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Ecotoxicological information

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:
25-03-2020 - 30-06-2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides

Batch number SPA19005

CAS No. 61789-77-3; 68391-05-9

Purity (certified) 95.7% m/m

Molecular formula Not specified

Molecular weight Not specified

Density 880 kg/m3 at 20 °C

Appearance Yellow, viscous liquid, paste

Water Solubility Dispersible

Stability under test
conditions Not specified

Expiry date 2021-03-12

Recommended storage Keep in a well ventilated place.
Analytical monitoring:
yes
Remarks:
via LC-MS/MS
Details on sampling:
Determination of the test item
Samples were analyzed with a LC-MS/MS method, implemented under non-GLP and documented finally in the GLP raw data. The method was validated according to Annex I.

Sampling schedule
All loading rates and the control were analytically verified via LC-MS/MS at the start (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) with algae. Additionally, the loading rate 6.32 µg/L was analytically verified via LC-MS/MS at the start (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) without algae. Additionally, adsorption to glass was analytically verified from all concentration levels at the end of the exposure. The peak distribution of the WAF was analyzed in fresh prepared medium in the highest test item concentration, at the start of the exposure. Therefore a high resolution MS (Q-ToF) was used.

Sampling and pre-treatment
At the start, after 24 and after 48 hours 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.
For the adsorption to glass the vessels were rinsed carefully before analysis to remove adhering algae.
Samples after 24, 48, 72 hours of exposure were centrifuged to separate the algae from the water phase. Both were analyzed separately to determine the truly dissolved test item fraction and the fraction adsorbed to algae. The centrifuge tubes were rinsed with the appropriate test solutions to minimize adsorption to the walls of the centrifuge tubes.
Vehicle:
no
Details on test solutions:
Water Soluble Fraction
Water accommodated fractions (WAFs) were prepared because the test item is a UVCB substance with compounds of different water solubility. This procedure is in accordance with the OECD guidance document No. 23 (2019).
Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

Preparation of the water accommodated fractions
Six water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 0.632 to 200 µg/L set up in a geometric series with a factor of √10: 0.632 – 2.00 – 6.32 – 20.0 – 63.2 – 200 µg/L.
The procedure according to ASTM D6081 (2019) as described below was carried out. The test item was melted at 70 °C for approximately 2h before preparation of the WAFs. For each loading rate an appropriate amount of stock solutions containing the test item with methanol was placed on a curved glass slide. The methanol was evaporated. The glass slide with the test item was inserted in a glass flask with an appropriate amount of dilution water (see Table 2). A slow stirring procedure was applied for 24 ± 1 hour at 20 ± 2 °C. The optimal stirring time was determined in a preliminary stirring test. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour, the aqueous phase of the WAF was removed by siphoning (from the approximate middle of the glass flask). The first 25 mL were discarded.
The WAFs were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative. The resulting water accommodated fractions (WAF) were used in the test.

Test loading
Per definition of the WAF all terms related to concentration level are 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
The control solution was prepared without test item following the same method as specified for the WAFs. Six replicates were exposed under the same conditions as the test concentrations.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test organism
Pseudokirchneriella subcapitata HINDÁK CCAP 278/4 (axenic)

Synonyms
Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)

Reason for the selection of the test organism
Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.

Origin
Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

Cultivation at test facility
Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux 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
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
See "Any other information on materilas and methods incl. tables" below
Test temperature:
See "Any other information on materilas and methods incl. tables" below
pH:
See "Any other information on materilas and methods incl. tables" below
Conductivity:
See "Any other information on materilas and methods incl. tables" below
Nominal and measured concentrations:
Water accommodated fractions (WAFs) were prepared because the test item is a UVCB ubstance with compounds of different water solubility.
Six water accommodated fractions (WAF) were prepared separately with nominal loading rates of the test item in the range of 0.632 to 200 µg/L set up in a geometric series with a factor of √10: 0.632 – 2.00 – 6.32 – 20.0 – 63.2 – 200 µg/L.

The test item concentrations of Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides were analytically verified via LC-MS/MS at the start (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) with algae despite the fact that a WAF approach is used. The test item is a UVCB substance with constituents of different water solubility which means that per definition of the WAF approach, the dose-response can only be based to loading rates because partly dissolved compounds and mixtures cannot be related to concentrations. Additionally, the loading rate 6.32 µg/L was analytically verified via LC-MS/MS at the start (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) without algae. Additionally, adsorption to glass was analytically verified from all concentration levels at the end of the exposure.
For the quantification of the fraction sorbed to algae samples after 24, 48, 72 hours of exposure were centrifuged to separate the algae from the water phase. Both were analyzed separately to determine the truly dissolved test item fraction and the fraction adsorbed to algae. As the test item is a UVCB substance, all evaluations are based on the nominal loading rates of Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides and time weighted average concentration of the test item.
The concentrations decreased from 0 hours to 72 hours. Test item could be observed on the algae and on the glass surface as well. But even in the replicates without algae a decreased concentration in the old medium (72 hours) could be observed.
Details on test conditions:
Reference item
Potassium dichromate is tested twice per year as a reference item. Results of the most recent test was provided in the report.

Test method
Static procedure

Duration of the test
72 hours

Replicates
Six replicates for the control, three replicates per concentration.

Test container and Pre-treatment
Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs were used. A coating phase (saturation of the test container) was carried out. The test containers were pre-treated with the appropriate test solution for at least 12 hours under test conditions. Before the start of the exposure, the test container was emptied and refilled with freshly prepared test solution. Preparation of the test solutions for coating was documented in the raw data and in the final report.

Test volume 100 mL

Ultrapure water
Ultrapure water was used to prepare the dilution water (conductivity max. 0.1 µS/cm).

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
Actual: 4886 cells/mL

Application
Application was carried out by adding an appropriate amount of algae inoculum to the test solutions.

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 (target)
Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

Light regime 24 hours/day light

Light homogeneity
Within ± 15% over incubation area

Biological Parameters

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 observed in the preliminary range finding test at the loading rate of 2 mg/L. A calibration curve with a satisfactory correlation was used to calculate the cell density from fluorescence and given in the report.

Microscopic evaluation
The algae cells were evaluated microscopically at the start and the end of the incubation period. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

Physico-chemical Properties

The pH-value at the start of the exposure was measured in one additional replicate of each test item loading rate and the control. At the end of the exposure, it was measured in a pooled sample of the test item loading rates and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of the test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is tested twice per year as a reference item. Results of the most recent test is provided in "Any other information on materials and methods incl. tables" below.
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
31.6 µg/L
95% CI:
ca. 25.5 - ca. 42.7
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Remarks:
Inhibition of yield
Remarks on result:
other: 30.2 (CI: 24.38-40.82) μg a.i./L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.53 µg/L
95% CI:
ca. 2.12 - ca. 3.2
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
yield
Remarks:
Inhibition of yield
Remarks on result:
other: 2.42 (CI: 2.03-3.06) μg a.i./L
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
58.2 µg/L
95% CI:
ca. 51 - ca. 61.1
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Inhibition of growth rate
Remarks on result:
other: 55.64 (CI: 48.76-58.41) μg a.i./L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.65 µg/L
95% CI:
ca. 2.21 - ca. 3.96
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks:
Inhibition of growth rate
Remarks on result:
other: 3.49 (CI: 2.11-3.79) μg a.i./L
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
30 µg/L
95% CI:
ca. 21.5 - ca. 43
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Inhibition of growth rate
Remarks on result:
other: 28.68 (CI: 20.55-41.1) μg a.i./L
Duration:
72 h
Dose descriptor:
EC10
Remarks:
EL10
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
yield
Remarks:
Inhibition of yield
Remarks on result:
not determinable
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.04 µg/L
95% CI:
ca. 1.82 - ca. 3.69
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks:
Inhibition of growth rate
Remarks on result:
other: 2.91 (CI: 1.74-2.93) μg a.i./L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
20 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Inhibition of growth rate
Remarks on result:
other: 19.12 μg a.i./L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.74 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks:
Inhibition of growth rate
Remarks on result:
other: 1.66 μg a.i./L
Details on results:
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 loading rates of Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides.

