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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25.01.2010 - 28.01.2010
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)
Remarks:
2009-03-04
Analytical monitoring:
yes
Details on sampling:
Chemical analysis of test concentrations at start and end of exposure
- at the beginning of the test, duplicate samples of each of the test solutions and the control were taken.
- at the end of the test, immediately after determination of the biological and physico-chemical parameters, the test solutions of the replicates of each treatment were mixed. Duplicate samples were taken from the control, each of the test solutions and from the test vessels without algae with the highest test item concentration.
Vehicle:
no
Details on test solutions:
The algal medium (Kuhl and Lorenzen, 1964) consists of trace elements and macronutrients in the following final concentrations:
KNO3: 1011.1 mg/L
NaH2PO4 * 1 H2O: 621.0 mg/L
Na2HPO4 * 2 H2O: 89.0 mg/L
MgSO4 * 7 H2O: 246.5 mg/L
CaCl2 * 2 H2O: 14.7 mg/L
FeSO4 * 7 H2O: 6.9 mg/L
Titriercomplex III: 9.3 mg/L
H3BO3: 0.061 mg/L
MnSO4 * 1 H2O: 0.169 mg/L
ZnSO4 * 7 H2O: 0.287 mg/L
CuSO4 * 5 H2O: 0.00249 mg/L
(NH4)6Mo7O24 * 4 H2O: 0.01235 mg/L
100 mg of the test item was mixed into 1,000 mL algal medium in a 1,000 mL measuring flask, resulting in a nominal concentration of 100 mg test item/L. The solution was shaken for 3 minutes at room temperature.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata (SAG 61.81) was supplied by Georg-August-Universität Göttingen, D-37073 Göttingen, Germany (Date of receipt: December 18, 2009)
Amount of liquid stock culture per pre-culture vessel: 100 mL
Pre-culture vessels: 300 mL Erlenmeyer flasks
Number of cells per mL in the pre-culture before inoculating the test solution: 88.5E4
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:
22.9 +/- 0.6 degrees Celcius (mean +/- SD)
pH:
start of test: 5.8-5.9 (all test concentrations)
end of test: 6.0-6.1 (all test concentrations)
Dissolved oxygen:
not relevant for algal testing
Nominal and measured concentrations:
nominal: 0, 1.0, 2.2, 10, 31.6 and 100 mg test item/L
dissolved Ba at t=0: below detection limit, 0.139, 0.213, 0.65, 1.92, 1.33 mg Ba/L; expressed as BaCl2: below detection limit, 0.247, 0.376, 1.15, 3.42, 2.37 mg test item/L
dissolved Ba at t=72h: below detection limit, 0.054, 0.111, 0.359, 0.839, 0.999/1.12 (double determination) mg Ba/L; expressed as BaCl2: below detection limit, 0.096, 0.20, 0.64, 1.49, 1.78/1.99 (double determination) mg test item/L
total Ba at t=0: below detection limit, 0.312, 0.211, 3.65, 2.18, 26.32 mg Ba/L; expressed as BaCl2: below detection limit, 0.555, 0.376, 6.49, 3.88, 46.83 mg test item/L
total Ba at t=72h: below detection limit, 0.196, 0.271, 3.21, 8.67, 34.31/30.10 (double determination) mg Ba/L; expressed as BaCl2: below detection limit, 0.348, 0.481, 5.70, 15.43, 61.05/53.56 (double determination) mg test item/L
Details on test conditions:
Test units: 300 mL Erlenmeyer flasks
Volume of test solution per test vessel: 100 ml
No. of test concentrations (incl. control): 6
No. of replicates in the control: 6
No. of replicats per test concentration: 3
Test medium: Algal medium (Kuhl & Lorenzen, 1964)
The concentration of the algal cells in the test solutions at the beginning of exposure was approximately 0.5 * 1E4 cells per mL.
Endpoints: EC50, NOEC, LOEC
Biological parameters: Inhibition of growth in relation to control (growth rate & yield)
Light regime: permanent at 93.6 µE/m2 * s (mean; required is 60-120 µE/m2 * s).
Types of light: fluorescent tubes of universal white type (Osram Lumilux 58W/865)
For chemical analysis at the end of the test, two additional test vessels without algae were prepared at the highest test item concentration in order to assess test item stability during exposure without the presence of algae (stability samples).
Determination of cell number: THOMA Counting Chamber with Microscope Ortholux II, Ernst Leitz KG, Wetzlar, Germany; Multiple Reader Tecan ULTRA, Tecan Deutschland GmbH, Crailsheim, Germany

After 24, 48 and 72 hours, the cell numbers were determined by measuring the fluorescence intensity in 4 samples of 200 µL of test solution per replicate using a fluorometer (Multiple Reader Tecan ULTRA). The results [RFU, relative fluorescence units] were converted into biomass concentration [cells/mL] using a calibration line. A dilution series of the pre-culture was used to determine the calibration line by comparison of cell numbers (counted using a microscope) and fluorescence intensity (measured using a fluorometer) in consideration of background fluorescence of the blank algal medium.
Reference substance (positive control):
yes
Remarks:
K2Cr2O7 (potassium dichromate)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
Ba
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 1.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
Ba
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 30.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
total Ba
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 30.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
total Ba
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
barium dichloride dihydrate
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
barium dichloride dihydrate
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 85.3 mg/L
Nominal / measured:
nominal
Conc. based on:
other: barium dichloride
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 85.3 mg/L
Nominal / measured:
nominal
Conc. based on:
other: barium dichloride
Basis for effect:
growth rate
Details on results:
No statistically significant differences (Table 1) were found between the control and the test item concentrations up to 100 mg test item/L. The EC50 (72 h) based on nominal concentrations was therefore determined to be higher than the highest concentration of 100 mg test item/L. The results of the study demonstrate that the no-observed effect concentration for Pseudokirchneriella subcapitata for the parameters yield and growth rate is ≥ 100 mg test item/L (nominal concentration).

