tetraammine palladium (II) hydrogen carbonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August to 5 December 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and all the test groups (replicates pooled) at 0 and 72 hours and stored at -20°C until despatch to Butterworth Laboratories Limited for analysis of palladium content. Analysis was also carried out on the 1.6, 16 and 160 mg/l aqueous stock solution used to prepare the test series at 0 hours.

Moreover, water samples from all the test groups were taken at 0 hours for the initial stability analysis and at 24 hours after storage at ambient temperature both in the light and the dark for the 24 hours’ stability analysis.

Samples were also taken at 0, 24, 48 and 72 hours and the absorbance measured using a spetrophotometer. The densities of the cells were confirmed by direct counting using a haemocytometer. Once the direct counting method confirmed the spectrophotometer absorbance values, only the spectrophotometer was used in the remainder of the experiment.
.

Vehicle:

no

Details on test solutions:

160 mg of test material was dispersed in OECD standard culture medium* and the volume adjusted to 1 litre to give a 160 mg/l stock solution. Serial dilutions were prepared form this to give further stock solutions of 0.010, 0.020, 0.040, 0.080, 0.16, 1.6 and 16 mg/l. An aliquot (500 ml) of the 0.010, 0.020, 0.040, 0.080 and 0.16 mg/l stock solution was each separately dispersed in algal suspension (500 ml) to give the test concentrations of 0.0050, 0.010, 0.020, 0.040 and 0.080 mg/l.

* The standard OECD culture medium was composed of:
15 mg/l of NH4Cl
12 mg/l of MgCl2·6H2O
18 mg/l of CaCl2·2H2O
15 mg/l of MgSO4·7H2O
1.6 mg/l of KH2PO4
0.08 mg/l of FeCl3·6H2O
0.1 mg/l of Na2EDTA·2H2O
0.185 mg/l of H3BO3
0.415 mg/l of MnCl2·4H2O
0.003 mg/l of ZnCl2
0.0015 mg/l of CoCl2·6H2O
0.00001 mg/l of CuCl2·2H2O
0.007 mg/l of Na2MoO4·2H2O
50 mg/l of NaHCO3

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: CCAP 276/20
- Source (laboratory, culture collection): liquid cultures of Scenedesmus subspicatus were obtained from the Culture Centre for Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria.

HOLDING CONDISTIONS PRIOR TO TEST
Cultures were maintained in the laboratory by the replenishment of culture medium approximately once per week. The culture was maintained in the laboratory at a temperature of 21 + 1 °C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

LIFE STAGE OF TEST SPECIES USED
Pre-culture conditions gave an algal suspension in log phase growth characterised by an absorbance of 1.238 (at 665 nm). This suspension was diluted to an absorbance of 0.029 prior to use. At initiation of the study the culture contained a nominal cell density of 1000 cells per ml.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

See 'Details on test solutions' box

Test temperature:

24 °C

pH:

From 7.8 to 10.6

Nominal and measured concentrations:

Measured concentrations could not be determined for all test concentrations, since, in many cases, they were below the limit of quantitation of the analytical method. Considering stability tests performed for other studies using palladium, the author assumes that no significant change in palladium concentration occurred over the 72h test period. Therefore, the results have been based on nominal test concentrations only.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks
- Type (delete if not applicable): the flasks were covered with aluminium foil
- Fill volume: 100 ml of test solution
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: 10000
- Control end cells density: 1000000
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes (OECD standard)

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: intensity approximately 7000 lux
The flasks were covered with aluminium foil and incubated (Gallenkamp INR-401- 010W) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at 100 rpm for 72 hours.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
A range-range finding study was initially performed at 0.1, 1, 10 and 100 mg/l test concentrations. The results showed severe inhibition of growth at all the test concentrations employed. Therefore, a second range-finding study was conducted using test concentrations of 0.0010, 0.010, 0.10 and 1.0 mg/l. The results showed no effect on growth at the test concentrations of 0.0010 and 0.010 mg/l. However, growth was observed to be reduced at 0.10 and 1.0 mg/l. Based on this information, test concentrations of 0.0050, 0.010, 0.020, 0.040 and 0.080 mg/l were selected for the definitive study

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.066 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.078 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Extrapolated value

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.04 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Absorbance values appeared to be well correlated with cal density values and therefore it was considered justifiable to monitor the growth of test cultures by absorbance values only.
All test and control cultures were inspected microscopically at 72 hours. there were no abnormalities detected in any of the control or test cultures.