The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test media were clear throughout exposure period (possible turbidity related to algae growth not taken into account).

The test item concentrations of Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides were analytically verified via LC-MS/MS at the start (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) with algae. Additionally, the loading rate 6.32 µg/L was analytically verified via LC-MS/MS at the start (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) without algae. Additionally, adsorption to glass was analytically verified from all concentration levels at the end of the exposure.
For the quantification of the fraction sorbed to algae samples after 24, 48, 72 hours of exposure were centrifuged to separate the algae from the water phase. Both were analyzed separately to determine the truly dissolved test item fraction and the fraction adsorbed to algae. As the test item is a UVCB substance, all evaluations are based on the nominal loading rates of Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides and time weighted average concentration of the test item.
The concentrations decreased from 0 hours to 72 hours. Test item could be observed on the algae and on the glass surface as well. But even in the replicates without algae a decreased concentration in the old medium (72 hours) could be observed.

Fingerprint of the test item (non-GLP)

The peak distribution of the WAF was analyzed in fresh prepared medium in the highest test item concentration. An analytical standard of the test item was prepared in methanol and diluted to 100 µg/L with acetonitrile : ultra-pure water (50 : 50) containing 1% formic acid. The highest test item concentration was diluted factor 2 with acetonitrile containing 2% formic acid to avoid an inhomogeneous sample. The standard dilution and the test item dilution were analytical verified via high resolution MS (Q-ToF) and evaluated by the software. The detected signals of the analytical standard and of the test item solution were compared. In both solutions 6 masses, related to the test item could be observed. No additional signals, than analyzed during the study, related to the test item could be observed in the analytical standard or test item.

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

NOEL, LOEL and analyses
The NOEL / LOEL was determined by calculation of statistical significance of growth rate and yield. The following statistical tests were conducted:

Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.

Levene’s test on variance homogeneity was done with a significance level of 0.01.

Monotonicity of Concentration/Response was calculated by Trend Analysis by Contrasts (significance level 0.05) for growth rate.

Williams Multiple Sequential t-test Procedure was performed with a significance level of 0.05 for growth rate.

Monotonicity of Concentration/Response was tested by non-parametric trend analysis by contrast with a significance level of 0.05 for yield.

Step-down Jonckheere-Terpstra Test procedure and Williams Multiple Sequential t-test procedure with a significant level of 0.05 for yield.

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.

Measured Concentrations of the compound C24H52N of the test substance, C12 -18 DAQ during the Definitive Test (0, 24, 48, 72 hours)

 

Sampling date Fresh medium, Old medium, Old medium, Old medium,
0 hours 24 hours 48 hours 72 hours
Nominal Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
loading rate of the C24H52N
test item active ingredient Meas. % Meas. % Meas. % Meas. %
[µg/L] [µg a.i./L] conc.  conc.  conc.  conc. 
    [µg a.i./L] [µg a.i./L] [µg a.i./L] [µg a.i./L]
200 13.8 5.39 39 0.268 2 0.230 2 0.697 5
63.2 4.36 1.93 44 0.131 3 0.460 11 0.326 7
20.0 1.38 0.411 30 0.124 9 0.172 12 0.0787 6
6.32 0.436 0.0821 19 0.01272) 3 0.0159 4 < LOQ  
6.321) 0.4361) 0.129 30 0.0328 8 0.01342) 3 0.0315 7
2.00 0.138 0.0147 11 < LOQ   0.01122) 8 < LOQ  
0.632 0.0436 < LOQ   < LOQ   < LOQ   < LOQ  
Control < LOQ < LOQ < LOQ < LOQ

Meas. conc. = measured concentration of the compound C24H52N of the test item, dilution factors taken into account
a.i. = active ingredient
% = percent of the nominal concentration of the test item
LOQ = limit of quantification (0.200 µg/L of the test item, corresponding to 0.0138 µg a.i./L)
1) = test item concentration without algae
2) = < LOQ but > 70% LOQ

 

Measured Concentrations of the compound C24H52N of the test substance, C12 -18 DAQ (Algae and glass adsorption) (24, 48, 72 hours)

Sampling date Old medium, Old medium, Old medium, Old medium,
24 hours 48 hours 72 hours 72 hours
Nominal Algae adsorption Glass Adsorption
loading rate of the Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
  C24H52N
test item active ingredient Meas. % Meas. % Meas. % Meas. %
[µg/L] [µg a.i./L] conc.  conc.  conc.  conc. 
    [µg a.i./L] [µg a.i./L] [µg a.i./L] [µg a.i./L]
200 13.8 4.72 34 4.57 33 6.85 50 5.26 38
63.2 4.36 1.41 32 0.932 21 2.52 58 4.39 101
20.0 1.38 0.164 12 0.340 25 0.493 36 0.938 68
6.32 0.436 0.0484 11 0.0427 10 0.0932 21 0.198 45
2.00 0.138 0.0179 13 0.0141 10 0.0256 19 < LCL  
0.632 0.0436 < LCL   < LCL   < LCL   < LCL  
Control < LCL < LCL < LCL < LCL

Meas. conc. = measured concentration of the compound C24H52N of the test item, dilution factors taken into account
a.i. = active ingredient
% = percent of the nominal concentration of the test item
LCL = lowest calibration level (0.500 µg/L of the test item)

 

Measured Concentrations of the compound C26H56N of the test substance, C12 -18 DAQ during the Definitive Test (0, 24, 48, 72 hours)

 

Sampling date Fresh medium, Old medium, Old medium, Old medium,
0 hours 24 hours 48 hours 72 hours
Nominal loading rate of  Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
C26H56N
test item active ingredient Meas. % Meas. % Meas. % Meas. %
[µg/L] [µg a.i./L] conc.  conc.  conc.  conc. 
    [µg a.i./L] [µg a.i./L] [µg a.i./L] [µg a.i./L]
200 51.2 10.4 20 0.247 0 0.709 1 0.788 2
63.2 16.2 4.12 25 0.325 2 1.60 10 0.645 4
20.0 5.12 1.12 22 0.243 5 0.364 7 0.244 5
6.32 1.62 0.269 17 0.04071) 3 0.04892) 3 < LOQ  
6.321) 1.621) 0.402 25 0.0819 5 < LOQ   0.0645 4
2.00 0.512 0.04792) 9 < LOQ   < LOQ   < LOQ  
0.632 0.162 < LOQ   < LOQ   < LOQ   < LOQ  
Control < LOQ < LOQ < LOQ < LOQ

