Dihydrogen hexahydroxyplatinate

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

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other: GLP compliant, guideline study, available as an unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dihydrogen hexahydroxyplatinate

EC Number:

257-471-2

EC Name:

Dihydrogen hexahydroxyplatinate

Cas Number:

51850-20-5

Molecular formula:

H6O6Pt.2H

IUPAC Name:

hexahydroxyplatinate(2-)

Test material form:

solid: crystalline

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

All test solutions were sampled prior to the beginning of the exposure period and the addition of algae. After 72 hours, 20-mL samples for chemical analysis were taken from the pooled replicates of each treatment level and controls. All clear samples were analysed at the day of sampling.

Test solutions

Vehicle:

no

Details on test solutions:

Due to the low solubility of the test substance the test solutions were individually prepared by dissolving 1.35, 2.4, 5.11, 10.00 and 19.98 mg of test item to 1 L sterile growth medium under sterile conditions. After stirring for 1 hour at room temperature (at approximately 20 °C), any surplus of test item was allowed to settle for 1 hour and aliquots of the clear supernatants were used for the test.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular green algae
- 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.
- Age of inoculum (at test initiation): exponentially growing algae
- Method of cultivation: the stock cultures were maintained fulfilling the criteria of the OECD 201 guideline (culture medium recommended by Bringmann und Kühn, 1980)

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: prior to testing, a pre-culture was established in standard OECD growth medium to obtain exponentially-growing algae for the test
- Any deformed or abnormal cells observed: none

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.0 - 22.1 °C

pH:

7.74 - 8.12

Nominal and measured concentrations:

Nominal concentrations: 1.35, 2.40, 5.11, 10.0 and 20.0 mg test item/L (corresponding to 0.875, 1.56, 3.31, 6.48 and 12.95 mg Pt/L)
Measured (geometric mean) concentrations: 0.364, 0.946, 1.76, 5.06 and 11.3 mg test item/L (corresponding to 0.236, 0.613, 1.14, 3.28 and 7.31 mg Pt/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL conical glass flasks, containing 100 mL test medium
- Type: covered with air-permeable silicon sponge caps
- Initial cells density: 10000 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 OECD medium was used. In this medium, the molar ratio of EDTA to iron slightly exceeds unity with EDTA in excess (molar ratio EDTA:Fe(III) = 1.14). However, considering the molar ratio and the high stability constant of EDTA with Fe(III) ions, the chelation of platinum remains negligible in comparison with nominal test concentrations.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile ultrapure water
- Intervals of water quality measurement: the temperature was measured daily, and the pH was measured at the beginning and at the end of the test

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Light intensity and quality: OSRAM cool white light adjusted to 4440 - 8880 Lux (60 - 120 µE/m2/s), close to the surface of the liquid, and measured daily
- The cultures were oscillated by continuous stirring on a laboratory shaker with 150 rpm

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter, whose correctness was checked by microscope counts. The cell concentration was determined after 24, 48 and 72 hours in samples taken directly from the test vessels, except for the highest treatment. At the highest concentration, precipitates were observed after one day of exposure and the algal cells were counted microscopically.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study (2 preliminary tests)
- Test concentrations: a) 0.1, 1.0, 3.16 and 10.0 mg test item/L and b) 1, 10, 100 mg test item/L
- Results used to determine the conditions for the definitive study: a) after 72 hours of exposure, the inhibition of growth was -4.3, -0.3, 1.3 and 1.6 % at nominal test concentrations of 0.1, 1.0, 3.16 and 10.0 mg/L, with respective inhibition of yield of -24.8, -1.8, 6.4 and 7.8 %. b) At loadings of 10 and 100 mg test item/L, the highest recoveries were found for 1 h stirring duration.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% Confidence Interval: 2.96 - 4.03 mg test item/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.24 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

Pt

Basis for effect:

other: yield

Remarks on result:

other: 95% Confidence Interval: 1.92 - 2.61 mg Pt/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% Confidence Interval: 0.84 - 1.52 mg test item/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.778 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

Pt

Basis for effect:

other: yield

Remarks on result:

other: 95% Confidence Interval: 0.546 - 0.989 mg Pt/L

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.946 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.613 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

Pt

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Interval: 9.66 - 11.0 mg test item/L

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.63 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

Pt

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Interval: 6.25 - 7.08 mg Pt/L

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Interval: 2.63 - 3.45 mg test item/L

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.98 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

Pt

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Interval: 1.70 - 2.23 mg Pt/L

Effect concentrations 11

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.76 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 12

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Remarks:

Pt

Basis for effect:

growth rate

Details on results:

- The mean of the replicate coefficients of variations in the section-by-section growth rate of controls was 6.6 % during the 72-hour test period.
- Observation of abnormalities (for algal test): at test concentrations of 5.06 and 11.3 mg test item/L, about one third of the cells and 70 - 80 % of the cells, respectively, were damaged/broken after 48 hours of exposure.
- The LOEC for growth rate was determined to be 1.76 mg test item/L (1.14 mg Pt/L)

Results with reference substance (positive control):

The nominal EC50 value for growth rate was determined to be 3.78 mg/L (3.64 - 3.92 95% confidence limits) and was found to be in good agreement with the results of an international ring test with EC50 values for growth rate of 3.38 ± 1.30 mg 3,5-dichlorophenol/L.

