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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 - 27 April 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Minor deviation to the guideline (pH varied by more than 1.5 units in certain treatment groups), conducted according to GLP.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
pH varied by more than 1.5 units in certain treatment groups
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
pH varied by more than 1.5 units in certain treatment groups
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:
- Concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/mL
- Sampling method: During the final test duplicate samples were taken from all concentrations and the blank-control. 10 mL samples were taken at the start(t=0) and end (t=72 hr) of the final test.
- Sample storage conditions before analysis: Samples were stored at room temperature until transportation to the analysis laboratory.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 100 mg/L test solution was prepared by dissolving the test material (white crystals) in test medium with no special treatment other than careful mixing. The lowest test concentrations were prepared by subsequent dilutions of the highest test solution in test medium.
- Eluate: No data
- Differential loading: Not applicable
- Controls: Test medium without test material or other additives
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No, the test material was completely soluble at all concentrations.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): in-house laboratory culture
- Age of inoculum (at test initiation): no data
- Method of cultivation: algal 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 deg C.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
0.24 mmol/L as CaCO3
Test temperature:
23-24 deg C
pH:
Beginning of test = 8.4
End of Test = 8.1-10.2
Dissolved oxygen:
Not determined
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations were 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. Analysis of the samples taken during the final test showed that measured concentrations, based on the platinum content, were stable and in agreement with the nominal concentrations throughout the test period (90-105%).
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL all-glass vessels
- Type (delete if not applicable): no data
- Material, size, headspace, fill volume: 50 mL test solution in 100 mL vessel
- Aeration: continuously aerated
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 10000 cells/mL
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Millipore corp., Bedford Mass., USA) containing NaNO3 (500 mg/L), K2HPO4 (40 mg/L), MgSO4.7H2O (76 mg/L), Na2CO3.10H2O (54 mg/L), C6H8O7.H2O (6 mg/L), NH4NO3 (330 mg/L), CaCl2.H2O (36 mg/L), C6H5FeO7.xH20 (6 mg/L), H3BO3 (2.9 mg/L), MnCL2.4H2O (1.81 mg/L), ZnCL2 (0.11 mg/L), CuSO4.5H2O (0.08 mg/L) and (NH4)6Mo7O24.4H2O (0.018 mg/L).

OTHER TEST CONDITIONS
- Sterile test conditions: no data
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 85-92 μE/m^2/s
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell densities, inhibition of cell growth and reduction of growth rate
- Determination of cell concentrations: At the beginning of the test cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength=20 mm). Algal medium was used as a blank.
- Chlorophyll measurement: no data

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Yes
- Test concentrations: 0.1, 1, 10 and 100 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate
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.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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% CL = 2.1 - 11 mg/L
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% CL = 35 - 73 mg/L
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% CL = 0.18 - 0.99 mg/L
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% CL = 0.21 - 9.2 mg/L
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No data
- Unusual cell shape: No data
- Colour differences: No data
- Flocculation: No data
- Adherence to test vessels: No data
- Aggregation of algal cells: No data
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not applicable
- Effect concentrations exceeding solubility of substance in test medium: Not applicable
Results with reference substance (positive control):
- Results with reference substance valid? Yes, the EC50 values for cell growth inhibition and growth rate reduction were both within the respective historical control ranges.
- EC50: The EC50 for cell growth inhibition was 0.74 mg/L with a 95% confidence level from 0.58 - 0.93 mg/L. The EC50 for growth rate reduction was 1.5 mg/L with a 95% confidence level from 1.0 - 2.1 mg/L.
Reported statistics and error estimates:
Analysis of the samples taken during the final test showed that measured concentrations based on the platinum content were stable and in agreement with nominal throughout the test (90-105%).

Statistically significant inhibition of cell growth was found at test concentrations of 1.0 mg/L and higher (Bonferroni t- and Tukey tests, alpha = 0.05). Statistically significant reduction of growth rate was found at test concentrations of 3.2 mg/L and higher (Bonferroni t- and Tukey tests, alpha = 0.05).