Meas. conc. = measured concentration of the compound C26H56N of the test item, dilution factors taken into account
a.i.. = active ingredient
% = percent of the nominal concentration of the test item
LOQ = limit of quantification (0.200 µg/L of the test item, corresponding to 0.0512 µg a.i./L)
1) = test item concentration without algae
2) = < LOQ but > 70% LOQ

 

Measured Concentrations of the compound C26H56N of the test substance, C12 -18 DAQ (Algae and glass adsorption) (24, 48, 72 hours)

 

Sampling date Old medium, Old medium, Old medium, Old medium,
24 hours 48 hours 72 hours 72 hours
Nominal Algae adsorption Glass Adsorption
loading rate of the Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
  C26H56N
test item active ingredient Meas. % Meas. % Meas. % Meas. %
[µg/L] [µg a.i./L] conc.  conc.  conc.  conc. 
    [µg a.i./L] [µg a.i./L] [µg a.i./L] [µg a.i./L]
200 51.2 5.53 11 6.22 12 16.2 32 20.2 39
63.2 16.2 1.87 12 1.67 10 4.66 29 9.37 58
20.0 5.12 0.227 4 0.676 13 1.18 23 2.82 55
6.32 1.62 0.118 7 0.122 8 0.243 15 0.556 34
2.00 0.512 0.0453 9 0.0466 9 0.0817 16 < LCL  
0.632 0.162 < LCL   < LCL   < LCL   < LCL  
Control < LCL < LCL < LCL < LCL

Meas. conc. = measured concentration of the compound C26H56N of the test item, dilution factors taken into account
a.i. = active ingredient
% = percent of the nominal concentration of the test item
LCL = lowest calibration level (0.500 µg/L of the test item)

 

Measured Concentrations of the compound C28H60N of the test substance, C12 -18 DAQ during the Definitive Test (0, 24, 48, 72 hours)

 

Sampling date Fresh medium, Old medium, Old medium, Old medium,
0 hours 24 hours 48 hours 72 hours
Nominal loading rate of  Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
C28H60N
test item active ingredient Meas. % Meas. % Meas. % Meas. %
[µg/L] [µg a.i./L] conc.  conc.  conc.  conc. 
    [µg a.i./L] [µg a.i./L] [µg a.i./L] [µg a.i./L]
200 35.8 < LOQ   0.0601 0 0.105 0 0.120 0
63.2 11.3 0.979 9 0.218 2 0.430 4 0.163 1
20.0 3.58 0.492 14 0.125 3 0.155 4 0.0958 3
6.32 1.13 < LOQ   0.03472) 32) 0.02922) 3 < LOQ  
6.321) 1.131) 0.227 20 0.0822 7 < LOQ   0.03152) 3
2.00 0.512 0.0510 14 0.02702) 8 < LOQ   < LOQ  
0.632 0.162 < LOQ   < LOQ   < LOQ   < LOQ  
Control < LOQ < LOQ < LOQ < LOQ

Meas. conc. = measured concentration of the compound C28H60N of the test item, dilution factors taken into account
a.i. = active ingredient
% = percent of the nominal concentration of the test item
LOQ = limit of quantification (0.200 µg/L of the test item, corresponding to 0.0358 µg a.i./L)
1) = test item concentration without algae
2) = < LOQ but > 70% LOQ

 

Measured Concentrations of the compound C28H60N of the test substance, C12 -18 DAQ (Algae and glass adsorption) (24, 48, 72 hours)

Sampling date Old medium, Old medium, Old medium, Old medium,
24 hours 48 hours 72 hours 72 hours
Nominal Algae adsorption Glass Adsorption
loading rate of the Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
  C28H60N
test item active ingredient Meas. % Meas. % Meas. % Meas. %
[µg/L] [µg a.i./L] conc.  conc.  conc.  conc. 
    [µg a.i./L] [µg a.i./L] [µg a.i./L] [µg a.i./L]
200 35.8 0.524 1 1.11 3 2.97 8 4.58 13
63.2 11.3 0.274 2 0.374 3 0.882 8 2.18 19
20.0 3.58 0.0906 3 0.211 6 0.392 11 1.56 44
6.32 1.13 0.0596 5 0.0750 7 0.113 10 0.374 33
2.00 0.358 0.0263 7 0.0279 8 0.0508 14 0.0981 27
0.632 0.113 < LCL   < LCL   < LCL   < LCL  
Control < LCL < LCL < LCL < LCL

Meas. conc. = measured concentration of the compound C28H60N of the test item, dilution factors taken into account
a.i. = active ingredient
% = percent of the nominal concentration of the test item
LCL = lowest calibration level (0.500 µg/L of the test item)

 

Measured Concentrations of the compound C30H64N of the test substance, C12 -18 DAQ during the Definitive Test (0, 24, 48, 72 hours)

 

Sampling date Fresh medium, Old medium, Old medium, Old medium,
0 hours 24 hours 48 hours 72 hours
Nominal loading rate of  Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
C30H64N
test item active ingredient Meas. % Meas. % Meas. % Meas. %
[µg/L] [µg a.i./L] conc.  conc.  conc.  conc. 
    [µg a.i./L] [µg a.i./L] [µg a.i./L] [µg a.i./L]
200 26.4 < LOQ   0.0453 0 0.0362 0 0.0380 0
63.2 8.34 < LOQ   0.0916 1 0.135 2 0.0521 1
20.0 2.64 0.659 25 0.117 4 0.157 6 0.0601 2
6.32 0.834 0.265 32 0.0669 8 0.0347 4 < LOQ  
6.321) 0.8341) 0.290 35 0.376 45 0.0305 4 0.0313 4
2.00 0.264 0.104 39 0.0318 12 < LOQ   < LOQ  
0.632 0.0834 < LOQ   < LOQ   < LOQ   < LOQ  
Control < LOQ < LOQ < LOQ < LOQ

Meas. conc. = measured concentration of the compound C30H64N of the test item, dilution factors taken into account
a.i. = active ingredient
% = percent of the nominal concentration of the test item
LOQ = limit of quantification (0.200 µg/L of the test item, corresponding to 0.0264 µg a.i./L)
1) = test item concentration without algae

 

Measured Concentrations of the compound C30H64N of the test substance, C12 -18 DAQ (Algae and glass adsorption) (24, 48, 72 hours)

Sampling date Old medium, Old medium, Old medium, Old medium,
24 hours 48 hours 72 hours 72 hours
Nominal Algae adsorption Glass Adsorption
loading rate of the Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
  C30H64N
test item active ingredient Meas. % Meas. % Meas. % Meas. %
[µg/L] [µg a.i./L] conc.  conc.  conc.  conc. 
    [µg a.i./L] [µg a.i./L] [µg a.i./L] [µg a.i./L]
200 26.4 0.238 1 0.507 2 0.886 3 2.86 11
63.2 8.34 0.156 2 0.150 2 0.231 3 0.863 10
20.0 2.64 0.190 7 0.257 10 0.256 10 1.36 52
6.32 0.834 0.109 13 0.0994 12 0.106 13 0.502 60
2.00 0.264 0.0418 16 0.0339 13 0.0482 18 0.0946 36
0.632 0.0834 < LCL   < LCL   < LCL   < LCL  
Control < LCL < LCL < LCL < LCL

