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EC number: 243-797-2 | CAS number: 20407-84-5
- Life Cycle description
- Uses advised against
- Endpoint summary
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- Endpoint summary
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
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- Biotransformation and kinetics
- Additional ecotoxological information
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- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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Toxicity to aquatic algae and cyanobacteria
Administrative data
Link to relevant study record(s)
- Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- The study was conducted in accordance with international guidelines (OECD TG 201) and in accordance with GLP. The study design was modified in accordance with OECD 23 (2000) and ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b. The study met all relevant validity criteria.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Alga, Growth Inhibition Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.3 (Algal Inhibition test)
- Deviations:
- no
- Principles of method if other than guideline:
- In addition to compliance with OECD TG 202 and EU Method C.3., modifications to the testing approach were conducted in accordance with OECD 23 (2000) and ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b. The aqueous half-life of the test item was determined to <48h in aqueous testing medium, so it was concluded that the test should be conducted on the transformation products rather than the parent substance (see "Details on Test Solutions"); as environmental exposure will be to the transformation products.
- GLP compliance:
- yes (incl. QA statement)
- Analytical monitoring:
- yes
- Details on sampling:
- Separate replicates for the test item analysis at the beginning of the exposure were prepared without algae. For the test item analysis after 72 hours, separate replicates were prepared with algae at the beginning of the exposure and incubated under test conditions.
- Vehicle:
- no
- Details on test solutions:
- A saturated solution with a nominal loading of 10.0 mg/L of the test item was prepared with dilution water two days before the start of the exposure phase. An appropriate amount of the test item was weighed out and transferred with dilution water into a 1 L glass flask with a side-arm, which was nearly completely filled. The stock solution was stirred at approximately 1100 rpm for 48 hours at 30 °C. The stock solution had nearly no headspace during the stirring period to reduce losses of the test item by evaporation. After completion of stirring, the stock solution was allowed to stand for 1 hour and cool down at room temperature for separation of undissolved test item. The saturated solution was removed via side-arm of the flask (from the approximate bottom of the glass flask). Due to the density of the test item, any undissolved material would be expected to float on the surface.
The saturated solution was checked for a Tyndall effect with a laser pointer and used as stock solution for preparation of further loading levels by diluting with dilution water. - Test organisms (species):
- Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
- Details on test organisms:
- Test Organism: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
Origin: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µEm-2s-1 for 24 hours per day.
Culture medium: Nutrient medium Z according to Lüttge et al. (1994) - Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 72 h
- Post exposure observation period:
- Not applicable
- Hardness:
- Not applicable.
- Test temperature:
- The test temperature ranged from 21.5 to 23.0 °C
- pH:
- Refer to Table 4.
- Dissolved oxygen:
- Not applicable
- Salinity:
- Not applicable
- Nominal and measured concentrations:
- 5 loading levels of the test item in a geometric series with a separation factor of 3.16, prepared by diluting the saturated solution with dilution water, were tested as follows: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 % of the saturated solution.
Measured concentrations of the test item are presented in Table 5. - Details on test conditions:
- Control: Six replicates (without test item) were tested under the same test conditions as the test vessels.
Test method: Static procedure
Performance of the test: A static test was carried out. With regard to the volatility of the test item, glass flasks without headspace were used to reduce losses of the test item.
Replicates: Three replicates per concentration and six for the control were tested. Separate replicates for each measuring time were prepared.
Test containers: Sterile Erlenmeyer flasks, volume: 119 mL, with aluminium tops with PFTE seals
Test volume: 119 mL
Dilution water: According to the guidelines
Preculture: A four days old preculture, prepared in dilution water, was used as inoculum.
Initial cell density:
Nominal: approximately 5 x 103 - 104 cells/mL
Current: : 6153 cells/mL
Application: Application was carried out by adding appropriate volumes of the test solutions to the replicate test vessels.
Incubation : The flasks were positioned randomly and repositioned daily.
