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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:
72 hours
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes
Specific details on test material used for the study:
Chemical Name Titanium citrate
KIT Code No. K-7042
Batch/Lot No. 115829/70
Appearance Clear, particulate free liquid
Purity (wt.%) 54.7
Storage conditions Room temperature (in sealed container)
Expiry date December, 2019
Analytical monitoring:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 183 mg of the test item (purity: 54.7%) was added to 1.2 L volume of glass bottle and 1000 mL dilution water was added up to give the 100 mg/L of test solution. 300 mL of 100 mg/L of test solution was mixed with 700 mL of dilution water to prepare 30 mg/L of test solution firstly and then test solutions of 0.028-9.4 mg/L were prepared by serial dilution method.
- Controls: Dilution water (algae culture medium)
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
Pseudokirchneriella subcapitata (Strain No. ATCC 22662)
Source: Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology (KIT), originally purchased from American Type Culture Collection (ATCC), Manassas, VA, USA on May 2, 2017.

ACCLIMATION
Three days before the test, sterile OECD nutrient medium (OECD, 2006) was inoculated with about 5.0×103 cells/mL from a stock culture and incubated in an incubator under continuous illumination (shanking rate: about 100 times/min, and light intensity: 60-120 μE/m2/s at 21- 24 °C).

Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Test temperature:
22.1 ± 0.0 ºC
pH:
pH: 6.34-8.87
Details on test conditions:
TEST SYSTEM
- Test vessel:250 mL volume Erlenmeyer flasks
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Light intensity and quality: 4642-4991 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples (50 μL) were taken at 24, 48 and 72 hours and the cell densities were determined by an optical microscope.

TEST CONCENTRATIONS
- The range-finding test was conducted at the nominal concentrations of 0.01, 0.1, 1, 10 and 100 mg/L. 5% inhibition (of growth rate and yield) was observed in 0.02-0.04 mg/L and 75% inhibition (of growth rate and yield) was observed in 0.7-27 mg/L by statistical analysis using rangefinding
test result. Therefore, the definitive test was performed with a control and a nominal concentration of 0.028, 0.088, 0.28, 0.91, 2.9, 9.4, and 30 mg/L in three replicates.

Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.043 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape:
- Colour differences:
- Flocculation:
- Adherence to test vessels:
- Aggregation of algal cells:
- Other:
- Any stimulation of growth found in any treatment:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium:
Reported statistics and error estimates:
Yield and average specific growth rate were analyzed by CETIS Version 1.8.7.15 (Tidepool Scientific Software, USA). Linear Interpolation methods were applied to obtain the effective concentrations, EC10 (and/or EC20) and EC50 for each approach. LOECs and NOECs were determined using the Dunnett Multiple Comparison Test.

Table 4. Cell densities ofPseudokirchneriella subcapitataduring the test period

Nominal concentration (mg/L)

Mean measured concentration (mg/L)

Replicate No.

