Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2019-08-26 to 2019-08-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
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:
None
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Chemical Analysis of the Test Item Concentrations

Determination of
Samples were analyzed with an LC-MS method, implemented the test item under non-GLP and documented finally in the GLP raw data.

Sampling schedule
All concentration levels and the control were analytically verified via LC-MS at the start (0 hours) of the exposure (72 hours) with algae.

Sampling and pre-treatment
At the start, samples were taken from one additional replicate of each test item concentration and the control and at the end samples were taken from pooled replicates.

Test solutions

Vehicle:
no
Details on test solutions:
Test item solution, media preparation
200 mg test item/L was prepared with dilution water. The dispersion was shaken manually. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used in the test. During filtration, the filter was always kept covered. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was positive.

Preparation of the test item solutions
Out of the stock solution five concentrations were tested in a geometric series with a dilution factor of √10: 2.00 - 6.32 - 20.0 - 63.2 - 200 mg/L.
The dilution levels are based on the results of a preliminary range finding test (non-GLP).

Control
Six replicates (without test item) were exposed under the same conditions as the test concentrations.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK, CCAP 278/4
- Strain: CCAP 278/4
- Source (laboratory, culture collection):
Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.


Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No

Test conditions

Hardness:
Not specified
Test temperature:
min.: 21.5 max.: 22.5 mean value: 22.0
pH:
Nominal test item concentration pH-values
[mg/L] Start; 0 hours End; 72 hours
200 7.71 8.20
63.2 7.82 8.54
20.0 7.87 9.05
6.32 7.93 9.25
2.00 7.98 9.34
Control 8.10 9.46
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
2.00 - 6.32 - 20.0 - 63.2 - 200 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 5367 cells/mL
- Control end cells density: Mean 2191749 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:

Ultrapure water was used to prepare the dilution water (conductivity max. 0.1 µS/cm)
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064a2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, measured: 5059 - 6039 lux with a mean of 5567 lux



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. Self-fluorescence was observed in the preliminary range finding test at the concentration of 100 mg/L. A separate replicate of each test item concentration were prepared without algae and treated like the other test replicates. The self-fluorescence of the measuring points between 24 and 72 hours were taken into account in the evaluation.
Reference substance (positive control):
yes
Remarks:
(Potassium dichromate)

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
63.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
44.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI: 40.3 - 48.3
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
78.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI: 72.9 - 85.4
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
14 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield inhibition
Remarks on result:
other: CII: 11.3 - 17.2
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
22.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield inhibition
Remarks on result:
other: CI: 19.6 - 26.0
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
48.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield inhibition
Remarks on result:
other: CI: 44.9 - 53.3
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: The test media were clear throughout exposure period (possible turbidity related to algae growth not taken into account).
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2019-03-05 to 2019-03-08. The reference item toxicity is in the valid range which was established by calculation of the average of the historic reference data since 2006, and the limits were set using the threefold standard deviation of these values.

EC50-Values of the Reference Item
based on nominal concentrations [mg/L], (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.701 0.752 ± 0.534
95% confidence interval 0.660 – 0.750
Yield inhibition
EyC50 0.353 0.405 ± 0.308
95% confidence interval 0.312 – 0.394
SD = Standard deviation
Reported statistics and error estimates:
EC-values and statistical analyses
EC10-,EC20- and EC50- values with confidence intervals of growth rate inhibition and the EC50- value for yield inhibition after 72 hours were calculated by sigmoidal dose-response regression with the GraphPad Prism Software.

NOEC, LOEC and statistical analyses
The NOEC / LOEC were determined by calculation of statistically significant differences of growth rate and yield using ANOVA. A Shapiro-Wilk’s Normality test and a Brown-Forsythe’s Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is  = 0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
•Excel, MICROSOFT CORPORATION
•SigmaPlot, SPSS INC.
•GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Any other information on results incl. tables

Microscopic evaluation of the cells at the start and the end of exposure revealed no morphological abnormalities. All effect values given are based on the nominal test item concentration of cyclic Glucamide C8- C110

Cell Densities

Nominal test item concentration [mg/L]  Replicate       Cell density [cells/mL]

                                                            No.       0 hours       24 hours       48 hours       72 hours

200                                                         1       5367       4859       21274       204442

                                                              2       5367       5165       20290       221694

                                                              3       5367       5010       22912       242480

                                                         Mean       5367       5011       21492       222872

 63.2                                                       1       5367       8556       66081       913919

                                                              2       5367       10564       64499       822181

                                                              3       5367       11513       72981       813979

                                                        Mean       5367       10211       67854       850026

 20.0                                                        1       5367       24508       191305       1750263

                                                              2       5367       24148       204060       1861545

                                                              3       5367       24197       196144       1889390

                                                         Mean       5367       24284       197170       1833733

   6.32                                                     1       5367       33441       271223       2132739