Temperature: Nominal range: 21 - 24°C, controlled at ± 2°C
Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
Light intensity: 60 - 120 µE m-2 s-1
Light regime: 24 hours/day light
Light homogeneity: Within ± 15 % over incubation area - Reference substance (positive control):
- yes
- Remarks:
- Potassium dichromate
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- > 100 other: % saturated solution
- Nominal / measured:
- nominal
- Conc. based on:
- other: Transformation products
- Basis for effect:
- growth rate
- Remarks on result:
- other: Not applicable
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- >= 100 other: % saturated solution
- Nominal / measured:
- nominal
- Conc. based on:
- other: Transformation products
- Basis for effect:
- growth rate
- Remarks on result:
- other: Not applicable
- Details on results:
- Cell density values are presented in Table 1. Evaluations of growth rate inhibition and yield inhibition are given in Tables 2 and 3 (calculated based on cell density values).
Microscopic evaluation of the cells at the start of the incubation period and at test end revealed no morphological abnormalities in the test item concentration or control.
All effect values are given based on nominal loading levels of the test item. - Results with reference substance (positive control):
- The toxicity of potassium dichromate (Sigma, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2015-04-14 to 2015-04-17 (with headspace) (batch number MKBQ9179V, purity 100 %) and 2014-11-10 to 2014-11-13 (without headspace) (batch number MKBH9127V, purity 99.9 %), respectively. The results of testing with the reference item are presented in table 6.
- Reported statistics and error estimates:
- Statistical Evaluation: EC-values and statistical analyses: EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were estimated by sigmoidal dose-response regression.
NOEC, LOEC and Statistical Anlyses: The NOEC/LOEC was determined by calculation of statistically significant differences of growth rate and yield inhibition. As a standard, One Way Analysis of Variance (ANOVA) and Dunnett’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. Thea-value (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05.
Software: Calculations were carried out using software:
- Excel,Microsoft Corporation
- SigmaPlot,Spss Inc.
- GraphPad Prism,GraphPad Software Inc. - Validity criteria fulfilled:
- yes
- Conclusions:
- The alga growth inhibition test with Pseudokirchneriella subcapitata produced the following effect values (nominal loading levels): EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were estimated to be > 100% of the saturated solution concentration, respectively. The corresponding NOEC values were, in both cases, >= 100% of the saturated solution concentration.
- Executive summary:
The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD TG 201 (2011) and Commission Regulation (EC) No. 761/2009, C.3. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6153 cells/mL. A saturated solution with a nominal loading of 10.0 mg/L of the test item was prepared with dilution water. Five loading levels were tested in a geometrical series with a separation factor of 3.16: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 % of the saturated solution. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. All test concentrations of the test item and the control were analytically verified by LC-MS/MS at the start of the exposure (0 h) and at the end of the exposure (72 h). As the test design intended to test the effects of the transformation products of the test item, the measured concentrations of the test item at the start and at the end of the exposure were < LOQ, which was determined to be 15 µg/L. All effect values were based on the nominal loading levels of the test item and are related to the transformation products. All validity criteria of the test guideline were fulfilled (Increase of cell growth in the control cultures, mean coefficients of variation for specific growth rates and coefficient of variation of average specific growth rates). The alga growth inhibition test with Pseudokirchneriella subcapitata produced the following effect values (nominal loading levels): EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were estimated to be > 100% of the saturated solution concentration, respectively. The LOEC in terms of both growth rate and yield was > 100% of the saturated solution concentration and the corresponding NOEC values were, in both cases, >= 100% of the saturated solution concentration.