Meas. conc. = measured concentration of the compound C30H64N of the test item, dilution factors taken into account
a.i. = active ingredient
% = percent of the nominal concentration of the test item
LCL = lowest calibration level (0.500 µg/L of the test item)

 

Measured Concentrations of the compound C32H68N of the test substance, C12 -18 DAQ during the Definitive Test (0, 24, 48, 72 hours)

 

Sampling date Fresh medium, Old medium, Old medium, Old medium,
0 hours 24 hours 48 hours 72 hours
Nominal loading rate of  Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
C32H68N
test item active ingredient Meas. % Meas. % Meas. % Meas. %
[µg/L] [µg a.i./L] conc.  conc.  conc.  conc. 
    [µg a.i./L] [µg a.i./L] [µg a.i./L] [µg a.i./L]
200 20.8 5.44 26 1.05 5 0.553 3 0.172 1
63.2 6.57 2.65 40 0.676 10 0.518 8 0.230 3
20.0 2.08 1.50 72 0.374 18 0.265 13 0.136 7
6.32 0.657 0.389 59 0.329 50 0.0432 7 0.0393 6
6.321) 0.6571) 0.496 76 0.485 74 0.0785 12 0.0543 8
2.00 0.208 0.104 50 0.0387 19 0.01602) 8 < LOQ  
0.632 0.0657 < LOQ   < LOQ   < LOQ   < LOQ  
Control < LOQ < LOQ < LOQ < LOQ

Meas. conc. = measured concentration of the compound C32H68N of the test item, dilution factors taken into account
a.i. = active ingredient
% = percent of the nominal concentration of the test item
LOQ = limit of quantification (0.200 µg/L of the test item, corresponding to 0.0208 µg a.i./L)
1) = test item concentration without algae
2) = < LOQ but > 70% LOQ

 

Measured Concentrations of the compound C32H68N of the test substance, C12 -18 DAQ (Algae and glass adsorption) (24, 48, 72 hours)

Sampling date Old medium, Old medium, Old medium, Old medium,
24 hours 48 hours 72 hours 72 hours
Nominal Algae adsorption Glass Adsorption
loading rate of the Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
  C32H68N
test item active ingredient Meas. % Meas. % Meas. % Meas. %
[µg/L] [µg a.i./L] conc.  conc.  conc.  conc. 
    [µg a.i./L] [µg a.i./L] [µg a.i./L] [µg a.i./L]
200 20.8 3.44 17 2.35 11 3.03 15 9.02 43
63.2 6.57 1.25 19 0.893 14 1.14 17 3.96 60
20.0 2.08 0.647 31 0.659 32 0.449 22 1.54 74
6.32 0.657 0.122 19 0.138 21 0.119 18 0.410 62
2.00 0.208 0.0570 27 0.0411 20 0.0506 24 0.0594 29
0.632 0.0657 0.0126 19 < LCL   < LCL   < LCL  
Control < LCL < LCL < LCL < LCL

Meas. conc. = measured concentration of the compound C32H68N of the test item, dilution factors taken into account
a.i. = active ingredient
% = percent of the nominal concentration of the test item
LCL = lowest calibration level (0.500 µg/L of the test item)

 

Measured Concentrations of the compound C34H72N of the test substance, C12 -18 DAQ during the Definitive Test (0, 24, 48, 72 hours)

Sampling date Fresh medium, Old medium, Old medium, Old medium,
0 hours 24 hours 48 hours 72 hours
Nominal loading rate of  Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
C34H72N
test item active ingredient Meas. % Meas. % Meas. % Meas. %
[µg/L] [µg a.i./L] conc.  conc.  conc.  conc. 
    [µg a.i./L] [µg a.i./L] [µg a.i./L] [µg a.i./L]
200 6.80 2.97 44 0.520 8 0.239 4 0.0738 1
63.2 2.15 1.27 59 0.299 14 0.229 11 0.0949 4
20.0 0.680 0.585 86 0.147 22 0.0884 13 0.0751 11
6.32 0.215 0.135 63 0.154 72 0.0184 9 0.0292 14
6.321) 0.2151) 0.169 79 0.160 75 0.0317 15 0.0295 14
2.00 0.0680 0.0343 50 0.0114 17 < LOQ   < LOQ  
0.632 0.0215 < LOQ   < LOQ   < LOQ   < LOQ  
Control < LOQ < LOQ < LOQ < LOQ

Meas. conc. = measured concentration of the compound C34H72N of the test item, dilution factors taken into account
a.i. = active ingredient
% = percent of the nominal concentration of the test item
LOQ = limit of quantification (0.200 µg/L of the test item, corresponding to 0.00680 µg a.i./L)
1) = test item concentration without algae

 

Measured Concentrations of the compound C34H72N of the test substance, C12 -18 DAQ (Algae and glass adsorption) (24, 48, 72 hours)

Sampling date Old medium, Old medium, Old medium, Old medium,
24 hours 48 hours 72 hours 72 hours
Nominal Algae adsorption Glass Adsorption
loading rate of the Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
  C34H72N
test item active ingredient Meas. % Meas. % Meas. % Meas. %
[µg/L] [µg a.i./L] conc.  conc.  conc.  conc. 
    [µg a.i./L] [µg a.i./L] [µg a.i./L] [µg a.i./L]
200 6.80 2.12 31 1.35 20 1.41 21 3.77 56
63.2 2.15 0.663 31 0.517 24 0.524 24 1.62 75
20.0 0.680 0.267 39 0.294 43 0.161 24 0.466 69
6.32 0.215 0.0347 16 0.0551 26 0.0327 15 0.116 54
2.00 0.0680 0.0211 31 0.0177 26 0.0186 27 0.0189 28
0.632 0.0215 < LCL   < LCL   < LCL   < LCL  
Control < LCL < LCL < LCL < LCL

Meas. conc. = measured concentration of the compound C34H72N of the test item, dilution factors taken into account
a.i. = active ingredient
% = percent of the nominal concentration of the test item
LCL = lowest calibration level (0.500 µg/L of the test item)

 

Conditions of analysis

Gradient Table

Time [min] A [%] B [%]
0.00 60 40
0.50 60 40
1.00 5 95
3.00 5 95
3.10 60 40
3.50 60 40

 

Dilution steps

test item Factor* volume volume
loading rate   [mL] [mL]
[µg/L]      
200 40 0.05 1.0
200 4 0.5 1.0
200 2 1.0 1.0
63.2 10 0.2 1.0
63.2 4 0.5 1.0
63.2 2 1.0 1.0
20.0 4 0.5 1.0
20.0 2 1.0 1.0
6.32 2 1.0 1.0
2.00 2 1.0 1.0

*including factor 2

 

Dilution steps (Enrichment)

Nominal Dilution Total Sample Final
test item Factor Enrichment volume volume
loading rate   Factor* [mL] [mL]
[µg/L]        
200 1 10 1.0 1.0
200 5 2 0.2 1.0
200 10 1 0.1 1.0
63.2 1 10 1.0 1.0
63.2 5 2 0.2 1.0
63.2 10 1 0.1 1.0
20.0 1 10 1.0 1.0
20.0 5 2 0.2 1.0
20.0 10 1 0.1 1.0
6.32 1 10 1.0 1.0
6.32 5 2 0.2 1.0
2.00 1 10 1.0 1.0
0.632 1 10 1.0 1.0
Control 1 10 1.0 1.0