Reported statistics and error estimates:

Statistical analysis of area under the growth curve data was carried out using one way ANOVA. When calculating the NOEC, no statistically significant differences (P ≥ 0.05) were observed between the control and the ≤ 0.4 mg/l test groups. The 0.08 mg/l test group was shown to be significantly different from the control (P < 0.001).

Analysis of the test solutions at 0 and 72 h showed measured concentrations to be low and variable with concentrations ranging frrm < LoQ and 63% of nominal. This was considered to be due to the concentrations being at or close to the LoQ of the analytical method. A value of 447% of nominal was calculated for the 0.005 mg/l test group at 0 h. This value was considered to be due to sampling/analytical variation.

Validity criteria fulfilled:

yes

Remarks:

However, when evaluating if the validity criteria were fulfilled, the absorbance values were used instead of the biomass values suggested by the OECD guideline.

Conclusions:

In a OECD- and GLP-compliant study, a 72-hr EbC50 of 0.066 mg/L and a 24-48-hr ErC50 of 0.078 mg/L were determined for tetraamminepalladium(II) hydrogen carbonate. The NOEC was 0.040 mg/L

Executive summary:

The effect of tetraamminepalladium(II) hydrogen carbonate on the growth of green alga Scenedesmus subspicatus was evaluated over a 72hr period using OECD test guideline 201 and conducted to GLP.

Following preliminary range-finding studies, S. subspicatus was exposed to an aqueous dispersion of the test material at concentrations of 0.0050, 0.010, 0.020, 0.040 and 0.080 mg/L (three replicate flasks per concentrations) for 72 hr, under constant illumination and shaking at a temperature of 24 °C. Samples of the algal populations were removed daily and absorbance values determined for each control and treatment group.

The 72-hr EbC50, 0-24-hr ErC50 and NOEC were determined to be 0.066 mg/L, 0.078 mg/l and 0.04 mg/L, respectively, based on nominal concentrations. Analytical confirmation of test concentrations was not possible due to the concentrations being at or close to the LoQ of the analytical method. The validity criteria are fulfilled although their evaluation is based on the absorbance values rather than on biomass values (not available for each replicate).

Reference 2

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:

other: GLP compliant, guideline study, available as an unpublished report, no restrictions, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the test media at test start and after 72 hours. At test start a sample of fresh control media and freshly prepared test media of each treatment were taken prior to distributing the test media to the test containers. After 72 hours samples of the replicate vessels from each treatment were pooled prior to analysis. Samples were taken and filtered (0.2µm). Solutions were acidified with nitric acid (target concentration 3% HNO3) and stored at 4°C until analysis.

Vehicle:

no

Details on test solutions:

A stock solution of 19.87 mg diamminedichloropalladium/L was prepared by dissolving adequate amounts of test item in sterile growth medium in a sterile glass bottle and stirring for one hour. The stock solution was diluted with 10-fold concentrated growth medium and ultrapure water under sterile conditions to obtain the required test concentrations.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: freshwater alga
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University of Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

21 - 22 °C

pH:

At test start: 7.88 - 8.09
At end of test: 7.29 - 8.12

Dissolved oxygen:

Not reported

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal concentrations: Control, 1.24, 2.48, 4.97, 9.94 and 19.87 g DDP/L
corresponding to Control, 0.625, 1.25, 2.5, 5.0 and 10.0 µg palladium/L,
Mean measured (geometric mean) concentrations: Control, 0.140, 0.286, 0.765, 1.33 and 3.56 µg palladium/L,
corresponding to Control, 0.278, 0.569, 1.52,2.65 and 7.08 µg DDP/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flask
- Type: covered with air-permeable silicon sponge caps
- Material, size, headspace, fill volume: Glass, 250 mL containing 100 mL test medium
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile ultrapure water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Light intensity and quality: OSRAM "day light" 4440 - 8880 Lux (60 - 120 µE/m2/s)
- Shaken at a rate of 100 rpm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

- Range finding study: Yes
- Test concentrations: Not reported

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.03 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Diamminedichloropalladium

Basis for effect:

other: yield

Remarks on result:

other: 95% CL 3.58 - 4.85

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.84 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Diamminedichloropalladium

Basis for effect:

other: yield

Remarks on result:

other: 95% CL 2.50 - 3.16

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.64 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Diamminedichloropalladium

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.88 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Diamminedichloropalladium

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 5.27 - 6.22

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.74 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Diamminedichloropalladium

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 2.56 - 4.45

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.64 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

