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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
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 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
>99.9% Palladium (II) di(4-oxopent-2-en-2-oate) (Pd(C5H7O2)2) (based on analyzed Pd of 34.52%)
Analytical monitoring:
yes
Details on sampling:
Dissolved Pd-concentrations were analysed in samples of all fresh test solutions at the beginning of the test prior to addition of test organisms and at each media renewal immediately after preparation of test solutions before distributing to the test vessels. Aged samples were taken before each media renewal and at test end. Analysis was performed in representative replicates per treatment level.

For this, duplicate samples were taken (10 mL), filtered (0.2 µm polyether sulphone membrane (PES) syringe filter) and afterwards acidified with conc. HNO3 (addition of 0.1 mL conc. HNO3 to the 10 mL sample). Samples were sealed after acidification.

Chemical analysis of the processed samples was performed immediately after sampling and within 24 hours.
Vehicle:
no
Details on test solutions:
Stock solutions were prepared by dissolving adequate amounts of test item in dilution water. A stirring bar were added to the bottle and stirred vigorously for 24 hours at room temperature (about 20°C). The stock solution was serially diluted with dilution water to obtain the test concentrations. The stock solutions were freshly prepared before each renewal.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain: unknown
- Source: Origin of the cladocerans was 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
- Age at study initiation: 4-24 hours old
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
purified drinking water was enriched with an appropriate amount of CaCl2 to increase the hardness, resulting in values of about 250 – 350 mg CaCO3/L
Test temperature:
Temperature: 19.0 – 20.5 °C throughout the test, with the permitted range of 18 – 22 °C (with a variance of less than 2 °C)
pH:
pH: 7.71 – 9.51 at all treatment levels. Usually the pH should be between 6 and 9. The pH surpassed the range up to a pH of 9.5 in aged media of all assays. However, this is not unusual due to the activity of the feed algae in the vessels. Since all validity criteria are kept, this deviation is assumed to be without effect on the plausibility of the results and the integrity of the study.
Dissolved oxygen:
The dissolved oxygen saturation was between 8.38 mg/L and 11.86 mg/L at the beginning and during the test, which is above the required 3 mg/L
Nominal and measured concentrations:
Dose-range finding test:
nominal concentrations: 3.0-30.0-300.0 µg Pd/L (8.60-86.0-860 µg test item/L)

Definitive test: nominal concentrations: Control-4.6-11.5-28.8-72.0-180 µg Pd/L,
equivalent to control-13.3-33.4-83.4-209-521 µg test item/L.

Mean measured (time weighted average) concentrations: control-1.60-4.29-11.3-28.4-84.4 µg Pd/L,
equivalent control-4.6-12.4-32.6-82.4-245 µg test item/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beaker (50 mL nominal volume)
- Type: beakers covered with glass pane to prevent from evaporation as much as possible, but also permit gaseous exchange between the medium and the atmosphere and access of light
- 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 limestone column, and aeration. In spite of the passage through a lime-stone column, hardness of the Cu-reduced water was below the recommended range (>140 mg/L as CaCO3) for Daphnia magna indicated in the guideline. Therefore, the dilution water was enriched with an appropriate amount of CaCl2 to increase the hardness, resulting in values of about 250 – 350 mg CaCO3/L.
- Food: the daphnids were fed during the test with a mixture of suspensions of unicellular alga Desmodesmus subspicatus and JBL ArtemioFluid in a ratio of 9 : 1 (vol/vol). The content of food in the test suspensions, measured at 585 nm, was kept constant at 0.2 mg C/(Daphnia * day).
- Light intensity: light intensity was measured using an illuminance meter (MINOLTA) with photometric sensor in Lux. With 846-975 lx (corresponding to 11.3 – 13.0 µE/(m² *s)) the light intensity was below the threshold value of about 1000 lx (15 µE/(m² *s)) as permitted by OECD guideline 211
- Aeration: No
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified drinking water
- Conductivity: 323 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: 0 and 48/72 hours

