cyclic C12/14-Glucamide;cyclic N-methyl-N-dodecanoyl/tetradecanoyl-glucamine;anhydro-N-dodecanoyl/tetradecanoyl-N-methylglucamine;Amides, C12-C14, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2021-01-25 to 2021-01-29, with the definitive exposure phase from 2021-01-25 to 2021-01-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

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

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

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

Vehicle:

no

Details on test solutions:

Preparation of the water accommodated fractions
Five water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 1.00 to 100 mg/L set up in a geometric series with a factor of √10: 1.00 – 3.16 – 10.0 – 31.6 – 100 mg/L. For each loading rate, an appropriate amount of the test item was weighed and transferred with an appropriate amount of the dilution water into a measuring flask. The WAFs were treated with ultrasound for 5 minutes at room temperature and manual agitation. The WAFs were checked via laser beam (Tyndall effect) for undissolved test item.
The test media were visually clear throughout the test period. A positive Tyndall effect were observed at the WAFs 10.0 to 100 mg/L. The Tyndall effect was negative at the WAFs 1.00 and 3.16 mg/L. The WAFs showed a concentration related foaming.

Test item loading rates
Per definition of the WAF all terms related to concentration level have to be given as loading rates because partly dissolved compounds and mixtures cannot be related to concentrations. The loading rates are based on the results of a preliminary range finding test (non-GLP, open system with headspace).

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

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- 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): 4 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 23.0 mean value: 22.5

pH:

Nominal test item loading pH-values
[mg/L] Start; 0 hours End; 72 hours
100 7.96 7.80
31.6 7.98 7.90
10.0 8.04 8.05
3.16 8.05 8.28
1.00 8.13 8.44
Control 8.08 8.55

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

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 5375 cells/mL
- Control end cells density: Mean 776762 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:
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 0.064
Na2EDTA 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, range of the measured values: 5006 - 6389 / equalling -12.2 to 12.06%, mean value: 5701 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. No self-fluorescence was found in the definitive test in the saturated solution.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

6.17 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Cl: 4.77-7.54

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

2.49 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: Cl: 2.19-2.81

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

7.88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Cl: 6.63-8.75

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: Cl: 2.90-3.53

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

11.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 11.0 - 13.8

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

5.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI:4.88 - 5.81

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 clear throughout the exposure period.
- 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 BCCC1619, purity 100.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2020-10-20 to 2020-10-23. 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 0.903 0.775 ± 0.558
95% confidence interval 0.858 – 0.946
Yield inhibition
EyC50 0.441 0.421 ± 0.339
95% confidence interval 0.421 – 0.462
SD = Standard deviation

Reported statistics and error estimates:

EL-values and statistical EL10-,EL20- and EL50- values with confidence intervals of growth
analyses rate inhibition and for yield inhibition after 72 hours were calculated by sigmoidal dose-response regression with the GraphPad Prism Software.

NOEL, LOEL and The coefficients of variation of the control group are within the valid
analyses ranges but indicate that significance tests are not sensitive. Therefore, no NOEC/LOEC values are given.

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
• GraphPad Prism, GRAPHPAD SOFTWARE, INC.
• ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH

Cell Densities

Nominal test item loading rates       Replicate       Cell density [cells/mL]

[mg/L]       No.       0 hours       24 hours       48 hours       72 hours

100       1       5375       <LOQCell density       <LOQCell density       <LOQCell density

      2       5375       <LOQCell density       <LOQCell density       <LOQCell density

      3       5375       <LOQCell density       <LOQCell density       <LOQCell density

      Mean       5375       n.a.       n.a.       n.a.

31.6       1       5375       <LOQCell density       3853       6262

      2       5375       <LOQCell density       3469       6164

      3       5375       <LOQCell density       2848       5067

      Mean       5375       n.a.       3390       5831

10.0       1       5375       8986       23872       156369

      2       5375       9421       28542       166446

      3       5375       4078       14162       81793

      Mean       5375       7495       22192       134869

3.16       1       5375       16007       90685       644866

      2       5375       14912       74597       645232

      3       5375       15645       79132       589472

      Mean       5375       15521       81471       626523

1.00       1       5375       18822       103460       779530

      2       5375       17751       97380       789670

      3       5375       20476       112129       844918

      Mean       5375       19016       104323       804706

Control       1       5375       18152       62778       555006

      2       5375       16957       97417       901470

      3       5375       18255       66678       907579

      4       5375       19762       67430       852087

      5       5375       21016       112420       906552

      6       5375       18377       116476       537877

      Mean       5375       18753       87200       776762

n.a.       = not applicable                            LOQCell density       = 2280 cells/mL

 

 

 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 loading rates       Replicate       Growth rate       Inhibition of growth rate       Yield       Inhibition of yield