Diamminedichloropalladium

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.03 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

Palladium

Basis for effect:

other: yield

Remarks on result:

other: 95% CL 1.80 - 2.44

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.43 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

Palladium

Basis for effect:

other: yield

Remarks on result:

other: 95% CL 1.26 - 1.59

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.33 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

Palladium

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.96 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

Palladium

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 2.65 - 3.13

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.88 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

Palladium

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 1.29 - 2.24

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.33 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

Palladium

Basis for effect:

growth rate

Results with reference substance (positive control):

The sensitivity of the test organism is routinely checked using 3,5-dichlorophenol. The nominal ErC50 value of 2.91 mg/L (95% confidence limits 2.35 - 3.63 mg/L) is in good agreement with the results of an international ring test with ErC50 of 3.38 ± 1.30 mg/L.

Reported statistics and error estimates:

Test results were statistically analysed to determine EC values together with 95 % confidence intervals using Probit-analysis assuming log-normal distribution of the values. Individual replicate responses were used for the regression analysis.
The NOEC and LOEC values for growth rate were determined by the Mann-Whitney U-test with Bonferroni correction and for yield by the Williams Multiple Sequential t-test.

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour ErC50 and ErC10 for growth rate were 5.88 and 3.74 μg test item/L, respectively based on mean measured concentrations of diamminedichloropalladium. For yield the EYC50 and EYC10 values were 4.03 and 2.84 μg /L. NOEC for both growth rate and yield were 2.64 μg test item/L.

Based on the mean measured concentrations of dissolved palladium, the EC50 values for growth rate and yield were 2.96 and 2.03 μg Pd/L. The EC10 values were 1.88 and 1.43 μg Pd/L for growth rate and yield, respectively. The NOEC values for both growth rate and yield were calculated to be 1.33 μg Pd/L.

Executive summary:

A GLP compliant, OECD guideline study to assess algal growth was carried out (Fraunhofer 2012). It is considered to be suitable for use as the key study for this endpoint.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant; guideline study; available as unpublished report; minor restrictions in design and/or reporting but otherwise adequate for assessment.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

stock solution stirred for 5 hours rather than 4; introduction of algae cells, samples from 2 day culture taken

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

stock solution stirred for 5 hours rather than 4; introduction of algae cells, samples from 2 day culture taken

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from all test concentrations and control at 0 and 72 hours

Vehicle:

no

Details on test solutions:

Concentrated stock solution of test item of nominal 100 mg test item/L prepared by dissolving 60 mg of test item into 600 mL test water by intense stirring for 5 hours to obtain the hydrolysis products of the rapidly degrading test item. Following filtration (0.45 um cellulose nitrate filter) stock solution diluted with test water. Test media prepared just before introduction of algae.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Species: Desmodesmus subspicatus
Source: "Sammlung von Algenkulturen, Pflanzenphysiologisches Institu der Universitat Gottingen" 37073 Gottingen Germany
Culture conditions prior to test: The algae were cultivated in the laboratories of IBACON under standardised conditions according to the test guidelines. For the evaluation of the quality of the algae and the experimental conditions the substance potassium dichromate p.A. is tested at least twice a year to demonstrate satisfactory test conditions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

calculated water hardness 0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

23° C

pH:

pH 7.9 at test start pH 8.2 to 9.9 at test end

Dissolved oxygen:

No data

Salinity:

Not applicable

Nominal and measured concentrations:

1:100; 1:250: 1:625: 1:1562.5 and 1: 3906.25 filtrate of a 100 mg test item /L test solution

Details on test conditions:

5000 algal cells per mL test medium. Cells taken from an exponentially growing pre-culture, which was set up 2 days prior to the test start at the same conditions as in the test. Light regime: Continuous Illumination. with a maximum deviation of + 15%

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 5 µg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

other: biomass and yield

Remarks on result:

other: EC values recalculated based on reported data

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

< 8.5 µg/L

Nominal / measured:

estimated

Conc. based on:

element

Basis for effect:

other: biomass and yield

Remarks on result:

other: EC values recalculated based on reported data

Reported statistics and error estimates:

EC50s calculated by Probit Analysis. LOEC and NOEC calculated using ToxRat Version 2.09 2001-2005

The EC10 and EC50 values are recalculated using Trap v1.3 using the observed responses vs the estimated exposure concentration (only measured Pd concentration for highest concentration available, all other Pd treatments were <LOD – as a best alternative, a theoretical prediction of the exposure concentration was done based on the measured Pd concentration of the high dose and assuming that a similar fraction of nominal added Pd got into solution for the lower doses).