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours dark

Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
4.3 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element (dissolved fraction)
Remarks:
Pd
Basis for effect:
reproduction
Remarks:
offspring per introduced parent ('reproduction)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
12.4 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
Palladium (II) di(4-oxopent-2-en-2-oate)
Basis for effect:
reproduction
Remarks:
offspring per introduced parent ('reproduction')
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.2 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
element (dissolved fraction)
Remarks:
Pd
Basis for effect:
reproduction
Remarks:
reproduction determined as offspring per introduced parent
Remarks on result:
other:
Remarks:
95%CI: not determined due to mathematical reasons or inappropriate data
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
9.3 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
Palladium (II) di(4-oxopent-2-en-2-oate)
Basis for effect:
reproduction
Remarks:
reproduction determined as offspring per introduced parent
Remarks on result:
other:
Remarks:
95% CI: not determined due to mathematical reasons or inappropriate data
Details on results:
Based on the evaluation performed on TWM concentrations, for immobilisation a LC10 of 1.9 µg Pd/L, corresponding to 5.5 µg test item/L and a NOEC of 4.3 µg Pd/L, corresponding to 12.4 µg test item/L, were calculated. The EC50 value was 4.8 µg Pd/L (95%CI: ND).
For reproduction per survived daphnid an EC10 could not be derived. The NOEC was >=4.3 µg Pd/L, corresponding to >=12.4 µg test item/L.
For length, the NOEC was calculated to be lower than 1.60 μg Pd/L. However, due to a low inhibition of 9.1% at a concentration of 1.60 μg Pd/L, the NOEC was set to be 1.60 μg Pd/L, since effects below 10% compared to control are generally not considered to be ecotoxicologically relevant and it is generally recommended by OECD and EFSA to use the EC10 approach in preference to the NOEC approach for the environmental risk assessment. However, in this case, ECx determination was not possible due to limited data.
For age of first reproduction, the data do not allow to calculate ECx-values because it is not possible to define the effect size x (maximum possible increase in age not known). The variable was only evaluated by hypothesis testing to determine an LOEC/NOEC. The NOEC was >=11.3 µg Pd/L, corresponding to >=32.6 µg test item/L.
For development rate, the NOEC was determined at >=11.3 µg Pd/L, corresponding to >=32.6 µg test item/L. The EC10 value was 29.9 µg Pd/L, corresponding to 86.7 µg test item/L.
For intrinsic rate, he NOEC was determined at >=4.3 µg Pd/L, corresponding to >=12.4 µg test item/L
Reported statistics and error estimates:
For each endpoint, the NOEC and LOEC, and, if possible, the EC50 and EC10 were determined. A LOEC was calculated by using ANOVA followed by Student-t test, Fisher`s Exact Binomial test, Dunnett’s or Williams’ test or an appropriate non-parametric test.
When the test results show a concentration-response relationship, the data were analysed by regression to determine the EC50 including the 95% confidence interval as well as the EC10 using Probit-analysis assuming log-normal distribution of the values. The computer software ToxRat® [6] was used for statistical evaluations.
The evaluation of the concentration-effect-relationships and the calculations of effect concentrations was based on the mean measured concentrations (time weighted mean, TWM) of palladium, since the deviation from the nominal concentrations was greater than ± 20%.

The five test concentrations showed time weighted mean measured concentrations of freshly prepared test solutions between 39.1 – 52.2 % of nominal concentrations. During the time interval until renewal of the test solution, test item concentrations decreased to 30.9 – 41.2%

of nominal (Table 3). In the control, very low Pd concentration of 0.04 – 0.11 μg Pd/L were measured.

Since recovery rates did not remain in a range of ± 20% of nominal concentrations throughout the test, the evaluation of the effects was performed on basis of the time weighted mean test concentrations, which were calculated for the evaluation of the biological parameters and the endpoints LOEC/NOEC.

In the second and third test concentration, all exposed daphnids showed discoloration than daphnids in the control. In the third and fourth test concentration the daphnids were smaller than daphnids in the control. No other clinical signs were observed.

TWM Pd Conc.  Parental immobilisation Mean length on TD21 Mean cumm. Offspring per survived parent Mean age of first brood Mean development rate  Mean intrinsic rate  Mean cumm. offspring per introduced parent 
[µg/L] [mm] [Ind] [d] [Ind]  [Ind]
Control 10 4,59 86.8 9.2 0,109 0,314  86.8
1.6 8 4,18 82 9.3 0,109 0,293  66.7
4.29 9 3.61 79 8.7 0,115 0,329  73.6
11.25 0 - - 9.9 0,102 -  25.8
28.42 0

-

-

-

-

-

 -

84.42

0

-

-

-

-

-

 -

Hypothesis testing in order to determine statistically significant differences was performed by comparing treatments against the control (NOEC/LOEC determination).

There was a significant difference in immobilisation of individuals between the control and the three highest treatment. In the three highest treatments all individuals were immobile. Thus, a concentration related immobility of the adults was observed. In addition, a concentration-effect relationship was observed for the adult body length of individuals with a significant difference between the length of daphnids in the control and all test concentrations.

A significant and concentration dependent trend on immobility of individuals was observed. Based on a high mortality the cumulative offspring per survived and the cumulative offspring per introduced parent as reproduction endpoint were performed.

The development rate and the intrinsic rate of population increase was computed based on introduced parental daphnids.

For the assessment of effects and for calculation of corresponding effect concentrations the mean numbers of offspring per introduced parent per concentration were used. A concentration-effect relationship was observed for immobilisation, reproduction and length. For reproduction per

survived daphnid, length of daphnids and intrinsic rate r, no EC10 values could be calculated.