Reference
Table 1. Cell Densities (Definitive Test)
Nominal loading levels of the saturated solution | Replicate | Cell Density [cells/mL] | |||
[%] | No. | 0 hours | 24 hours | 48 hours | 72 hours |
100 | 1 | 6153 | 24894 | 181838 | 527833 |
2 | 6153 | 24706 | 186862 | 553203 | |
3 | 6153 | 24536 | 182873 | 542980 | |
Mean | 6153 | 24712 | 183858 | 541339 | |
31.6 | 1 | 6153 | 20071 | 155590 | 493055 |
2 | 6153 | 21566 | 151074 | 507574 | |
3 | 6153 | 21852 | 148427 | 529181 | |
Mean | 6153 | 21163 | 151697 | 509937 | |
10 | 1 | 6153 | 20782 | 134052 | 497351 |
2 | 6153 | 21805 | 141879 | 461005 | |
3 | 6153 | 21372 | 127936 | 497978 | |
Mean | 6153 | 21320 | 134622 | 485445 | |
3.16 | 1 | 6153 | 19936 | 96181 | 418637 |
2 | 6153 | 23332 | 100183 | 446642 | |
3 | 6153 | 21099 | 100666 | 403365 | |
Mean | 6153 | 21456 | 99010 | 422881 | |
1 | 1 | 6153 | 21215 | 99292 | 432373 |
2 | 6153 | 20425 | 101964 | 401107 | |
3 | 6153 | 20729 | 89012 | 387905 | |
Mean | 6153 | 20790 | 96756 | 407128 | |
0.316 | 1 | 6153 | 23821 | 106060 | 448962 |
2 | 6153 | 24480 | 114452 | 468782 | |
3 | 6153 | 23100 | 104761 | 447990 | |
Mean | 6153 | 23800 | 108424 | 455245 | |
Control | 1 | 6153 | 22357 | 118723 | 456363 |
2 | 6153 | 24238 | 122486 | 457743 | |
3 | 6153 | 24568 | 118911 | 444321 | |
4 | 6153 | 22526 | 112752 | 404431 | |
5 | 6153 | 22134 | 103482 | 353942 | |
6 | 6153 | 22695 | 119739 | 386650 | |
Mean | 6153 | 23086 | 116016 | 417242 |
Table 2. Evaluation after 72 hours (Definitive Test)
Nominal loading levels of the saturated solution | Replicate | Growth Rate | Inhibition of Growth Rate | Yield | Inhibition of Yield | ||
[%] | No. | [d-1] | [%] | [cells/mL] | [%] | ||
100 | 1 | 1.48 | -6 | 521680 | -27 | ||
2 | 1.5 | -7 | 547050 | -33 | |||
3 | 1.49 | -6 | 536827 | -31 | |||
Mean | (+)* | 1.49 | -6 | (+)* | 535186 | -30 | |
31.6 | 1 | 1.46 | -4 | 486902 | -18 | ||
2 | 1.47 | -5 | 501421 | -22 | |||
3 | 1.49 | -6 | 523028 | -27 | |||
Mean | (+)* | 1.47 | -5 | (+)* | 503784 | -23 | |
10 | 1 | 1.46 | -4 | 491198 | -19 | ||
2 | 1.44 | -2 | 454852 | -11 | |||
3 | 1.47 | -4 | 491825 | -20 | |||
Mean | (+)* | 1.46 | -4 | (+)* | 479292 | -17 | |
3.16 | 1 | 1.41 | 0 | 412484 | 0 | ||
2 | 1.43 | -2 | 440489 | -7 | |||
3 | 1.39 | 1 | 397212 | 3 | |||
Mean | (-) | 1.41 | 0 | (-) | 416728 | -1 | |
1 | 1 | 1.42 | -1 | 426220 | -4 | ||
2 | 1.39 | 1 | 394954 | 4 | |||
3 | 1.38 | 2 | 381752 | 7 | |||
Mean | (-) | 1.4 | 1 | (-) | 400975 | 2 | |
0.316 | 1 | 1.43 | -2 | 442809 | -8 | ||
2 | 1.44 | -3 | 462629 | -13 | |||
3 | 1.43 | -2 | 441837 | -7 | |||
Mean | (-) | 1.44 | -2 | (-) | 449092 | -9 | |
Control | 1 | 1.44 | 450210 | ||||
2 | 1.44 | 451590 | |||||
3 | 1.43 | 438168 | |||||
4 | 1.4 | 398278 | |||||
5 | 1.35 | 347789 | |||||
6 | 1.38 | 380497 | |||||
Mean | 1.4 | 411089 |
* Statistically significant due to increase of growth
Negative inhibition indicates increase in growth
Table 3. Section-By-Section and Average Specific Growth Rate of the Control Group (0 -72h) (Definitive Test)
Replicate No. | Specific Growth Rate [d-1] | Mean | SD | CV | Mean CV [%] | |||
section-by-section | (0 - 72 hours) | ± | [%] | |||||