Pawlowski and Wydra, 2005
Desmodesmus subspicatus
Dihydrogen tetrachloropalladate
Concentration Test Item (ug/L)		LOQ=25 ug/L			Concentration Pd (ug/L)		Pd=19,97%	LOQ=5 ug/L
	Measured		Estimated			Measured		Estimated
	0 h	72 h				0 h	72 h
Control	<LOQ	<LOQ	/		Control	<LOQ	<LOQ	/
Conc 1	<LOQ	<LOQ	1		Conc 1	<LOQ	<LOQ	0.20
Conc 2	<LOQ	<LOQ	3		Conc 2	<LOQ	<LOQ	0.60
Conc 3	<LOQ	<LOQ	7		Conc 3	<LOQ	<LOQ	1.40
Conc 4	<LOQ	<LOQ	17		Conc 4	<LOQ	<LOQ	3.39
Conc 5	43.5	41.5	42.5		Conc 5	8.69	8.29	8.5
				EC50 (ug/L)	EC10 (ug/L)
Reported values using nominal concentrations				4.2	2.5

Validity criteria fulfilled:

yes

Remarks:

cell density in control cultures increased by factor of 71.1 within 72 hrs; coefficient of variation on the sectional growth rates in the control cultures was 34.0%; coefficient of variation of average growth in replicate control cultures was 2.2%.

Conclusions:

The toxicity of dihydrogen tetrachloropalladate to Desmodesmus subspicatus was determined. The 72 hr EC50 values were <8.5 µg Pd/L for biomass and growth rate, respectively. The 72 hr NOEC values were <5 µg Pd/L. Recalculated values are 2.5 and 4.2 ug Pd/L for EC10 and EC50, respectively.

Executive summary:

This information is taken from a GLP compliant, guideline study and is considered reliable and suitable for use as the key study for this endpoint. The toxicity of dihydrogen tetrachloropalladate to Desmodesmus subspicatus was determined. The 72 hr EC50 values were <8.5 µg Pd/L for biomass and growth rate, respectively. The 72 hr NOEC values were <5 µg Pd/L. Recalculated values are 2.5 and 4.2 ug Pd/L for EC10 and EC50, respectively.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

not specified

Remarks:

Study is a publication, referring to academic work performed with no GLP compliance

Specific details on test material used for the study:

ICP standard solution in HCl

Analytical monitoring:

yes

Details on sampling:

at test setup, and after 24, 48, 72 and 96h.

Vehicle:

no

Details on test solutions:

HSHM-1 medium (Modified High Salt Medium; Macfie et al 1994)
10 mM MOPS buffer added
Media autoclaved before use.

Test organisms (species):

Chlamydomonas reinhardtii

Details on test organisms:

exponentially growing cells, added at 5000 cells/mL at test initiation

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

pH:

7

Nominal and measured concentrations:

nominal concentrations: 0.1, 1, 10, 13, 17,20,30,40,50, 100 µg Pd/L
measured concentrations: 0.06, 0.20, 5.4, 8.2, 8.9, 11.8 µg Pd/L for nominal concentrations 0.1-20 µg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

10.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

cell number

Remarks on result:

other: value as reported in publication

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

8.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

cell number

Remarks on result:

other: value as reported in publication

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.06 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

cell number

Remarks on result:

other: EC10 re-calculated using Graph & TRAP software; 95% CI: 4.5-5.8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

cell number

Key result

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

4.48 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

cell number

Remarks on result:

other: EC10 re-calculated using Graph & TRAP software; 95% CI: 4.1-4.9

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

5.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

cell number

Reported statistics and error estimates:

EC50 values derived using Excel REGTOX or SigmaPlot v 10.0
EC10 values re-calculated using Graph & TRAP software

Validity criteria fulfilled:

not specified

Conclusions:

The 96-h EC50 for algal cell density was 5.7 µg Pd/L.
The 96-h EC50 for algal cell density was calculated as 4.48 µg Pd/L.

Executive summary:

The effect of palladium (tested as PdCl2) on algal growth was determined. The 96-h EC10 and EC50 values (based on measured Pd concentrations) were 4.48 and 5.7 µg Pd/L, respectively.

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

not specified

Remarks:

Study is a publication, referring to academic work performed with no GLP compliance

Specific details on test material used for the study:

ICP standard solution in HCl

Analytical monitoring:

yes

Details on sampling:

at test setup, and after 24, 48, 72 and 96h.