Table 1: Mean algal cell densities during the test period of 72 hr

 Concentration test substance (mg/L)  Exposure time (hr)              
   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 during the test period

  Concentration test substance (mg/L)  Cell growth (0 -72 hr)  
   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) during the test period

Concentration test substance (mg/L)     Mean growth rate               

μ (0 -24 hr)

 Reduction (%)

μ (0 -48 hr)

  Reduction (%)

μ (0 -72 hr)

 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

Table 4: pH levels during the final test

 Concentration test substance (mg/L)     Exposure time (hr)
 0  72
 Blank-control  8.4  10.1
 0.32  8.4  10.2
 1.0  8.4  10.2
 3.2  8.4  10.1
 10  8.4  9.1
 32  8.4  8.3
 100  8.4  8.1

The pH varied by more than 1.5 units (specified as a maximum by the guideline) in the control and lower range of treatment levels, though this was related to a relatively high rate of algal growth. Hence, this did not affect the validity criterion for algal growth.

Validity criteria fulfilled:
yes
Conclusions:
In an OECD guideline study, to GLP, platinum (2+) tetraammine diacetate was tested for growth inhibition of freshwater algae (Selenastrum capricornutum) over a 72-hr exposure period. The NOECs for cell growth inhibition and growth rate reduction were 0.32 mg/L (0.15 mg Pt/L) and 1.0 mg/L (0.47 mg Pt/L) respectively. The EC50 values for cell growth inhibition and growth rate reduction were 4.9 (2.3 mg Pt/L) and 52 mg/L (24.4 mg Pt/L) respectively.
Executive summary:

The acute toxicity of platinum (2+) tetraammine diacetate to freshwater algae (Selenastrum capricornutum) was determined in a 72-hour static test conducted according to OECD guidelines (TG 201) and to GLP.

A full test was performed in parallel with a control, with nominal test substance concentrations of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. Test substance concentrations in excess of 100 mg/L were not tested (based on the test guidelines). The pH varied by up to 1.8 units (more than the 1.5 units specified as a maximum by the guideline) in the control and lower range of treatment levels, though this was related to a relatively high rate of algal growth and was therefore not considered to affect the validity criterion for algal growth.

The 72-hr NOEC (highest concentration tested without toxic affect) for cell growth inhibition was 0.32 mg/L, while the NOEC for growth rate reduction was 1.0 mg/L (0.47 mg Pt/L). The EC50 for cell growth inhibition was 4.9 mg/L (95% CL = 2.1 - 11 mg/L). The EC50 for growth rate reduction was 52 mg/L (95% CL = 35 - 73 mg/L), equivalent to 24.4 mg Pt/L.

Description of key information

In an OECD guideline study, to GLP, platinum (2+) tetraammine diacetate was tested for growth inhibition of freshwater algae (Selenastrum capricornutum) over a 72-hr exposure period. The NOECs for cell growth inhibition and growth rate reduction were 0.32 mg/L (0.15 mg Pt/L) and 1.0 mg/L (0.47 mg Pt/L) respectively. The EC50 values for cell growth inhibition and growth rate reduction were 4.9 (2.3 mg Pt/L) and 52 mg/L (24.4 mg Pt/L) respectively (Migchielsen, 2006b).

Key value for chemical safety assessment

Additional information

The acute toxicity of tetraammineplatinum(II) diacetate to freshwater algae (Selenastrum capricornutum) was determined in a 72-hour static test according to OECD guidelines (TG 201) and to GLP (rated Klimisch 2 as there were pH variations that exceeded the guideline maximum. This was related to a relatively high rate of algal growth and was therefore not considered to affect the validity criterion for algal growth).

A full test was performed in parallel with a control, with nominal test substance concentrations of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.Test substance concentrations in excess of 100 mg/L were not tested (based on the test guidelines). The highest test concentration that did not cause a toxic effect was 0.32 mg/L, and this was determined to be the 72-hr NOEC for cell growth reduction (with an associated EC50 of 4.9 mg/L). The NOEC for growth rate reduction was 1.0 mg/L (0.47 mg Pt/L), and the EC50 was 52 mg/L (95% CL = 35 - 73 mg/L), equivalent to 24.4 mg Pt/L (Migchielsen, 2006b).