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

100       1              n.a.       100              n.a.       100

      2              n.a.       100              n.a.       100

      3              n.a.       100              n.a.       100

      Mean       (+)       n.a.       100       (n.d.)       n.a.       100

31.6       1              0.05       97              887       100

      2              0.05       97              789       100

      3              -0.02       100              -308       100

      Mean       (+)       0.03       98       (n.d.)       456       100

10.0       1              1.12       32              150994       80

      2              1.14       31              161071       79

      3              0.91       45              76418       90

      Mean       (+)       1.06       36       (+)       129494       83

3.16       1              1.60       3              639491       17

      2              1.60       3              639857       17

      3              1.57       5              584097       24

      Mean       (/)       1.59       4       (/)       621148       19

1.00       1              1.66       -1              774155       0

      2              1.66       -1              784295       -2

      3              1.69       -2              839543       -9

      Mean       (/)       1.67       -1       (/)       799331       -4

Control       1              1.55                     549631       

      2              1.71                     896095       

      3              1.71                     902204       

      4              1.69                     846712       

      5              1.71                     901177       

      6              1.54                     532502       

      Mean              1.65                     771387       

n.a.       = not applicable

n.d.       = not determined, since inhibition was 100% at the two highest loading rates

Section-by-Section and Average Specific Growth Ratesof 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                            

Control       1       1.22       1.24       2.18       1.55       0.549       36       32.9

      2       1.15       1.75       2.23       1.71       0.539       32       

      3       1.22       1.30       2.61       1.71       0.781       46       

      4       1.30       1.23       2.54       1.69       0.735       43       

      5       1.36       1.68       2.09       1.71       0.363       21       

      6       1.23       1.85       1.53       1.54       0.309       20       

                    Mean       1.65       

                    SD ±       0.08       

                    CV [%]       5.14       

SD = Standard deviation       CV = Coefficient of variation

 

SD = Standard deviation CV = Coefficient of variation

Validity criteria fulfilled:

yes

Conclusions:

In this study, cyclic Glucamide C12-14 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 loadings): The EL50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 11.8 (95% CI: 11.0 – 13.8) mg/L and 5.32 (95% CI: 4.88 – 5.81) mg/L, respectively.

Executive summary:

The toxicity of cyclic Glucamide C12-14 (batch number: S148866) 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 at the test facility from 2021-01-25 to 2021-01-29, with the definitive exposure phase from 2021-01-25 to 2021-01-28. 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 5375 cells/mL. Five water accommodated fractions (WAFs) were freshly prepared at the start of exposure with nominal loading rates of the test item in the range of 1.00 to 100 mg/L set up in a geometric series with a factor of √10: 1.00 – 3.16 – 10.0 – 31.6 – 100 mg/L.

Three replicates were tested for each WAF and six replicates for the control. The environmental conditions were within the acceptable limits.

The test media were visually clear throughout the test period. A positive Tyndall effect were observed at the WAFs 10.0 to 100 mg/L. The Tyndall effect was negative at the WAFs 1.00 and 3.16 mg/L. The WAFs showed a concentration related foaming.

The concentrations of the test item cyclic Glucamide C12-14 and the control were determined in fresh media (0 hours) and old media (72 hours) of all tested concentration levels and the control via LC-MS/MS.

The measured concentrations of the test item cyclic Glucamide C12-14 in the fresh media were in the range of 91 to 106% of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of 88 to 113% of the nominal test item concentrations.

All effect values are given as nominal loading rates.

  NOEL, LOEL and ELx-values (with 95% Confidence Intervals) of cyclic Glucamide C12-14 after 72 hours of exposure

             based on the nominal loading rates of the test item [mg/L]

      cyclic Glucamide C12-14

Nominal loading rates of the test item [mg/L]

      Inhibition of Growth Rate

ErL10       6.17 (95% CI: 4.77 – 7.54)

ErL20       7.88 (95% CI: 6.63 – 8.75)

ErL50       11.8 (95% CI: 11.0 – 13.8)

      Inhibition of Yield

EyL10       2.49 (95% CI: 2.19 – 2.81)

EyL20       3.20 (95% CI: 2.90 – 3.53)

EyL50       5.32 (95% CI: 4.88 – 5.81)

The coefficients of variation of the control group are within the valid ranges but indicate that significance tests are not sensitive (for an example see part 15.3). Therefore, no NOEC/LOEC values are given.  

Description of key information

In this study, cyclic Glucamide C12-14 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 loadings): The EL50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 11.8 (95% CI: 11.0 – 13.8) mg/L and 5.32 (95% CI: 4.88 – 5.81) mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

11.8 mg/L

EC10 or NOEC for freshwater algae:

6.17 mg/L