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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Platinum(2+) tetraammine diacetate
Cas Number:
0127733-97-5
IUPAC Name:
Platinum(2+) tetraammine diacetate
Test material form:
solid
Remarks:
crystal powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
10-mL samples were taken in duplicate from all test concentrations and blank controls at the beginning and at the end of the test. Prior to analysis, samples were stored at room temperature.
An additional singular reserve samples of 10 mL were taken from all test solutions for possible analysis. If not used, these samples were stored at room temperature for a maximum of three months.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: preparation of test solutions started with a solution of 100 mg test item/L. The test item was completely dissolved in the test medium by careful mixing. The lower test concentrations were prepared by subsequent dilutions of the highest test solution in test medium. The final test solutions were all clear and colourless. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration.
- Controls: test medium without test substance or other additives (blank control) and the highest test concentration without algae (background control)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Selenastrum capricornutum, NIVA CHL 1
- Source (laboratory, culture collection): in-house laboratory culture
- Age of inoculum (at test initiation): not reported
- Method of cultivation: not reported

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2 °C. Then, 3 days prior to test start, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. This pre-culture was maintained at the same conditions as used in the test.
- Any deformed or abnormal cells observed: not reported

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg/L as CaCO3
Test temperature:
23 - 24 °C
pH:
8.1 - 10.2
Nominal and measured concentrations:
Nominal concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg test item/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100-mL glass vessels, containing 50 mL of test solution
- Aeration: not reported
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 196.5 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6 (blank control), 1 (background control)

GROWTH MEDIUM
- Standard medium used: yes, the M2-medium was used

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by reverse osmosis, and subsequently passed over activated carbon and ion-exchange cartridges
- Total Hardness: 24 mg CaCO3/L
- Intervals of water quality measurement: the pH of test solutions was measured at the beginning and at the end of the test. The temperature was continuously measured in a temperature control vessel.

OTHER TEST CONDITIONS
- Photoperiod: continuous lighting
- Light intensity and quality: light intensity within the range of 85 to 92 μE.m-2.s-1, using a cool-white TLD-lamp of 30 Watt

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: cells were counted using a microscope and a counting chamber at test start. Thereafter, cells densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength of 20 mm).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 0.1, 1.0, 10 and 100 mg test item/L
- Results used to determine the conditions for the definitive study: the expected EC50 for cell growth inhibition was between 1.0 and 10 mg test item/L, and the expected EC50 for growth rate reduction was significantly higher and exceeded 100 mg test item/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.15 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Pt
Basis for effect:
biomass
Remarks on result:
other: Pt concentrations calculated based on % Pt in the test material.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% Confidence Interval: 2.1 - 11 mg test item/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.3 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Pt
Basis for effect:
biomass
Remarks on result:
other: 95% Confidence Interval: 0.99 - 5.16 mg Pt/L. Pt concentrations calculated based on % Pt in the test material.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.47 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Pt
Basis for effect:
growth rate
Remarks on result:
other: Pt concentrations calculated based on % Pt in the test material.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
52 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% Confidence Interval: 35 - 73 mg test item/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
24.41 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Pt
Basis for effect:
growth rate
Remarks on result:
other: 95% Confidence Interval: 16.43 - 34.27 mg Pt/L. Pt concentrations calculated based on % Pt in the test material.
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.42 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% Confidence Interval: 0.18 - 0.99 mg test item/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Pt
Basis for effect:
biomass
Remarks on result:
other: 95% Confidence Interval: 0.085 - 0.46 mg Pt/L. Pt concentrations calculated based on % Pt in the test material.
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% Confidence Interval: 0.21 - 9.2 mg test item/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.41 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Pt
Basis for effect:
growth rate
Remarks on result:
other: 95% Confidence Interval: 0.1 - 4.32 mg Pt/L. Pt concentrations calculated based on % Pt in the test material.
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none reported
Results with reference substance (positive control):
Based on the 72-hour reference study with Selenastrum capricornutum, the EC50 of potassium dichromate for cell growth inhibition and growth rate reduction were determined to be 0.74 mg/L (95% Confidence Interval of 0.58 to 0.93 mg/L) and 1.5 mg/L (95% Confidence Interval of 1.0 to 2.1 mg/L), respectively.
Reported statistics and error estimates:
Statistically significant inhibition of cell growth and reduction of growth rate were both analysed using Bonferroni t-Test (two-tailed, alpha = 0.05) and Tukey's Test (alpha = 0.05).

