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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:
11 May 2010 - 22 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP test according to OECD guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples from control and the test item concentration were taken at the start end the end of the exposure period and analysed to determine the actual test item concentrations in comparison to the nominally applied concentrations.
Before analysis, the samples were stored in a refrigerator in polyethylene bottles.
Vehicle:
no
Details on test solutions:
A stock solution (water-accommodated fraction, WAF) at a loading rate of nominal 137 mg test item/L (=100 mg Bi/L) was prepared by dissolving 137.4 mg of the test item in 1004 mL of test medium. This stock solution was agitated for seven days by using a horizontal laboratory shaker at ambient temperature in the dark. The shaker was set to a frequency of 100 rpm to keep the test item in permanent suspension according to OECD guideline. After that, the stock solution was filtered (glass fibre filter, pore size 0.4 µm). The filtrate was used to prepare the desired test item concentrations.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
common name: green algae (SAG 61.81)
breeder: Georg-August-Universität Göttingen

To adjust the algae to the test conditions a pre-culture was inoculated by an agar stock culture and incubated under test conditions:
pH-value of the algal medium: 5.9
Amount of liquid stock culture per pre-culture vessel: 100 mL
Pre-culture vessels: 300 mL Erlenmeyer flasks
Number of replicates: 2
Light: light/dark (24/0 h); fluorescent tubes of universal white type
Light intensity: 60 – 120 µE/m2 * s
Shaker: 100 ± 5 oscillations/min
Temperature in the test room: 21 - 24°C, controlled at ± 2°C
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.3-24.0 °C
pH:
5.9-6.1
Nominal and measured concentrations:
nominal added concentrations: control, 1, 3, 10, 30 and 100% of water-accommodated fraction (100 mg Bi/L), corresponding to measured dissolved Bi concentrations of <0.003, <0.003, <0.003, 0.006, 0.02 and 0.046 mg Bi/L (calculated as geometric mean of measured concentrations at start and end of exposure).
Details on test conditions:
-Age of the pre-culture: 4 days
-Number of cells per mL in the pre-culture before inoculating the test solution: 277 x 10^4
-Number of cells per mL test solution at the beginning of the test: 0.5 x 10^4
-Number of replicates per test item concentration: 3
-Number of replicates in the control: 6
-Number of replicates for stability check (limit concentration without algae): 2
-Light cycle: 24/0 hours light/dark
-Types of light: fluorescent tubes of universal white type (Osram Lumilux 58W/865)
-Light intensity: mean value of six measurements: 94.6 µE/m2s (required 60 to 120 µE*m2s1 equivalent to 4440 – 8880 lx)
-Shaker: 100 ± 5 oscillations/min; the test vessels were placed randomly on the shaker
-Counting of algae: daily
-Effect parameter: yield and growth rate
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 137 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 137 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 137 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 137 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Effect parameter, EC50 and NOEC for yield and growth rate, values based on mean measured dissolved Bismut concentrations are all higher than 0.046 mg Bi/L.
Results with reference substance (positive control):
results of the reference test performed in january 2010: 72h-EC50 for growth rate is 0.85 mg/L. Based on an international ring test (ISO 2004) mentioned in OECD guideline 201, the 72h-EC50 value for potassium dichromate obtained from different laboratories should be 1.19 mg/L with a standard deviation of 0.27 mg/L. The experimentally obtained value of 0.85 mg/L is within the historical range of the results of the testing laboratory (mean EC50 value = 1.15 +- 0.42 mg/L) and within the upper (2.54 mg/L) and lower (0.44 mg/L) warning limit. The warning limits were calculated as two standard deviations from the historical mean of the EC50. Furthermore, the EC50 value is within the refined range of 1.10 +- 0.48 mg/L, recommended based on an international ring test (pattard et al. 2009). Therefore, the results of this reference test are acceptable and the test conditions are reliable.
Reported statistics and error estimates:
The data were evaluated for homogeneity of variances by Levene's Test, and by Williams' test to calculate whether there were significant differences between the growth of algae in the controls and the algae exposed to the test item concentrations.

 

Treatment

[% of WAF]

 

%Inhibition of

Yield

(72 h)

 

Significant difference from control (p≤0.05)

 

%Inhibition of

Growth Rate

(72 h)

 

Significant difference from control (p≤0.05)

Control  

0.0

No 0.0 No
  

-13.5

No  -2.3 No
3   

-12

No -2 No
10   

-10.6

No -1.7 No
30   

4.4

No 0.8 No
100   

2.0

No 0.4 No
Validity criteria fulfilled:
yes
Conclusions:
GLP test according to OECD 201 guideline. The 72h-EC50 for growth rate and biomass yield of Pseudokirchneriella subcapitata are both > 100 mg Bi/L.
Executive summary:

A GLP test conducted according to OECD 201 guideline. The 72h-EC50 for growth rate and biomass yield of Pseudokirchneriella subcapitata are both > 100 mg Bi/L.

Description of key information

The 72h-EC50 for growth rate is > 137 mg/L bismuth subnitrate.

Key value for chemical safety assessment

EC50 for freshwater algae:
137 mg/L

Additional information

A GLP test was conducted according to OECD 201 guideline using bismuth subnitrate.

The 72h-EC50 for growth rate and biomass yield of Pseudokirchneriella subcapitata are both > 100 mg Bi/L (137 mg/L test item).