0-hour1

24-hour

48-hour

72-hour

Control

NA

R1

5.0 × 103

2.0 × 104

9.8 × 104

3.5 × 105

R2

5.0 × 103

2.0 × 104

1.0 × 105

3.3 × 105

R3

5.0 × 103

2.2 × 104

1.1 × 105

3.4 × 105

mean

5.0 × 103

2.1 × 104

1.0 × 105

3.4 × 105

SD

0

1.2 × 103

5.0 × 103

1.1 × 104

0.028

0.043

R1

5.0 × 103

2.2 × 104

9.5 × 104

3.2 × 105

R2

5.0 × 103

2.0 × 104

1.1 × 105

3.2 × 105

R3

5.0 × 103

2.0 × 104

9.8 × 104

3.3 × 105

mean

5.0 × 103

2.1 × 104

1.0 × 105

3.2 × 105

SD

0

1.2 × 103

7.4 × 103

3.5 × 103

0.088

0.10

R1

5.0 × 103

1.8 × 104

6.4 × 104

1.1 × 105

R2

5.0 × 103

1.7 × 104

6.2 × 104

1.1 × 105

R3

5.0 × 103

1.9 × 104

6.4 × 104

1.1 × 105

mean

5.0 × 103

1.8 × 104

6.3 × 104

1.1 × 105

SD

0

1.0 × 103

1.2 × 103

4.4 × 103

0.28

0.25

R1

5.0 × 103

1.6 × 104

 4.2 × 104

5.9 × 104

R2

5.0 × 103

1.6 × 104

4.1 × 104

5.4 × 104

R3

5.0 × 103

1.7 × 104

4.2 × 104

5.3 × 104

mean

5.0 × 103

1.6 × 104

4.2× 104

5.5 × 104

SD

0

5.8 × 102

5.8 × 102

3.2 × 103

0.91

0.77

R1

5.0 × 103

1.6 × 104

2.7 × 104

3.3 × 104

R2

5.0 × 103

1.6 × 104

2.7 × 104

2.9 × 104

R3

5.0 × 103

1.7 × 104

2.3 × 104

2.8 × 104

mean

5.0 × 103

1.6 × 104

2.6 × 104

3.0 × 104

SD

0

5.8 × 102

2.3 × 103

2.6 × 103

2.9

2.6

R1

5.0 × 103

1.3 × 104

2.2 × 104

2.1 × 104

R2

5.0 × 103

1.4 × 104

2.4 × 104

2.1 × 104

R3

5.0 × 103

1.4 × 104

2.2 × 104

2.3 × 104

mean

5.0 × 103

1.4 × 104

2.3 × 104

2.2 × 104

SD

0

5.8 × 102

1.2 × 103

1.2 × 103

9.4

8.2

R1

5.0 × 103

1.3 × 104

1.7 × 104

1.6 × 104

R2

5.0 × 103

1.3 × 104

1.7 × 104

1.9 × 104

R3

5.0 × 103

1.2 × 104

1.7 × 104

1.5 × 104

mean

5.0 × 103

1.3 × 104

1.7 × 104

1.7 × 104

SD

0

5.8 × 102

0

2.1 × 103

30

26

R1

5.0 × 103

5.0 × 103

1.4 × 104

1.5 × 104

R2

5.0 × 103

7.0 × 103

1.5 × 104

1.3 × 104

R3

5.0 × 103

5.0 × 103

1.4 × 104

1.2 × 104

mean

5.0 × 103

5.7 × 103

1.4 × 104

1.3 × 104

SD

0

1.2 × 103

5.8 × 102

1.5 × 103

 

1) Nominally inoculated value

NA: Not available

SD: Standard deviation

 

Validity criteria fulfilled:
yes
Conclusions:
The 72-hr EC50 values (95% confidence limits) expressed in terms of the geometric mean measured concentration of Titanium citrate to Pseudokirchneriella subcapitata were 0.48 mg/L (0.40-0.56 mg/L) for average growth rate and 0.083 mg/L (0.080-0.086 mg/L) for yield. The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) values expressed in terms of the mean measured concentration were 0.043 mg/L, <0.043 mg/L and 0.010 mg/L, 0.043 mg/L for average growth rate and yield, respectively.
Executive summary:

It is likely that the inhibition in growth was due to the chelating properties of the substance with essential anions (sulphates, phosphates, carbonates etc) associating with the titanium at the elevated pH leading to precipitation of the titanium salts.

Microscopic analysis of the algal cells showed no indication of damage or stress.

Citrate salts are not considered toxic to plants and are present naturally in plant cells at high concentrations.

Titanium itself is not considered hazardous to aquatic organisms.

Description of key information

It is likely that the inhibition in growth was due to the chelating properties of the substance with essential anions (sulphates, phosphates, carbonates etc) associating with the titanium at the elevated pH leading to precipitation of the titanium salts.

Microscopic analysis of the algal cells showed no indication of damage or stress.

Citrate salts are not considered toxic to plants and are present naturally in plant cells at high concentrations.

Titanium itself is not considered hazardous to aquatic organisms.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.48 mg/L

Additional information