Vehicle:

no

Details on test solutions:

HSHM-1 medium (Modified High Salt Medium; Macfie et al 1994)
for assessment of pH effect: MES (pH6 medium), MOPS (pH7 medium) or HEPES (pH8 medium) buffer added
for assessment of NH4 effect: no NH4 (no NH4 added, replaced by Ca(NO3)2 and MgSO4 salt), normal NH4, high NH4 (94 µM, addition as NH4NO3)
for assessment of Ca effect: Ca(NO3)2 and KNO3 in alternating concentrations to target 0.068, 0.5, 1.0 and 2.0 mM Ca2+
Media autoclaved before use.

Test organisms (species):

Chlamydomonas reinhardtii

Details on test organisms:

exponentially growing cells, added at 10000 cells/mL at test initiation

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

20°C

pH:

6, 7 or 8

Nominal and measured concentrations:

nominal concentrations to asses pH effect: 0, 2,5, 10, 15, 20 and 30 µg Pd/L
nominal concentrations to assess effects of NH4: 0, 2, 5, 10, 15,20 and 30 µg Pd/L
nominal concentrations to assess effects of NH4: 20 µg/L

Details on test conditions:

light intensity 100 +/- 10 pE m-2 s-1

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

5.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

cell number

Remarks on result:

other: EC10 re-calculated using Graph & TRAP software; 95%CI: 4.8-5.6; pH6

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

10 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

cell number

Remarks on result:

other: pH6.0

Key result

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

3.05 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

cell number

Remarks on result:

other: EC10 re-calculated using Graph & TRAP software; 95%CI: 1.6-5.9; pH7

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

cell number

Remarks on result:

other: pH7.0

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

>= 30 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

cell number

Remarks on result:

other: pH8

Remarks:

no effect observed at 24, 48, 72 and 96h

Reported statistics and error estimates:

EC50 values derived using Excel REGTOX or SigmaPlot v 10.0

Validity criteria fulfilled:

not specified

Conclusions:

The 24-h EC50 for algal cell density was 10 µg Pd/L at pH6 and 7 µg Pd/L at pH 7.
The 24-h EC10 for algal cell density was recalculated as 5.2 µg Pd/L at pH6 and 3.05 µg Pd/L at pH7.

Executive summary:

The effect of palladium (tested as PdCl2) on algal growth was determined.

The 24-h EC50 for algal cell density was 10 µg Pd/L at pH6 and 7 µg Pd/L at pH 7. No effects were observed at nominal Pd concentrations of 30 µg/L at pH8.

The 24-h EC10 for algal cell density was recalculated as 5.2 µg Pd/L at pH6 and 3.05 µg Pd/L at pH7.

Reference 6

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: 97.01% Pd(NH3)4Cl2 (based on analyzed Pd content of 42.06%), rest water (Pd content of pure Pd(NH3)4Cl2 = 43.357%).

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, 10.34 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
1 hours at room temperature (about 20°C). 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:

22.5 - 23 °C

pH:

pH of the controls: mean pH 7.9 at test start, mean pH 7.92 at test end
pH in test solutions: pH 7.46 to 7.66 at test start, pH 7.85 to 8.31 at test end

Dissolved oxygen:

Not stated

Salinity:

Not stated

Conductivity:

Not stated

Nominal and measured concentrations:

range-finder test: Nominal concentration 0.01-0.1-1 mg test item/L
definitive test: Nominal concentrations: control, 6.25, 12.5, 25.0, 50.0 and 100 μg Pd(NH3)4Cl2/L, equivalent to: control, 2.71, 5.42, 10.84, 21.7 and 43.4 μg Pd/L.

Measured concentrations in control: 0.004 µg Pd/L at test start, 0.006 μg Pd/L at test end
Measured concentrations at test start: 1.30, 3.14, 7.38, 15.9 and 36.5 μg Pd/L
Measured concentration after 72 hours (test end): 0.941, 1.93, 4.93, 9.42 and 26.7 μg/L μg Pd/L
Geometric mean of the measured concentrations at test initiation and termination (mean measured): 1.11, 2.46, 6.03, 12.2 and 31.2 μg Pd/L (equivalent to 2.55, 5.68, 13.9, 28.2 and 72.0 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 42.06%

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: 84.6 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 93.25 to 101.70 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
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Yes
- Test concentrations:
Nominal concentration 0.01-0.1-1 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 ).