*including enrichment factor 10

 

Dilution steps of algae adsorption samples

Nominal test item Total  Total  Sample Final
loading rate Dilution Enrichment volume volume
[µg/L] Factor* Factor [mL] [mL]
200 10 - 0.1 5.0
200 2 - 0.1 1.0
200 - 2.5 0.5 1.0
200 - 5 1.0 1.0
63.2 4 - 0.05 1.0
63.2 - 2.5 0.5 1.0
63.2 - 5 1.0 1.0
20.0 2 - 0.1 1.0
20.0 - 2.5 0.5 1.0
20.0 - 5 1.0 1.0
6.32 - 5 1.0 1.0
2.00 - 5 1.0 1.0
0.632 - 5 1.0 1.0
Control - 5 1.0 1.0

*including enrichment factor 10 and dilution factor 2

 

Dilution steps (glass adsorption)

Nominal Dilution Sample Final
test item Factor* volume volume
loading rate   [mL] [mL]
[µg/L]      
200 20 0.05 1.0
63.2 10 0.1 1.0
20.0 2 0.5 1.0
6.32 1 1.0 1.0
2.00 1 1.0 1.0
0.632 11) 1.0 1.0
Control 1 1.0 1.0

*including total enrichment factor 1
1) including total enrichment factor 0.5

 

Method Validation (non-GLP

Parameter, Acceptance Criteria and Results of the Method Validation

Parameter Acceptance criteria Result
Linearity ≥ 5 standard concentrations, 0.50 to 10 µg test item/L (n = 7), ü
r ≥ 0.99 r ≥ 0.99
Lowest calibration level (LCL) S/N ≥ 9 for quantifier ion trace S/N for 0.5 µg test item/L ü
S/N ≥ 3 for qualifier ion trace  C24H52N:
  754 (Quantifier), 286 (Qualifier)
  C26H56N:
  8075 (Quantifier), 761 (Qualifier)
  C28H60N:
  4388 (Quantifier), 977 (Qualifier)
  C30H64N:
  1739 (Quantifier), 394 (Qualifier)
  C32H68N:
  1693 (Quantifier), 357 (Qualifier)
  C34H72N:
  316 (Quantifier), 93 (Qualifier)
Limit of Detection (LOD) Determination only necessary when S/N LCL ≤ 30. Not necessary -
S/N of 3-5 for the signal which is used for the identification. If significant blank values are observable, LOD has to be 3-5 times higher as the mean value plus standard deviation of 5 to 10 blank measurements. 
Limit of Quantification (LOQ) At least 20% above lowest calibration level after sample preparation 0.200 µg test item/L (1 x LOQ) ü
2.00 µg test item/L (10 x LOQ)
250 µg test item/L (1250 x LOQ)
Accuracy  Mean recovery rate of 70-110%  C24H52N: ü
(Fortified samples) (ideally 80-100%) 1 x LOQ: 106% (n = 5)
  per fortification level (2 levels) 10 x LOQ: 94% (n = 5)
    1250 x LOQ: 102% (n = 5)
    C26H56N:
    1 x LOQ: 77% (n = 5)
    10 x LOQ: 83% (n = 5)
    1250 x LOQ: 104% (n = 5)
    C28H60N:
    1 x LOQ: 61% (n = 5)
    10 x LOQ: 85% (n = 5)
    1250 x LOQ: 101% (n = 5)
    C30H64N:
    1 x LOQ: 59% (n = 5)
    10 x LOQ: 81% (n = 5)
    1250 x LOQ: 102% (n = 5)
    C32H68N:
    1 x LOQ: 63% (n = 5)
    10 x LOQ: 93% (n = 5)
    1250 x LOQ: 103% (n = 5)
    C34H72N:
    1 x LOQ: 64% (n = 5)
    10 x LOQ: 92% (n = 5)
    1250 x LOQ: 102% (n = 5)
Precision Relative standard deviation ≤ 20% per fortification level C24H52N: ü
1 x LOQ: 6.0%
10 x LOQ: 10% 
1250 x LOQ: 5.0%
C26H56N:
1 x LOQ: 8.6%
10 x LOQ: 5.4% 
1250 x LOQ: 1.7%
C28H60N:
1 x LOQ: 4.5%
10 x LOQ: 3.0% 
1250 x LOQ: 1.7%
C30H64N:
1 x LOQ: 3.4%
10 x LOQ: 4.2% 
1250 x LOQ: 1.9%
C32H68N:
1 x LOQ: 1.6%
10 x LOQ: 7.0% 
1250 x LOQ: 2.9%
C34H72N:
1 x LOQ: 1.3%
10 x LOQ: 6.9% 
1250 x LOQ: 3.3%
Specificity:  Multiple Reaction Mode (MRM) C24H52N: ü
LC-MS/(MS) Measurement of two transitions of the same precursor ion - one quantifier (used for evaluation) and one qualifier (for confirmation of the analyte identity). 6 compounds related to the test item will be analysed. quantifier [m/z]: 354.31 > 186.16
    qualifier [m/z]: 354.31 > 57.11
    C26H56N:
    quantifier [m/z]: 382.35 > 214.16
    qualifier [m/z]: 382.35 > 57.11
    C28H60N:
    quantifier [m/z]: 410.38 > 214.17
    qualifier [m/z]: 410.38 > 57.11
    C30H64N:
    quantifier [m/z]: 438.41 > 214.17
    qualifier [m/z]: 438.41 > 57.11
    C32H68N:
    quantifier [m/z]: 466.44 > 214.21
    qualifier [m/z]: 466.44 > 57.11
    C34H72N:
    quantifier [m/z]: 494.47 > 242.21
    qualifier [m/z]: 494.47 > 57.11
  Blank values < 30% of the LOQ Blank values < 30% of LOQ ü
Procedural recovery Procedural recoveries with experimental samples. Ideally, the recovery should be in the range of the mean recovery +/- 2x relative standard deviation of the 1xLOQ in comparison to the method validation. If the criterion is not matched, the recovery has to be at least 70-110 % of the nominal value. See section 17.1 ü

 

Preparation of Fortified Samples of the Test Item

 

LOQ Level Control 1 10 1250
Stock solution - 1000 mg test item/L in Methanol
Spiking solution - 10.0 0.100 25.0
[mg test item/L] (dilution medium) (dilution medium) (methanol)
(Medium)      
Replicates 2 5 5 5
Concentration of the LOQ - 0.200 2.00 250
[µg test item/L]
Medium for preparation algae dilution medium
Volume of spiking solution [mL] - 1.0 0.1 0.05
Volume of medium [mL] 50 49 4.90 4.95
Enrichment factora) 10 10 - -
Dilution factor - - 2 40
Dilution medium Acetonitrile containing 2% formic acid 1)
Dilution medium 2)
Sample volume [mL] 5.0 5.0 5.01) 5.01)
0.052)
Finale volume [mL] 5.0 5.0 101) 101)
1.02)

1) First dilution step
2) Second dilution step
a) For details, see sample preparation for enrichment
Dilution medium = Acetonitrile : ultra-pure water (50 : 50) containing 1% formic acid

 

Nominal Concentrations of the Fortified Samples of the active ingredients of C12 -18 DAQ
Fortified concentrations*: 0.208 µg test item/L (1 x LOQ), 2.10 µg test item/L (10 x LOQ) and 262 µg test item/L (1250 x LOQ).

