-Whitney’s tests) after testing for normal distribution and variance homogeneity using the Shapiro-Wilk test and Levene test, respectively (p≤ 0.05).

Applicant's summary and conclusion

Validity criteria fulfilled:
Diamminedichloropalladium (DDP) had no adverse chronic effect on Daphnia magna in a 21 day reproduction study. Neither adults nor juveniles were affected up to and including 28.4 μg DDP/L TWA (corresponding to 14.3 μg Pd/L TWA) . Accordingly, the NOEC was found to be ≥ 28.4 μg DDP/L (corresponding to 14.3 μg Pd/L).
Executive summary:

The effects of Diamminedichloropalladium (DDP) in a 21 day Daphnia reproduction study conducted according to OECD 211 were considered (Fraunhofer, 2014). The study was a semi-static test design with test solutions renewals three times per week. The nominal test item concentrations applied were 0.101, 0.324, 1.04, 3.32, 10.63, and 34.0 μg test item per litre. The chemical analysis conducted at the start and end of each test solution renewal was based on the concentration of palladium. The mean measured palladium concentrations in the freshly prepared test solution were between 54% and 105% of the nominal concentrations. At the end of the renewal period palladium concentrations were between 3 and 65% of nominal at the five highest test concentrations and were below the mean background levels of palladium in the lowest test concentration of 0.101 test item per litre treatment. The time weighted average (TWA) mean measured concentrations of 0.025, 0.062, 0.275, 1.16, 4.05, and 14.3 μg Pd per litre, corresponding with 0.049, 0.124, 0.546, 2.31, 8.04, and 28.4 μg DDP per litre were used for reporting of results.

No significant effects were found due to the test item up to and including the highest concentration tested. Survival (viability), growth (body length), and reproduction of adults, age to first brood and intrinsic rate were unaffected by the test item up to and including 28.4 μg DDP per litre (TWA) corresponding with 14.3 μg Pd per litre (TWA). The NOEC was found to be ≥28.4 μg DDP per litre or ≥14.3 μg Pd per litre.

The study was conducted in accordance with GLP and the validity criteria were met. The study was well-documented and adhered to guideline procedures and has been given a reliability rating of 1. This is considered to be a key study for this endpoint.