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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:
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:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
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. certificate)
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):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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.

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.

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.

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.