Active ingredient 1 x LOQ 10 x LOQ 1250 x LOQ
[µg a.i./L] [µg a.i./L] [µg a.i./L]
C24H52N 0.0143 0.145 18.1
C26H56N 0.0531 0.537 67.1
C28H60N 0.0372 0.375 46.9
C30H64N 0.0274 0.277 34.6
C32H68N 0.0216 0.218 27.2
C34H72N 0.00706 0.0713 8.91

* = weighing factor taken into account
a.i. = active ingredient

 

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of C12 -18 DAQ
Active ingredient: C24H52N

Replicate Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
C24H52N
1 x LOQ 10 x LOQ 1250 x LOQ
Meas. % Meas. % Meas. %
conc. conc. conc.
[µg a.i./L] [µg a.i./L] [µg a.i./L]
1 0.0143 100 0.148 103 18.5 102
2 0.0146 102 0.146 101 18.2 100
3 0.0153 107 0.126 87 19.4 108
4 0.0151 106 0.143 99 19.3 107
5 0.0167 116 0.117 81 17.2 95
Mean 0.0152 106 0.14 94 18.5 102
SD ± 0.0009   0.01   0.9  
CV [%] 6.0   10   5.0  

Meas. conc. = measured concentration of each component of the test item, dilution factor taken into account
a.i. = active ingredient
% = percent concentration of the fortified sample
SD = standard deviation
CV = coefficient of variation

 

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of C12 -18 DAQ
Active ingredient: C26H56N and C28H60N

Replicate Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
C26H56N
1 x LOQ 10 x LOQ 1250 x LOQ
Meas. % Meas. % Meas. %
conc. conc. conc.
[µg a.i./L] [µg a.i./L] [µg a.i./L]
1 0.0354 67 0.426 79 68.6 102
2 0.0402 76 0.427 80 69.6 104
3 0.0422 79 0.432 81 70.7 105
4 0.0423 80 0.474 88 68.7 102
5 0.0447 84 0.469 87 71.3 106
Mean 0.041 77 0.45 83 70 104
SD ± 0.004   0.02   1  
CV [%] 8.6   5.4   1.7  
Replicate Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides          
C28H60N          
1 x LOQ   10 x LOQ   1250 x LOQ  
Meas. % Meas. % Meas. %
conc.   conc.   conc.  
[µg a.i./L]   [µg a.i./L]   [µg a.i./L]]  
1 0.0211 57 0.333 89 47.6 101
2 0.0232 62 0.314 84 46.3 99
3 0.0230 62 0.320 85 48.1 102
4 0.0220 59 0.320 85 47.2 101
5 0.0236 64 0.307 82 48.3 103
Mean 0.023 61 0.32 85 47.5 101
SD ± 0.001   0.01   0.8  
CV [%] 4.5   3.0   1.7  

Meas. conc. = measured concentration of each component of the test item, dilution factor taken into account
% = percent concentration of the fortified sample
SD = standard deviation
CV = coefficient of variation

 

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of C12 -18 DAQ
Active ingredient: C30H64N and C32H68N

Replicate Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
C30H64N
1 x LOQ 10 x LOQ 1250 x LOQ
Meas. % Meas. % Meas. %
conc. conc. conc.
[µg a.i./L] [µg a.i./L] [µg a.i./L]
1 0.0162 59 0.228 82 34.9 101
2 0.0158 58 0.234 85 35.6 103
3 0.0168 61 0.211 76 36.2 105
4 0.0170 62 0.227 82 34.4 100
5 0.0157 57 0.216 78 35.0 101
Mean 0.0163 59 0.22 81 35.2 102
SD ± 0.0006   0.01   0.7  
CV [%] 3.4   4.2   1.9  
Replicate Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
C32H68N
1 x LOQ   10 x LOQ   1250 x LOQ  
Meas. % Meas. % Meas. %
conc.   conc.   conc.  
[µg a.i./L]   [µg a.i./L]   [µg a.i./L]  
1 0.0134 62 0.211 97 27.9 102
2 0.0133 62 0.192 88 28.5 104
3 0.0136 63 0.218 100 29.2 107
4 0.0136 63 0.183 84 27.0 99
5 0.0138 64 0.207 95 27.8 102
Mean 0.0136 63 0.20 93 28.1 103
SD ± 0.0002   0.01   0.8  
CV [%] 1.6   7.0   2.9  

Meas. conc. = measured concentration of each component of the test item, dilution factor taken into account
% = percent concentration of the fortified sample
SD = standard deviation
CV = coefficient of variation

 

Measured Concentrations and Percent of Nominal Concentrations of the Fortified Samples of C12 -18 DAQ
Active ingredient: C34H72N

Replicate Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
C34H72N
1 x LOQ 10 x LOQ 1250 x LOQ
Meas. % Meas. % Meas. %
conc. conc. conc.
[µg a.i./L] [µg a.i./L] [µg a.i./L]
1 0.00451 64 0.0587 82 8.94 100
2 0.00458 65 0.0711 100 9.31 105
3 0.00457 65 0.0662 93 9.13 103
4 0.00449 64 0.0678 95 8.68 97
5 0.00444 63 0.0654 92 9.43 106
Mean 0.00452 64 0.066 92 9.1 102
SD ± 0.00006   0.005   0.3  
CV [%] 1.3   6.9   3.3  

Meas. conc. = measured concentration of each component of the test item, dilution factor taken into account
% = percent concentration of the fortified sample
SD = standard deviation
CV = coefficient of variation

 

The stability of the fortified samples at the 10xLOQ was checked after 14 days and was between 89 and 120% of the initially measured concentration for each component.

 

Procedural Recovery

A procedural recovery (Quality Control) on 10x LOQ Level was freshly prepared on each day of analysis. It was treated in parallel to the test samples.

Measured Concentrations and Percent of Nominal Concentration of the Quality Control during the Definitive Test

Sampling 0 hours 24 hours 48 hours 72 hours
date
Component Meas. % Meas. % Meas. % Meas. %
conc. conc. conc. conc.
[µg a.i./L] [µg a.i./L] [µg a.i./L] [µg a.i./L]
C24H52N 0.146 102 0.154 107 0.134 93 0.144 100
C26H56N 0.535 101 0.620 116 0.525 99 0.545 103
C28H60N 0.369 99 0.376 101 0.347 93 0.397 107
C30H64N 0.251 91 0.275 100 0.261 95 0.268 98
C32H68N 0.199 92 0.217 101 0.206 95 0.205 95
C34H72N 0.0731 103 0.0789 112 0.0792 112 0.0721 102
Sampling 72 hours1)  
date
Component Meas. %  
conc.
[µg a.i./L]
C24H52N 0.144 100  
C26H56N 0.558 105  
C28H60N 0.369 99  
C30H64N 0.257 94  
C32H68N 0.218 101  
C34H72N 0.0694 98  

Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the nominal concentration
a.i. = active ingredient
Quality Control = 0.200 µg test item/L, weighing factor taken into account
1) = sample set for glass adsorption samples

 

