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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3341/00-F9
- Expiration date of the lot/batch: August 06, 2020
- Purity test date: Certificate of analysis dated August 06, 2019
- Content: 10.94 % Pt
- Form: liquid
- Appearance: clear orange liquid
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.251, 0.792, 2.50, 7.91 and 25.0 mg test item/L, equivalent to 0.027, 0.087, 0.274, 0.866 and 2.74 mg Pt/L.
- Sampling method:
Samples (2 of about 20 mL) were taken from all test solutions and controls at the beginning of the test prior to addition of the algae. At the end of the growth test, samples were taken directly from representative replicates per test concentration and controls.
At test initiation and test termination one set of samples was filtered through 0.45 µm polyethersulfone filters (6 samples) and one set of samples was not filtered (6 samples). In addition, for each set a set of retain samples was taken (2 x 6 samples).
- Sample storage conditions before analysis:
All samples were stabilised by acidification and stored at 4 ± 3 °C until further analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The highest test concentration was prepared by adding 25.564 mg test item to 1 litre growth medium, and stirred for two hours at room temperature. This was then diluted in order to prepare the other test concentrations
- Controls: Dilution water
- 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.): After 24 hours, yellow precipitates in the three highest test concentrations were observed. After 48 hours, those precipitates could not be observed in the highest concentration, but in the two lower concentrations. This remained until test termination.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: unicellular green alga
- Strain: Chlorophycea, Chlorophyta
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D- 37073 Göttingen, Catalog No 61.81 SAG.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Not stated
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not applicable
Test temperature:
21 - 24 °C
pH:
The pH of the controls increased during the test from 7.03 at test start to a mean value (n = 8) of 7.74 at test end. In the test concentrations, the initial pH in the treatments ranged from 7.00 to 7.05 and the mean pH value was between 7.71 and 7.77 at the end of the test.
Dissolved oxygen:
not stated
Salinity:
not applicable
Conductivity:
not stated
Nominal and measured concentrations:
NOMINAL: 0.027, 0.087, 0.274, 0.866 and 2.74 mg Pt/L (equivalent to 0.251, 0.792, 2.50, 7.91 and 25.0 mg test item/L)
MEASURED test initiation: 0.025, 0.080, 0.265, 0.860 and 3.12 mg Pt/L (89.3 –112% of nominal)
MEASURED test termination: 0.020, 0.056, 0.088, 0.165 and 2.663 mg Pt/L (18.6 – 95.2% of nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flasks
- Type: open
- Material, size, headspace, fill volume: 250 mL conical glass flasks covered with air-permeable silicone-sponge caps.
- Aeration: Not stated
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: 1 x 10 (4) cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8
- 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

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Not stated
- Light intensity and quality: 96.6 – 100.5 µE(1)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: 3.16
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Yes
- Test concentrations: 0.2, 2.0 and 20 mg test item
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
The sensitivity of the test organism is routinely checked using 3,5-dichlorophenol following internal SOPs in a non-GLP test twice a year. The latest (September 2019) test is in good agreement with the results of an international ring test.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.36 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.73 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
yield
Details on results:
- Table 1: The respective 72-hour ErC50 for the inhibition of growth rate was determined to be at 1.36 mg test item/L. For yield, an EyC50 of 0.73 mg test item/L was determined. The EC10 values for growth rate and yield were at 0.47 and 0.46 mg test item/L, respectively.
The NOEC for growth rate and yield was determined to be at the lowest test concentration of 0.21 mg test item/L.

- Other observations - Table 2: 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.
After 24 hours, yellow precipitates in the three highest test concentrations were observed. After 48 hours, those precipitates could not be observed in the highest concentration, but in the two lower concentrations. This remained until test termination.
Results with reference substance (positive control):
Test results from September 2019: ErC50 value of 2.78 mg/L (nominal; 95% confidence limits: 2.69 – 2.87 mg/L) is in good agreement with the results of an international ring test with an ErC50 of 3.38 ± 1.30 mg/L.
Reported statistics and error estimates:
Since the measured concentrations were within a range of 80 – 120% of the nominal concentrations at test start, but did not remain stable throughout the test period of 72 hours biological evaluations were based on geometric mean measured concentrations of the test item.
Biological data were statistically analysed to determine EC50 and EC10 values together with 95 % confidence intervals, where possible. The EyC50 was determined using the 3-parametric non-linear regression procedure. The ErC50 was calculated based on linear regression procedures (Probit).
The NOEC for yield was determined by the Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm (significance level of 0.05, one-sided smaller).
The NOEC for growth rate was determined by a Williams Multiple Sequential t-test Procedure (significance level of 0.05, one-sided smaller).
The computer program ToxRat® was used for statistical evaluations.