Any other information on results incl. tables

Table 1. Mean cell densities (x 10^4 cells/mL) measured at 0, 24, 48, and 72 hours of exposure to platinum(2 +) tetraammine diacetate for Selenastrum capricornutum

 Nominal concentration (mg test item/L)     Exposure time (hours)         
 0 24  48  72 
 Blank control  1.0 11.5 69.2 196.5
 0.32  1.0 11.8 70.5 186.8
 1.0  1.0 7.2 46.4 155.8
 3.2  1.0 6.9 37.3 112.7
 10  1.0 5.7 26.1 82.0
 32  1.0 5.3  10.5 18.4
 100  1.0 4.7 5.7 5.5

Table 2. Percentage inhibition of cell growth and mean area under the growth curve (A) after 72 hours of exposure to platinum(2 +) tetraammine diacetate for Selenastrum capricornutum

 Nominal concentration (mg test item/L)    Cell growth (0 - 72 hours)    
 Mean area (A)  % Inhibition
 Blank control 4235.08 
0.32  4156.64  1.9 
1.0  3096.60  26.9 
3.2  2354.80  44.4 
10  1686.28  60.2 
32  537.80  87.3 
100  257.44  93.9 

Table 3. Percentage reduction of growth rate (μ) at 24, 48 and 72 hours of exposure to platinum(2 +) tetraammine diacetate for Selenastrum capricornutum

 Nominal concentration (mg test item/L)    Mean growth rate                

μ (0 - 24 hours)

% reduction 

μ (0 - 48 hours)

% reduction

μ (0 - 72 hours)

% reduction
Blank control 0.10163 - 0.08825 - 0.07329 -
0.32 0.10276 -1.1 0.08864 -0.4 0.07262 0.9
1.0 0.08251 18.8 0.07994 9.4 0.06991 4.6
3.2 0.08064 20.6 0.07541 14.6 0.06546 10.7
10 0.07246 28.7 0.06776 23.2 0.06117 16.5
32 0.06913 32.0 0.04887 44.6 0.04042 44.8
100 0.06478 36.3 0.03629 58.9 0.02349 67.9

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Based on nominal concentrations, the 72-hour EC50 of platinum(2+) tetraammine diacetate for biomass and growth rate of Selenastrum capricornutum were estimated to be 4.9 mg test item/L (corresponding to 2.3 mg Pt/L) and 52 mg test item/L (corresponding to 24.41 mg Pt/L), respectively. The 72-hour EC10 value for growth rate was 3 mg test item/L corresponding to 1.41 mg Pt/L.
Executive summary:

A 72-hour algal growth inhibition test was conducted with Selenastrum capricornutum for platinum(2+) tetrammine diacetate. The study is reliable without restriction, being GLP-compliant and having followed standard test guidelines (OECD 201 and EU Method C.3).

Based on a range-finding test, six test concentrations were used and these were analysed using ICP-MS. Measured test concentrations, based on the Pt content, were found to be stable and in agreement with nominal concentrations throughout the test (90 – 105 % of nominal). All validity criteria were met.

The 72-hour EC50 for biomass and growth rate were estimated to be 4.9 mg test item/L (2.3 mg Pt/L) and 52 mg test item/L (corresponding to 24.41 mg Pt/L), respectively. The 72-hour EC10 for growth rate was 3.00 mg test item/L corresponding to 1.41 mg Pt/L.