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the test substance 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 substance loadings and TWA concentration of the test substance):
The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were determined to be 58.2 μg/L (55.64 μg a.i./L) and 31.6 μg/L (30.2 μg a.i./L) based on the nominal loading rates of the test substance and 3.65 μg/L (3.49 μg a.i./L) and 2.53 μg/L (2.42 μg a.i./L) based on the time-weighted mean measured test item concentrations respectively. The NOEL for both inhibition of growth rate and yield after 72 hours were determined to be 20 μg/L (19.12 μg a.i./L) and <0.632 μg/L (<0.604 μg a.i./L) based on the nominal loading rates of the test substance and 1.74 μg/L (1.66 μg a.i./L) and <0.101 μg/L (<0.096 μg a.i./L) based on the time-weighted mean measured test item concentrations respectively (Scheerbaum, 2021).
Executive summary:

 


A study was conducted to determine the fresh water algal growth inhibition of the test substance, C12-18 DAQ(95.7% active), according to the OECD Guideline 201 and EU method C.3, in compliance with GLP. 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 4886 cells/mL. The test substance is a UVCB substance with constituents of different water solubility and therefore in agreement with OECD guidance 23 the Water Accommodated Fractions (WAF) were prepared. Six WAFs were prepared 24 ± 1 hour prior to the start of exposure with nominal loading rates of the test substance in the range of 0.632 to 200 µg/L set up in a geometric series with a factor of √10: 0.632 - 2.00 - 6.32 - 20.0 - 63.2 - 200 µg/L. For the preparation of the loading rates, the test substance was melted at 70°C and resuspended in methanol (2 g/L). The test substance with methanol was applied onto a curved glass slide and the methanol was evaporated. The glass slide with the test substance was inserted in a glass flask with an appropriate amount of dilution water. A slow stirring procedure was applied for 24 ± 1 hour at room temperature. The magnetic stirrer bar was placed with a fish-clip® system a few centimetres above the bottom of the flask to prevent direct contact with the test substance on the bottom. After a separation phase of 1 hour, the aqueous phase of the WAF was removed by siphoning (from the approximate middle of the glass flask). The WAFs were checked via laser beam (Tyndall effect) for undissolved test substance (formation of an emulsion). The Tyndall effect was negative. The resulting water accommodated fractions (WAF) were used in the test. The test vessels were pre-saturated with the WAFs for at least 12 hours. Three replicates were tested for each test substance 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 substance C12-18 DAQ was determined at the start of the exposure (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) of all tested concentration levels and the control via LC-MS/MS with algae. Additionally, the loading rate of 6.32 µg/L was analytically verified without algae determined at the start of the exposure (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) via LC-MS/MS. The time-weighted average concentration of the test substance 0.101 – 0.159 – 0.488 – 1.74 – 3.71 – 3.97 μg/L were calculated despite the fact that a WAF approach is used. The test substance is a UVCB substance with constituents of different water solubility which means that per the definition of the WAF approach, the dose-response can only be based on loading rates because partly dissolved compounds and mixtures cannot be related to concentrations.


 


Table 1: NOEL, LOEL and ELx-values and 95% Confidence Intervals of C12-18 DAQ (0 - 72 hours) based on the nominal test substance loadings and time-weighted average (TWA) concentrations [µg/L]


 


































































 



InhibitionofGrowthRate (µg/L) (nominal)



InhibitionofGrowthRate (µg/L) (TWA measured)



NOEL



20.0



1.74



LOEL



63.2



3.71



ErL10



30.0(21.5–43.0)



3.04(1.82–3.69)



ErL20



39.1(29.1–48.1)



3.30(1.99–3.69)



ErL50



58.2(51.0–61.1)



3.65(2.21–3.96)



 



InhibitionofYield


(µg/L) (nominal)



InhibitionofYield


(µg/L) ((TWA measured)



NOEL



<0.632



<0.101



LOEL



≤0.632



≤0.101



EyL10



n.d.



n.d.



EyL20



19.7(14.9–30.9)



1.72(1.32–2.67)



EyL50



31.6(25.5–42.7)



2.53(2.12–3.20)



 


 


Under the study conditions, the test substance was found to inhibit the growth of the freshwater green algae Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the nominal test substance loadings and TWA concentration of the test substance):


The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were determined to be 58.2 μg/L (55.64 μg a.i./L) and 31.6 μg/L (30.2 μg a.i./L) based on the nominal loading rates of the test substance and 3.65 μg/L (3.49 μg a.i./L) and 2.53 μg/L (2.42 μg a.i./L) based on the time-weighted mean measured test item concentrations respectively. The NOEL for both inhibition of growth rate and yield after 72 hours were determined to be 20 μg/L (19.12 μg a.i./L) and <0.632 μg/L (<0.604 μg a.i./L) based on the nominal loading rates of the test substance and 1.74 μg/L (1.66 μg a.i./L) and <0.101 μg/L (<0.096 μg a.i./L) based on the time-weighted mean measured test item concentrations respectively (Scheerbaum, 2021).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 23, 2010 to February 26, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Bulk approach applied, Natural water used
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Bulk approach applied, Natural water used
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- A sample of the stock solution was taken and a sample from each replicate of each concentration including the control was pooled at the beginning
of the test just prior to addition of the algae.
- At the end of the test the 0.19 mg/L replicate was sampled and filtered prior to analysis.
- Samples were analyzed immediately applying the analytical procedure as described below. Using a validated extraction procedure also described in the report , the test chemical bound to the glassware at 0.19 mg/L was determined analytically.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 50.6 mg/L of the test substance was prepared by accurately weighing 50.6 mg of test substance, on an analytical balance. The test chemical was then added to approximately 80 mL the test media and left to stir thoroughly. To ensure a homogeneous dispersion the mixture was sonicated for 2 min. A slightly opaque homogeneous stock solution was achieved. After 1 h cooling whilst stirring this solution was transferred to a 1 L volumetric flask and was filled to the mark with the test media.

- The test solutions were prepared by addition of the adequate amounts of stock solution to the test vessels containing dilution water. The final volume of all vessels was 40 mL. A control containing only test medium was included in the test.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: P. subcapitata (CCAP 278/4)
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland, UK
- Method of cultivation: After purchasing this strain was cultured and maintained. Cultures on sloped agar tubes were stored at 4°C until required. The initial stock culture was inoculated with P. subcapitata from a sloped agar tube and checked for purity by microscopic means. This algal stock culture (40 mL) of P. subcapitata was regularly transferred to fresh medium to act as inoculum for testing. The absorbance of an exponentially growing stock culture was measured. The cell density was determined using the calibration curve described below. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1x10(4) cells/mL in the test medium.


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
- At the end of the test 5 random samples were microscopically checked for purity of the algal culture. The 5 random samples checked at the end of the test pure and were not significantly contaminated with bacteria, although due to natural river water being used some bacteria were present.
Hardness:
ºdH = 6.62
Ca = 40.6mg/L
Mg=4.03 mg/L

Test temperature:
21.5- 22.35 °C
pH:
8.0

Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
- Nominal concentrations: 0, 0.02, 0.06, 0.19, 0.625 and 2.0 mg/L.
- Measured concentrations were >80% of the nominal concentrations in the highest 3 concentrations and the stock solution. The recovery fell slightly below 80% in the other concentrations. Enough evidence is present to demonstrate that the correct amount of test substance was added to the test vessels. Loss can be accounted by adhesion to suspended matter and is expected.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type of flow-through (e.g. peristaltic or proportional diluter): Test vessels were swirled continuously at a speed sufficient to prevent sedimentation of the algae.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vehicle per control(replicates): 3 (without algae)

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Light intensity and quality: 60 to 120 µm-2.s-1, continuous uniform illumination was provided in the spectral range of 400 to 700 nm by using fluorescent lamps at a distance of about 0.36 - 0.02 m from the algal cultures.
- The test was carried out in a temperature controlled illuminated orbital incubator, in which the temperature was maintained at 23 ± 2°C. The test vessels were swirled continuously at a speed sufficient to prevent sedimentation of the algae.