Samples from the test solutions were analysed to determine the actual test item concentration in comparison to the nominally applied concentration. Measured concentrations of dissolved test item in 0.45 µm-filtered test solutions were between 2 and 25% of the nominal concentrations. Analysis of total Barium in non-filtered, acidified test solutions resulted in recoveries of 12 and 61% of the nominal concentrations.
Results with reference substance (positive control):
The reference item potassium dichromate (K2Cr2O7) is tested at least twice a year in order to determine the sensitivity of the organisms from the culture, and to demonstrate that the test conditions are appropriate. Results of the reference test performed in January 2010:
- EbC50: 0.23 mg/L
- ErC50: 0.85 mg/L
Based on an international ring test (ISO 2004) mentioned in OECD guideline 201, the ErC50 (72h)-values for potassium dichromate obtained from different laboratories should be 1.19 mg/L with a standard deviation of 0.27 mg/L.
The EC50 value for the toxic reference item, potassium dichromate, was determined as 0.85 mg/L. This value is within the historical range of the results of our 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 (all calculated using the log concentration; according to Environment Canada 2005). 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:
Range-to-standard-deviation-ratio Test on Normal Distribution; Cochran´s Test on Variance Homogeneity; One-way Analysis of Variance (ANOVA)
The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.

Summary of tests results based on statistical evaluation of biological results and nominal concentrations of the test item.

 Treatment [mg test item/L]

 %Inhibition of Yield (72 h)  Significant difference to control (p≤0.05)  %Inhibition of Growth Rate (72 h) Significant difference to control (p≤0.05
 Control 0.0  No  0.0  No 
 1.0 0.6    No  0.1   No
 3.2 6.9   No    1.3  No  
 10.0 17.3    No  4.6    No 
 31.6 4.6   No   0.9   No  
 100 5.9   No   1.1 

 No  

 EC50 > 100.0 > 100.0    >100.0    
 NOEC  >= 100.0    >= 100.0    
 LOEC > 100.0     > 100.0    

Validiy of test:

- Mean biomass increase in the control cultures: 252.0 (required: factor of at least 16 within the 72h test period

- Mean coefficient of variation for section-by-section specific growth rates in the control cultures: 22.8% (required: up to 35%)

- Coefficient of variation of average specific growth rates during test period in replicate control cultures: 1.4% (required: up to 7%)

Validity criteria fulfilled:
yes
Conclusions:
No statistically significant differences were found between the control and any test item concentration up to and including 100 mg barium dichloride dihydrate/L.

Hence the recovery rates (RR) are < 80% the results are given as actual (geometric mean measured) concentrations.

The results of the study demonstrate that the EC50 for Pseudokirchneriella subcapitata is >1.74 mg BaCl2 (1.15 mg Ba/L). The no-observed effect concentration (NOEC) is ≥ 1.74 mg BaCl2/L (1.15 mg Ba/L) based on dissolved barium concentrations.

However, based on total barium concentrations the results of the study demonstrate that the EC50 for Pseudokirchneriella subcapitata is > 45.6 mg BaCl2/L (30.1 mg Ba/L). The no-observed effect concentration (NOEC) is ≥ 45.6 mg BaCl2/L (30.1 mg Ba/L).

These data can be used for hazard and risk assessment purposes (classification; PNEC-derivation)
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The justification for read across is attached in IUCLID Section 13.
Reason / purpose:
read-across source
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 d
Dose descriptor:
NOEC
Effect conc.:
>= 1.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 d
Dose descriptor:
EC50
Effect conc.:
> 2.19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
Ba(NO3)2
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 2.19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
Ba(NO3)2
Basis for effect:
growth rate

Description of key information

A read across approach was followed to cover the endpoint for barium nitrate. The study from Egeler and Kiefer (2010), performed with barium chloride, yielded a growth rate-based 72-h EC50 for Pseudokirchneriella subcapitata of > 30.1 mg Ba/L (total Ba) or > 1.15 mg Badiss/L (dissolved Ba). The no-observed effect concentration (NOEC) was >= 30.1 mg Ba/L (total Ba) or >= 1.15 mg Badiss/L (dissolved Ba). No adverse effects were observed up to and including the highest test concentration (nominal: 100 mg hydrated BaCl2/L).

Key value for chemical safety assessment

Additional information

A single relevant and reliable study was identified for the read across substance barium chloride (Egeler and Kiefer, 2010). An algal growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata yielded a growth rate-based 72-h EC50 and NOEC of > 100 and >= 100 mg/L, expressed as test item concentration (hydrated barium chloride). When expressed as measured total barium the EC50 and NOEC were > 30.1 and >= 30.1 mg Ba/L, respectively (geometric mean measured concentration). When expressed as measured dissolved barium the EC50 and NOEC were > 1.15 and >= 1.15 mg Badiss/L, respectively. Most of the added barium precipitated from solution. These results were recalculated to barium dinitrate based on the barium content of both substances (i.e. EC50 and NOEC were > 2.19 and >= 2.19 mg barium trinitrate/L).