Table 1:       Effective concentrations based on geometric mean measured concentrations of the test item for the exposure ofR. subcapitatafor 72 hours.

Parameter

 

EC50

EC20

EC10

LOEC

NOEC

 

 

[mg test item/L]

Growth rate

Value

1.36

0.67

0.47

0.61

0.21

 

Lower 95 %-cl

1.03

0.35

0.18

-

-

 

Upper 95 %-cl

1.82

0.91

0.69

-

-

Yield

Value

0.73

0.54

0.46

0.61

0.21

 

Lower 95 %-cl

0.39

0.34

0.29

-

-

 

Upper 95 %-cl

1.34

0.84

0.71

-

-


 

Table 2:       Microscopic observation of the algae cells at test initiation and termination of the 72 h growth test.

Date

Geometric mean
measured conc.
[mg test item/L]

Observations

Test start

Normal appearance of inoculum culture for all test concentrations.

Test end,
72 h

Control

Normal appearance of algal cells.

0.21

Normal appearance of algal cells.

0.61

Normal appearance of algal cells.

1.40

Reduced appearance of normal algal cells. Appearance of yellow precipitates.

3.44

Reduced appearance of normal algal cells. Appearance of yellow precipitates.

26.3

Mainly cell debris.

Validity criteria fulfilled:
yes
Conclusions:
Based on test item concentrations calculated from measured Pt concentrations, the 72 hour ErC50 and the ErC10 values (growth rate) were 1.35 and 0.47 mg test item/L, respectively. The 72 hour EyC50 and the EyC10 values (yield) were 0.73 and 0.46 mg test item/L, respectively. The NOEC for growth rate and yield was determined to be at the lowest test concentration of 0.21 mg test item/L.
Executive summary:

A 72-h algal growth inhibition study was conducted for Di(tetraethylammonium)hexahydroxoplatinate(IV) (aqueous solution). The study is reliable without restrictions, being GLP-compliant and having followed standard test guidelines (OECD 201).

There were concentration-dependent effects on the growth of the green algae over the range of the tested concentrations.

The respective 72-hour ErC50for the inhibition of growth rate was determined to be at 1.36 mg test item/L. For yield, an EyC50of 0.73 mg test item/L was determined. The EC10values for growth rate and yield were at 0.47 and 0.46 mg test item/L, respectively.

The NOEC for growth rate and yield was determined to be at the lowest test concentration of 0.21 mg test item/L.

Description of key information

Based on test item concentrations calculated from measured Pt concentrations, the 72 hour ErC50 and the ErC10 values (growth rate) were1.35 and 0.47 mg test item/L, respectively. The 72 hour EyC50 and the EyC10 values (yield) were 0.73 and 0.46 mg test item/L, respectively.The NOEC for growth rate and yield was determined to be at the lowest test concentration of 0.21 mg test item/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
1.35 mg/L
EC10 or NOEC for freshwater algae:
0.47 mg/L

Additional information

An algal toxicity study with Raphidocelis subcapitata (Pseudokirchneriella subcapitata) following OECD test guideline 201 is available for this substance (Schlich 2020). Five test concentrations were included, with four replicates of each and eight replicates of the control. Platinum concentrations were analysed in the test solutions at the start and end of the test, by ICP-OES. Both filtered and unfiltered test solutions were analysed for platinum.The measured concentrations in the unfiltered test solutions were used for the evaluation of effects.

At test initiation, the measured concentrations ranged from 89.3 –112% of nominal concentrations and therefore were within a range of 80 – 120% of the nominal concentrations. However, the measured concentrations did not remain within ± 20% throughout the test period of 72 hours (% of initial: 19.2 – 85.4%; % of nominal: 18.6 – 95.2%) and the evaluation of biological results was based on the geometric mean measured concentrations of 0.21, 0.61, 1.40, 3.44 and 26.3 mg test item/L.

Based on test item concentrations calculated from measured Pt concentrations, the 72 hour ErC50 and the ErC10 values (growth rate) were1.35 and 0.47 mg test item/L, respectively. The 72 hour EyC50 and the EyC10 values (yield) were 0.73 and 0.46mg test item/L, respectively. The NOEC for growth rate and yield was determined to be at the lowest test concentration of 0.21 mg test item/L.