Validity criteria fulfilled:
yes
Remarks:
The test fulfils all validity criteria as: mortality in controls (0%) is <20% & mean n° of offspring in control 21 d was >60/female. In addition, dissolved oxygen conc was >3 mg/L & pH was 7.7-9.5 and did not vary by more than 1.5 units.
Conclusions:
During the 21-day exposure period to the test item Palladium (II) di (4-oxopent-2-en-2-oate) significant effects on reproduction of introduced parent daphnids, immobilization and length
of the test organisms Daphnia magna were observed. No significant effect on reproduction of survived parent daphnids, age of first reproduction, the development rate and the intrinsic rate could be observed.
Based on the evaluation performed on TWM concentrations for reproduction per introduced parent daphnid and immobility a NOEC of 4.3 μg Pd/L and a LOEC of 11.3 μg Pd/L were
determined, corresponding to 12.4 μg test item/L and 32.6 μg test item/L, respectively. For introduced parent daphnid an EC10 of 3.2 μg Pd/L, corresponding to 9.3 μg test item/L was
calculated. For immobilisation of parant daphnids, a LC10 of 1.9 μg Pd/L, corresponding to 5.5 μg test item/L, and for development rate, an EC10 of 29.9 μg Pd/L, corresponding to
86.7 μg test item/L, were determined.
Executive summary:

The effects of Palladium (II) di(4-oxopent-2-en-2-oate) in a 21 day Daphnia magna reproduction study conducted according to OECD 211 were considered. The study was a semi-static test design with test solutions renewals three times per week. The nominal test item concentrations applied were control, 4.6, 11.5, 28.8, 72.0 and 180 µg Pd/L, equivalent test item concentrations of control, 13.3, 33.4, 83.4, 209 and 521µ g test item/L.

The chemical analysis conducted at the start and end of each test solution renewal was based on the concentration of palladium. Mean measured concentrations in fresh test media were between 39.1 and 52.2% of nominal concentrations. During the time interval until renewal of the test solution, mean concentrations were between 30.9 and 41.2 % of nominal concentrations. Thus, chemical analysis revealed that the deviation of the measured Pd-concentrations from the nominal test concentrations was greater than 20%. Therefore, the evaluation of the results was based on the time weighted mean of Pd-concentration: control, 1.60, 4.29, 11.25, 28.42 and 84.42 μg Pd/L, corresponding to 4.64, 12.43, 32.61, 82.38 and 244.70 μg test item/L.

If the parental mortality follows a concentration-response pattern, according to the guideline OECD 211, it is required to report the NOEC and ECX for the cumulative offspring per introduced parent provided these values are lower than in the cumulative offspring per survivor. The Cochran-Armitage test revealed a statistically significant trend in immobility of individuals between the control and the treatments. However, since the NOEC and ECX values for the cumulative offspring per survivor were lower than for the cumulative offspring per introduced parent daphnid, the evaluation for reproduction was based on the cumulative offspring per survivor.

During the 21-day exposure period to the test item Palladium (II) di (4-oxopent-2-en-2-oate) significant effects on reproduction of introduced parent daphnids, immobilization and length of the test organisms Daphnia magna were observed. No significant effect on reproduction of survived parent daphnids, age of first reproduction, the development rate and the intrinsic rate could be observed.

Based on the evaluation performed on TWM concentrations for reproduction per introduced parent daphnid and immobility a NOEC of 4.3 μg Pd/L and a LOEC of 11.3 μg Pd/L were determined, corresponding to 12.4 μg test item/L and 32.6 μg test item/L, respectively. For introduced parent daphnid an EC10 of 3.2 μg Pd/L, corresponding to 9.3 μg test item/L was calculated. For immobilisation of parant daphnids, a LC10 of 1.9 μg Pd/L, corresponding to 5.5 μg test item/L, and for development rate, an EC10 of 29.9 μg Pd/L, corresponding to 86.7 μg test item/L, were determined.

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.

Description of key information

The 21-day EC10 based on reproduction was 9.3 µg test item/L, corresponding to 3.2 µg Pd/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
9.3 µg/L

Additional information

The effects of Palladium (II) di(4-oxopent-2-en-2-oate) in a 21 day Daphnia magna reproduction study conducted according to OECD 211 were considered. The study was a semi-static test design with test solutions renewals three times per week. The time weighted mean of Pd-concentration were control,1.60, 4.29, 11.25, 28.42 and 84.42 μg Pd/L, corresponding to 4.64, 12.43, 32.61, 82.38 and 244.70 μg test item/L. For reproduction, an EC10 of 3.2 μg Pd/L, corresponding to 9.3 μg test item/L was calculated.