Reported statistics and error estimates:

The EC50 and EC10 values for growth rate and yield were calculated using Probit-analysis and a linear maximum likelihood regression. The NOEC and LOEC values for growth rate and yield were determined by the Williams' Multiple Sequential t-test (alpha = 0.05).

Any other information on results incl. tables

Table 1. Mean cell number (x10^4) per mL and per treatment (4 replicates per concentration) and control (8 replicates) at 24, 48 and 72 hours of exposure to dihydrogen hexahydroxyplatinate for Pseudokircheneriella subcapitata.

	Geometric mean measured concentration (mg test item/L)
Time of exposure	Control	0.364	0.946	1.76	5.06	11.3
0*	1.000	1.000	1.000	1.000	1.000	1.000
24	5.082	4.902	5.439	5.162	3.083	0.777
48	27.778	23.485	29.125	27.425	13.330	1.350
72	153.788	137.675	151.275	122.175	51.448	9.963

* Cell number at test start: 10000 cells/mL

Table 2. Mean yield (as cell number increase) per treatment (8 replicates per concentration) and control (8 replicates) at 24, 48 and 72 hours of exposure to dihydrogen hexahydroxyplatinate for Pseudokircheneriella subcapitata.

Geometric mean measured concentration (mg test item/L)	Mean yield values (CV)
	24 h	48 h	72 h
Control	4.082 (13.7)	26.777 (9.1)	152.787 (13.4)
0.364	3.902 (10.0)	22.485 (12.6)	136.675 (12.2)
0.946	4.439 (18.5)	28.125 (16.3)	150.275 (13.7)
1.76	4.161 (15.9)	26.425 (14.5)	121.175 (7.3)
5.06	2.083 (26.0)	12.330 (18.3)	50.447 (19.2)
11.3	-0.223 (32.4)	0.350 (110.0)	8.963 (20.5)

CV = Coefficient of Variation

Table 3. Mean growth rate (1/d) and % Inhibition per treatment (8 replicates per concentration) and control (8 replicates) at 24, 48 and 72 hours of exposure to dihydrogen hexahydroxyplatinate for Pseudokircheneriella subcapitata.

Geometric mean measured concentration (mg test item/L)	Mean growth rate (CV)	% Inhibition	Mean growth rate (CV)	% Inhibition	Mean growth rate (CV)	% Inhibition
	24 h		48 h		72 h
Control	1.620 (6.8)	0.0	1.660 (2.6)	0.0	1.676 (2.7)	0.0
0.364	1.587 (4.9)	2.0	1.576 (3.7)	5.1	1.640 (2.4)	2.2
0.946	1.685 (8.9)	-4.0*	1.681 (5.1)	-1.2*	1.671 (2.8)	0.3
1.76	1.635 (7.7)	-0.9*	1.652 (4.3)	0.5	1.601 (1.5)	4.5
5.06	1.113 (16.9)	31.3	1.289 (7.0)	22.4	1.309 (5.0)	21.9
11.3	-0.256 (36.4)	115.8	0.134 (10.9)	91.9	0.762 (7.6)	54.5

CV = Coefficient of Variation

* Negative inhibition indicate increase in the observed parameter

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on geometric mean measured concentrations, the 72-hour EC50 values of dihydrogen hexahydroxyplatinate for inhibition of growth rate and yield of Pseudokirchneriella subcapitata were determined to be 10.225 mg/L (6.626 mg Pt/L) and 3.45 mg/L (2.239 mg Pt/L), respectively. The 72-hour EC10 values for inhibition of growth rate and yield were determined to be 3.06 mg/L (1.98 mg Pt/L) and 1.20 mg/L (0.778 mg Pt/L), respectively. The NOEC was determined to be 0.946 mg/L for yield, equivalent to 0.613 mg Pt/L.

Executive summary:

An algal growth inhibition study was conducted with Pseudokirchneriella subcapitata for dihydrogen hexahydroxyplatinate. The study is reliable without restrictions, being GLP-compliant and having followed the standard test guideline (OECD 201).

Five test concentrations were used and these were analysed using ICP-OES. Results were based on geometric mean measured concentrations, since the concentration of the test item was found to be decreased more than 20 % of the measured initial concentration throughout the test. The 72-hour EC50 values of dihydrogen hexahydroxyplatinate for inhibition of growth rate and yield of Pseudokirchneriella subcapitata were determined to be 10.225 mg/L (6.626 mg Pt/L) and 3.45 mg/L (2.239 mg Pt/L), respectively. The 72-hour EC10 values for inhibition of growth rate and yield were determined to be 3.06 mg/L (1.98 mg Pt/L) and 1.20 mg/L (0.778 mg Pt/L), respectively. The NOEC was determined to be 0.946 mg/L for yield, equivalent to 0.613 mg Pt/L.