0 - 24 hours | 24 - 48 hours | 48 - 72 hours | ||||||
Control | 1 | 1.29 | 1.67 | 1.35 | 1.44 | 0.205 | 14.3 | 13.2 |
2 | 1.37 | 1.62 | 1.32 | 1.44 | 0.161 | 11.2 | ||
3 | 1.39 | 1.58 | 1.32 | 1.43 | 0.134 | 9.42 | ||
4 | 1.3 | 1.61 | 1.28 | 1.4 | 0.187 | 13.4 | ||
5 | 1.28 | 1.54 | 1.23 | 1.35 | 0.168 | 12.4 | ||
6 | 1.31 | 1.66 | 1.17 | 1.38 | 0.254 | 18.4 | ||
Mean | 1.4 | |||||||
SD ± | 0.03 | |||||||
CV [%] | 2.47 |
SD = Standard deviation
CV = Coefficient of variation
Table 4. Environmental Conditions (Definitive Test)
Nominal loading levels of the saturated solution | pH-value | |||
[%] | Start; 0 hours | End; 72 hours | ||
100 | 7.92 | 8.86 | ||
31.6 | 8.08 | 8.91 | ||
10 | 8.15 | 9.06 | ||
3.16 | 8.2 | 8.93 | ||
1 | 8.22 | 8.95 | ||
0.316 | 8.29 | 9.02 | ||
Control | 8.24 | 9.16 | ||
Light intensity [µE∙m-2∙s-1] |
n = 28 | mean value: 75.1 range of the measured values: 64.5 to 82.6 equalling -14.2 to 9.93 % |
CV [%]: 7.17 | |
Table 5. Measured Concentrations and Percent the Nominal Concentration of the Test Item
Sampling date | 0 h (start of exposure) |
72 h (End of exposure) |
Start of analysis | 29/09/2015 | 05/10/2015 |
Percent of the saturated solution* | Meas. Conc. [µg/L] |
Meas. Conc. [µg/L] |
100 | < LOQ | < LOQ |
31.6 | < LOQ | < LOQ |
10 | < LOQ | < LOQ |
3.16 | < LOQ | < LOQ |
1 | < LOQ | < LOQ |
0.316 | < LOQ | < LOQ |
Control | < LOQ | < LOQ |
Table 6. EC50 Values of the Reference Item (based on nominal concentrations [mg/L], (0 -72 h)
Set-up and Experimental Date | With Headspace 2015-04-14 to 2015-04-17 | Without Headspace 2014-11-10 to 2014-11-13 | Valid Range (average ± 3 x SD) | |
Growth Rate Inhibition | ||||
ErC50 | 0.613 | 0.774 | 0.810 ± 0.404 | |
95 % confidence interval | 0.589 - 0.638 | 0.711 - 1.08 | ||
Yield Inhibition | ||||
EyC50 | 0.281 | 0.502 | 0.421 ± 0.274 | |
95 % confidence interval | 0.234 - 0.314 | 0.400 - 0.583 |
Statistical Output
One Way Analysis of Variance
Normality Test (Shapiro-Wilk) Passed(P = 0.378)
Equal Variance Test: Passed(P = 0.377)
Group Name N Missing Mean Std Dev SEM
Control 6 0 1.404 0.0347 0.0142
0.316 % 3 0 1.435 0.00853 0.00492
1.00 % 3 0 1.397 0.0185 0.0107
3.16 % 3 0 1.410 0.0172 0.00993
10.0 % 3 0 1.456 0.0147 0.00850
31.6 % 3 0 1.472 0.0118 0.00684
100 % 3 0 1.492 0.00788 0.00455
Source of Variation DF SS MS F P
Between Groups 6 0.0273 0.004549.311 <0.001
Residual 17 0.00829 0.000488
Total 23 0.0356
The differences in the mean values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P = <0.001).
Power of performed test with alpha = 0.050: 0.998
Multiple Comparisons versus Control Group (Dunnett's Method) :
Comparisons for factor:
Comparison Diff of Means q' P P<0.050
Control vs. 100 % 0.0882 5.648 -- Yes
Control vs. 31.6 % 0.0682 4.368 -- Yes
Control vs. 10.0 % 0.0517 3.312 -- Yes
Control vs. 0.316 % 0.0305 1.951 -- No
Control vs. 1.00 % 0.00704 0.451 -- Do Not Test
Control vs. 3.16 % 0.00566 0.363 -- Do Not Test
Note: The P values for Dunnett's and Duncan's tests are currently unavailable except for reporting that the P's are greater or less than the critical values of .05 and .01.
