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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:
The study was conducted between 4 February and 6 March 2013.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The filter containing the undissolved residue was also retained for possible analyses.
Frequency: at t=0 h, t=24 h and t=72 h
Volume: 3 ml
Storage: Samples were stored in a freezer until analysis.
On the day of analysis, the samples were defrosted at room temperature. The test samples were diluted in a 3:1 (v:v) ratio with acetonitrile and analysed. If necessary, the samples were further diluted with 25/75 (v/v) acetonitrile/M2-medium to obtain concentrations within the calibration range.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

For the estimation of the algal growth, defined volumes of the algal suspensions from replicates and from the blanks were sampled at the beginning and after 24, 48 and 72 hours.
Vehicle:
no
Details on test solutions:
The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (e.g. film of the test substance on the water surface). No correction was made for the purity/composition of the test substance.
The test substance was a white crystalline powder with a purity of 100.0% and completely soluble in test medium at the highest concentration prepared for the final test.
Preparation of test solutions started with the highest concentration of 100 mg/l. Two days of magnetic stirring were applied to ensure reaching maximum dissolution in test medium. The resulting dispersions obtained in the combined limit/range-finding and final test were filtered through a 0.45 μm membrane filter (Whatman, RC55) to remove a limited fraction of undissolved material. The resulting clear and colourless filtrate was used to prepare the lower test concentrations by subsequent dilution in test medium. The final test solutions were all clear and colourless.
After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
The test was carried out using Pseudokirchnerella subcapitata, strain: NIVA CHL 1k, in-house laboratory culture.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg/l as CaCO3
Test temperature:
Temperature was maintained at 22.1 and 23.1°C throughout the test.
pH:
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. It was:
8.2-8.3 at test start and
8.1 to 8.3 at test end.
Nominal and measured concentrations:
Based on the results of the range-finding test, the following test concentrations were assigned to the definitive test:
Nominal test concentrations: 1.0, 3.2, 10, 32 and 100 mg/I.
Concentrations measured at t=0: 0.989, 3.10, 9.64, 31.6, 97.3, 101 mg/l.
Time Weighted Average exposure concentrations of the test item: 0.38, 1.1, 3.5, 23 and 93 mg/l, and a control.
Details on test conditions:
TEST SYSTEM
- Test vessel:
100 mL all-glass flasks were used.
The control group was maintained under identical conditions but not exposed to the test material.

- Initial cells density:
Pre-culture conditions gave an algal suspension in log phase growth, 1 x 104 cells/ml.

- No. of vessels per concentration (replicates): 3

- No. of vessels per control (replicates): 6

Additionally, the following were used:
1 extra replicate of each test group for sampling purposes,
2 replicates of the highest test concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes
The culture medium was prepared using analytical grade salts dissolved in sterile purified water.

- Photoperiod:
The flasks were incubated under continuous illumination at a measured light intensity within the range of 103 to 97 μE.m-2.s-1.

- Determination of cell concentrations:
Samples were taken at the beginning and after 24, 24 and 72 hours and the cell density was determined as described above.

- The water temperature was recorded continuously. The pH was recorded in each test concentration and the control at the start and at the end of the test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
31 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval ranging from 10 to 97 mg/l.
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.8 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval ranging from 1.5 to 16 mg/l.
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
At the start of the test, the measured levels of the test substance ranged between 96 and 101% of their nominal values, and after 72 h they ranged between below the LOQ and 99% of their nominal values.
Results with reference substance (positive control):
- Results with reference substance: valid
- Dose-response test: yes; tested concentrations: 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l
- The main test: 72h-EC50 = 0.97 mg/L (95% C.I.: 0.62-1.5 mg/l)
Reported statistics and error estimates:
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Bonferroni t-test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test substance.
Validity criteria fulfilled:
yes
Remarks:
Cell Density Increase in Control Cultures: 180-fold increase within 72 hours; Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 9 %; Coefficient of Variation of Average Growth between Control Replicates: 2 %
Conclusions:
Algae toxicity test (OECD TG 201): The ErC50, EC10 and NOEC were 31.0, 4.8 and 1.1 mg/l, respectively.
Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchnerella subcapitata. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, Pseudokirchnerella subcapitata was exposed to solutions of the test material at Time Weighted Average exposure concentrations: 0.38, 1.1, 3.5, 23 and 93 mg/l (3 replicate flasks per concentration), and a control for 72 hours, under constant illumination and shaking at a temperature of 22.1 and 23.1°C. At the start of the test, the measured levels of the test substance ranged between 96 and 101% of their nominal values, and after 72 h they ranged between below the LOQ and 99% of their nominal values. The mean measured values were used for the effect assessment. The ErC50, EC10 and NOEC were 31.0, 4.8 and 1.1 mg/l, respectively.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read-across was applied
Justification for type of information:
Read across from Piperonal was applied. The respective read-across document can be found in the endpoint summary, the accompanying files are also attached in the endpoint summary.
Reason / purpose:
read-across source
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
31 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval ranging from 10 to 97 mg/l.
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.8 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval ranging from 1.5 to 16 mg/l.
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Validity criteria fulfilled:
yes
Remarks:
Read acros documentation is available in accordance with Annex XI
Conclusions:
The Algae EC50 and EC10 for Veratraldehyde are 31.8 and 4.8, respectively

Description of key information

Read across from Piperonal was applied. The full read across justification can be found in the Endpoint Summary. Below is the summary of the study performed with Piperonal.

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchnerella subcapitata. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, Pseudokirchnerella subcapitata was exposed to solutions of the test material at Time Weighted Average exposure concentrations: 0.38, 1.1, 3.5, 23 and 93 mg/l (3 replicate flasks per concentration), and a control for 72 hours, under constant illumination and shaking at a temperature of 22.1 and 23.1°C. At the start of the test, the measured levels of the test substance ranged between 96 and 101% of their nominal values, and after 72 h they ranged between below the LOQ and 99% of their nominal values. The mean measured values were used for the effect assessment. The ErC50, EC10 and NOEC were 31.0, 4.8 and 1.1 mg/l, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
31 mg/L
EC10 or NOEC for freshwater algae:
4.8 mg/L

Additional information