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.94 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 9.02-10.7 µg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.25 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 5.14-7.15 µg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.67 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.:

7.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL 6.46-11.7 µg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.87 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95%CL 3.37-5.35 µg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

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 (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 two lowest dose treatments throughout test.
At mid dose level, few algal cells with normal appearance + some cell debris.
At highest two doses, very few algal cells with normal appearance + some cell debris.

Validity criteria fulfilled:

yes

Remarks:

cell number in control cultures increased by factor of 84.6 within 72h; mean of replicate CV in section-by-section growth rate of controls was 12.3% during test period; CV of average specific growth rate at test end in replicate control cultures was 2.4%

Conclusions:

The 72-hour ErC10 and ErC50 for the inhibition of growth rate were estimated with 2.71 μg Pd/L (6.25 μg Pd(NH3)4Cl2/L) and 4.31 μg Pd/L (9.94 μg Pd(NH3)4Cl2/L), based on mean measured Pd concentrations.

For yield, EyC10 and EyC50 values of 2.11 and 3.12 μg Pd/L (4.87 and 7.20 μg Pd(NH3)4Cl2/L) were determined, respectively, based on mean measured Pd concentrations.

The NOEC for the inhibition of both growth rate and yield was determined with 2.46 μg Pd/L (5.68 μg Pd(NH3)4Cl2/L), based on mean measured Pd concentrations.

Executive summary:

A GLP compliant study was performed to determine the toxicity of Tetraamminepalladium(2+) dichloride (Pd(NH3)4Cl2) 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, eight replicates of the control (test medium only) and four replicates of each treatment were tested at the nominal concentrations of 6.25, 12.5, 25.0, 50.0, 100 μg Pd(NH3)4Cl2/L plus control (equivalent to 2.71, 5.42, 10.84, 21.7, 43.4 μg Pd/L). The concentrations of dissolved palladium (Pd) in the media were measured at test initiation and test termination by ICP-MS (LOQ = 0.002 – 0.004 μg Pd/L). Measured palladium concentrations were within a range of 48.0 – 84.1% of the nominal concentrations at test start and between 34.7 – 61.6% of nominal at test termination. Thus, the evaluation was based on geometric mean measured Pd concentrations of 1.11, 2.46, 6.03, 12.2 and 31.2 μg Pd/L (equivalent to 2.55, 5.68, 13.9, 28.2 and 72.0 μg Pd(NH3)4Cl2/L).

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

The 72-hour ErC10 and ErC50 for the inhibition of growth rate were estimated with 2.71 μg Pd/L (6.25 μg Pd(NH3)4Cl2/L) and 4.31 μg Pd/L (9.94 μg Pd(NH3)4Cl2/L), based on mean measured Pd concentrations.

For yield, EyC10 and EyC50 values of 2.11 and 3.12 μg Pd/L (4.87 and 7.20 μg Pd(NH3)4Cl2/L) were determined, respectively, based on mean measured Pd concentrations.

The NOEC for the inhibition of both growth rate and yield was determined with 2.46 μg Pd/L (5.68 μg Pd(NH3)4Cl2/L), based on mean measured Pd concentrations.

Description of key information

Acute toxicity data are available for diamminedichloropalladium, dihydrogen tetrachloropalladate, palladium dichloride (2 studies), tetraamminepalladium di(hydrogencarbonate) and tetraamminepalladium dichloride. P. subcapitata, C. reinhardtii and S. subspicatus were used as test species, and inhibition of growth rate was used as endpoint of preference. The EC50 values for the above-mentioned test items as summarised in below table, were 2.96, <8.5 (recalculated to 4.2), 5.7 and 8.2, 8 and 4.31 µg Pd/L.

reference	test substance	effect	endpoint	value	unit	organism
Wenzel (2012)	diamminedichloropalladium	growth	EC50	2.96	µg Pd/L	P.subcapitata
Pawlowski (2005)	dihydrogen tetrachloropalladate	growth	EC50	4.2	µg Pd/L	S.subspicatus
Roy (2009)	palladium dichloride	growth	EC50	5.7	µg Pd/L	C.reinhardtii
Tetrault (2014)	palladium dichloride	growth	EC50	8.2	µg Pd/L	C.reinhardtii
Mead (1997)	tetraamminepalladium di(hydrogencarbonate)	growth	EC50	8	µg Pd/L	S.subspicatus
Wenzel (2018)	tetraamminepalladium dichloride	growth	EC50	4.31	µg Pd/L	P.subcapitata