                                                              2       5367       35461       264175       2108448

                                                              3       5367       30447       255782       1998236

                                                        Mean       5367       33116       263727       2079808

   2.00                                                      1       5367       35529       275992       2157586

                                                              2       5367       35240       282690       2076047

                                                              3       5367       38701       275709       2264828

                                                        Mean       5367       36490       278130       2166154

Control                                                    1       5367       36486       317937       2214782

                                                              2       5367       33283       288826       2127548

                                                              3       5367       35549       278769       2450296

                                                              4       5367       32095       268634       2029701

                                                              5       5367       30452       239134       2160163

                                                              6       5367       35211       277406       2168001

                                                        Mean       5367       33846       278451       2191749

 Evaluation after 72 hours

 Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal test item concentration       Replicate       Growth rate       Inhibition of growth rate       Yield       Inhibition of yield

[mg/L]                                                 No.              [d-1]                            [%]                [cells/mL]       [%]

200        1              1.21       39              199075       91

             2              1.24       38              216327       90

             3              1.27       37              237113       89

   Mean       (+)       1.24       38       (+)       217505       90

 63.2      1              1.71       15              908552       58

             2              1.68       16              816814       63

             3              1.67       16              808612       63

    Mean       (+)       1.69       16       (+)       844659       61

 20.0      1              1.93       4              1744896       20

             2              1.95       3              1856178       15

             3              1.96       2              1884023       14

 Mean       (+)*       1.94       3       (+)       1828366       16

   6.32    1              2.00       0              2127372       3

             2              1.99       1              2103081       4

             3              1.97       2              1992869       9

     Mean       (-)       1.99       1       (-)       2074441       5

   2.00    1              2.00       0              2152219       2

             2              1.99       1              2070680       5

             3              2.02       -1              2259461       -3

    Mean       (-)       2.00       0       (-)       2160787       1

Control       1              2.01                     2209415       

             2              1.99                     2122181       

             3              2.04                     2444929       

             4              1.98                     2024334       

             5              2.00                     2154796       

             6              2.00                     2162634       

       Mean              2.00                     2186382       

*       = statistically significant but biologically not relevant (inhibition < 5%)

 Section-by-Section and Average Specific Growth Rates of the Control Group       (0 - 72 hours)

      Replicate No.       Specific Growth Rate [d-1]       Mean (0 - 72 hours)       SD ±       CV [%]       Mean CV [%]             

 section-by-section                            

                           0 - 24 hours       24 - 48 hours       48 - 72 hours                            

     1       1.92       2.17       1.94       2.01       0.137       6.82       8.00

      2       1.83       2.16       2.00       1.99       0.168       8.43       

      3       1.89       2.06       2.17       2.04       0.142       6.97       

      4       1.79       2.13       2.02       1.98       0.172       8.71       

      5       1.74       2.06       2.20       2.00       0.239       11.9       

      6       1.88       2.06       2.06       2.00       0.103       5.17       

                    Mean       2.00       

                    SD ±       0.02       

                    CV [%]       1.04       

SD = Standard deviation       CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, cyclic Glucamide C8-C10 was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the nominal test item concentrations): The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 200 mg/L and 48.9 (44.9 - 53.3) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 20.0 and 6.32 mg/L.
Executive summary:

The toxicity of cyclic Glucamide C8-C10 (Batch no.: BP 1c (reactor mix)) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2019-08-26 to 2019-08-29 at the test facility. The aim of the study was the determination of 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 5367 cells/mL. A test item solution with a nominal concentration of 200 mg test item/L was prepared with dilution water. The dispersion was shaken manually. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used in the test. During filtration, the filter was always kept covered. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was positive. Out of the solution five concentrations were tested in a geometrical series with a dilution factor of √10: 2.00 - 6.32 - 20.0 - 63.2 - 200 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The test media were visually clear throughout the test period. The concentrations of the test item cyclic Glucamide C8-C10 in all concentration levels and the control were analytically verified by LC-MS at the start and at the end of exposure.

The measured concentrations of the test item cyclic Glucamide C8 in the fresh media were in the range of 98 to 101% of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of 101 to 112% of the nominal test item concentrations. The measured concentrations of the test item cyclic Glucamide C10 in the fresh media were in the range of 99 to 103% of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of 98 to 103% of the nominal test item concentrations. The test items remained stable during the course of the study. Therefore, all affect values are given as nominal concentrations.

 NOEC, LOEC and ECx-values and 95% Confidence Intervals of cyclic Glucamide C8-C10 (0 - 72 hours)

             based on the nominal test item concentration [mg/L]

      Inhibition of Growth Rate [mg/L]

NOEC       20.0

LOEC       63.2

ErC10       44.6 (40.3 – 48.3)

ErC20       78.2 (72.9 – 85.4)

ErC50       > 200

      Inhibition of Yield [mg/L]

NOEC       6.32

LOEC       20.0

EyC10       14.0 (11.3 – 17.2)

EyC20       22.7 (19.6 – 26.0)

EyC50       48.9 (44.9 – 53.3)