A result of "Do Not Test" occurs for a comparison when no significant difference is found between two means that enclose that comparison. For example, if you had four means sorted in order, and found no difference between means 4 vs. 2, then you would not test 4 vs. 3 and 3 vs. 2, but still test 4 vs. 1 and 3 vs. 1 (4 vs. 3 and 3 vs. 2 are enclosed by 4 vs. 2: 4 3 2 1). Note that not testing the enclosed means is a procedural rule, and a result of Do Not Test should be treated as if there is no significant difference between the means, even though one may appear to exist.
The program states a significant differences for
the nominal loading levels 10.0 % - 100% compared to the control. This
difference caused by growth stimulation.
Statistical Significance (NOEC/LOEC)–Yield
One Way Analysis of Variance
Normality Test (Shapiro-Wilk)Passed(P = 0.575)
Equal Variance Test: Passed(P = 0.461)
Group Name N Missing Mean Std Dev SEM
Control 6 0 411088.667 42452.180 17331.030
0.316 % GL 3 0 449091.667 11733.744 6774.480
1.00 % GL 3 0 400975.333 22837.318 13185.132
3.16 % GL 3 0 416728.333 21948.473 12671.957
10.0 % GL 3 0 479291.667 21167.694 12221.174
31.6 % GL 3 0 503783.667 18178.521 10495.374
100 % GL 3 0 535185.667 12764.392 7369.525
Source of Variation DF SS MS F P
Between Groups 6 52620516412.625 8770086068.771 11.316 <0.001
Residual 17 13175775823.333 775045636.667
Total 23 65796292235.958
The differences in the mean values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P = <0.001).
Power of performed test with alpha = 0.050: 1.000
Multiple Comparisons versus Control Group (Dunnett's Method) :
Comparisons for factor:
Comparison Diff of Means q' P P<0.050
Control vs. 100 % GL 124097.000 6.304 -- Yes
Control vs. 31.6 % GL 92695.000 4.709 -- Yes
Control vs. 10.0 % GL 68203.000 3.465 -- Yes
Control vs. 0.316 % GL 38003.000 1.930 -- No
Control vs. 1.00 % GL 10113.333 0.514 -- Do Not Test
Control vs. 3.16 % GL 5639.667 0.286 -- Do Not Test
Note: The P values for Dunnett's and Duncan's tests are currently unavailable except for reporting that the P's are greater or less than the critical values of .05 and .01.
A result of "Do Not Test" occurs for a comparison when no significant difference is found between two means that enclose that comparison. For example, if you had four means sorted in order, and found no difference between means 4 vs. 2, then you would not test 4 vs. 3 and 3 vs. 2, but still test 4 vs. 1 and 3 vs. 1 (4 vs. 3 and 3 vs. 2 are enclosed by 4 vs. 2: 4 3 2 1). Note that not testing the enclosed means is a procedural rule, and a result of Do Not Test should be treated as if there is no significant difference between the means, even though one may appear to exist. The program states a significant differences for the nominal loading levels 10.0 % - 100% compared to the control. This difference caused by growth stimulation.
Description of key information
72-h ErC50 = >100 % saturated solution (transformation products); 72h NOEC = >= >100 % saturated solution (transformation products); OECD TG 201 (modified to OECD 23); 2016
Key value for chemical safety assessment
Additional information
Key data : OECD TG 201, 2016 : The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD TG 201 (2011) and Commission Regulation (EC) No. 761/2009, C.3. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6153 cells/mL. A saturated solution with a nominal loading of 10.0 mg/L of the test item was prepared with dilution water. Five loading levels were tested in a geometrical series with a separation factor of 3.16: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 % of the saturated solution. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. All test concentrations of the test item and the control were analytically verified by LC-MS/MS at the start of the exposure (0 h) and at the end of the exposure (72 h). As the test design intended to test the effects of the transformation products of the test item, the measured concentrations of the test item at the start and at the end of the exposure were < LOQ, which was determined to be 15 µg/L. All effect values were based on the nominal loading levels of the test item and are related to the transformation products. All validity criteria of the test guideline were fulfilled (Increase of cell growth in the control cultures, mean coefficients of variation for specific growth rates and coefficient of variation of average specific growth rates). The alga growth inhibition test with Pseudokirchneriella subcapitata produced the following effect values (nominal loading levels):
- The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were estimated to be > 100% of the saturated solution concentration, respectively;
- The LOEC in terms of both growth rate and yield was > 100% of the saturated solution concentration;
- The corresponding NOEC values were, in both cases, >= 100% of the saturated solution concentration.
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