Reference substance (positive control):
yes
Remarks:
Pottasium dichromate conducted twice annually
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 0.13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 0.022-0.224 (95 % CL)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 0.386 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 0.236-0.618 (95 % CL)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 0.062 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.006-0.0985 (95 % CL)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 0.148 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.088-0.217 (95 % CL)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.06 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 0.19 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
A dose response curve with confidence limits was observed for both rate and biomass endpoints.
Results with reference substance (positive control):
- The sensitivity of the algae was checked by performing a growth inhibition test with a reference compound (potassium dichromate) twice a year,
when the cultures are transferred from the sloped agar tubes. The sensitivity was tested for compliance with the guidelines.
- The observed EC50 values were between 0.25 and 2.0 mg/L.
Reported statistics and error estimates:
- The EC10, 50 values were computed from the best fitted line (least-squares method) through the points given by the probit of the percentage of inhibition and the logarithm of the concentration of the test substance.
- The LOEC was determined by comparison of the growth at each concentration and the control using threshold values from the William’s test. - Confidence limits were computed on the basis of Fieller’s theorem if possible.
- All computations were performed using the TOXCALC version 5.0 program.

Results

Preliminary test

During the preliminary range finding test in natural surface water, performed as a non-GLP test, concentrations of 0.001, 0.01, 0.05, 0.1 and 1.0 mg/L were tested. Based on growth rate, a flat dose response effect was seen until 0.05 mg/L where effect was observed. A NOEC of 0.01 mg/L could be determined but an Erc10 & Erc50 could not accurately be estimated from these values

Definitive test

The results reported are based on nominal concentrations. The test substance caused inhibition of growth rate and therefore EC10 and EC50 values could be calculated by a maximum likely hood probit plot. NOEC and LOEC were also determined using a Dunetts test. The ErC10 (72h) was calculated 0.13mg/L. The ErC50 (72h) was calculated as 0.39 mg/L. The NOEC was determined as 0.06 mg/L and the LOEC as 0.19 mg/L. All values are reported for test chemical as received and are calculated based on growth rate.

Chemical analysis

All test concentrations and the stock solution, were analyzed at the beginning of the test. At the end of the test 0.19 mg/L replicate was also analyzed. The glassware from the same replicate was extracted to determine the loss of the substance to glass.

Chemical analysis

Samples

T=0 % of nominal

T=72h % of nominal

Glass % of nominal

Control

--

 

 

0.02 mg/L

72.0

 

 

0.06 mg/L

78.3

 

 

0.19 mg/L

85.9

24.5**

42.1**

0.625 mg/L

83.8

 

 

2.0 mg/L

96.3

 

 

Stock sol.

92.9

 

 

* Nominal stock concentration was 50.4 mg/L

**Averages of three results

Absorbance values and inhibition percentages in the algal growth inhibition test

Concentration

Time (hours)

Area

Inhibition

Specific

Inhibition

 

(mg/L)

 

0h

 

24h

 

48h

 

72h

 

 

(%)

Growth

rate

 

(%)

0.000

0.005

0.034

0.181

0.645

12.600

 

0.068

 

0.000

0.007

0.035

0.195

0.652

12.924

 

0.064

 

0.000

0.006

0.032

0.192

0.667

13.020

 

0.066

 

0.000

0.007

0.034

0.199

0.653

13.008

 

0.064

 

0.000

0.007

0.036

0.201

0.673

13.344

 

0.064

 

0.000

0.009

0.032

0.199

0.654

12.852

 

0.061

 

Mean

0.007

0.034

0.195

0.657

12.958

 

0.064

0.000

 

 

 

 

 

 

 

 

 

0.020

0.007

0.034

0.198

0.651

12.960

 

0.065

 

0.020

0.007

0.032

0.189

0.643

12.600

 

0.065

 

0.020

0.008

0.031

0.161

0.650

11.928

 

0.063

 

Mean

0.007

0.032

0.183

0.648

12.496

3.565

0.064

0.322

 

 

 

 

 

 

 

 

 

0.060

0.011

0.025

0.160

0.623

11.256

 

0.064

 

0.060

0.011

0.027

0.189

0.667

12.528

 

0.065

 

0.060

0.010

0.029

0.170

0.625

11.676

 

0.066

 

Mean

0.011

0.027

0.173

0.638

11.820

8.782

0.065

-0.862

 

 

 

 

 

 

 

 

 

0.190

0.008

0.015

0.050

0.237

3.924

 

0.050

 

0.190

0.008

0.012

0.059

0.308

4.920

 

0.055

 

0.190

0.008

0.013

0.066

0.302

5.040

 

0.054

 

Mean

0.008

0.013

0.058

0.282

4.628

64.285

0.053

17.798

 

 

 

 

 

 

 

 

 

0.625

0.007

0.009

0.015

0.025

0.456

 

0.018

 

0.625

0.006

0.006

0.010

0.020

0.264

 

0.017

 

0.625

0.007

0.007

0.009

0.021

0.216

 

0.015

 

Mean

0.007

0.007

0.011

0.022

0.312

97.592

0.016

74.380

 

 

 

 

 

 

 

 

 

2.000

0.005

0.000

0.000

0.010

-0.180

 

0.007

 

2.000

0.004

0.000

0.000

0.009

-0.132

 

0.007

 

2.000

0.005

0.000

0.000

0.009

-0.192

 

0.006

 

Mean

0.005

0.000

0.000

0.009

-0.168

101.296

0.007

89.891

*No growth occurred values nominalized at 100% negative measurements due to suspended solid absorbance.

Validity Criteria

 

Average specific growth rates and coefficients of variation for average specific growth rates during the whole test period in the control:

0-24

24-48

48-72

72

0.08

0.07

0.05

0.07

0.07

0.07

0.05

0.06

0.07

0.07

0.05

0.07

0.07

0.07

0.05

0.06

0.07

0.07

0.05

0.06

0.05

0.08

0.05

0.06

 

 

mean

0.06

 

 

stdev

0.00

 

 

CV%

4.21

 

Mean coefficient of variation for section by section specific growth rates in the control:

 

 

mean

stdev

%CV

control 1

0-24 and 24-48

0.07

0.01

9.65

control 2

0-24 and 24-48

0.07

0.00

4.60

control 3

0-24 and 24-48

0.07

0.00

4.81

control 4

0-24 and 24-48

0.07

0.01

7.88

control 5

0-24 and 24-48

0.07

0.00

3.46

control 6

0-24 and 24-48

0.06

0.02

25.54

mean

 

0.07

0.01

9.32

control 1

24-48 and 48-72

0.06

0.01

19.29

control 2

24-48 and 48-72

0.06

0.02

24.69

control 3

24-48 and 48-72

0.06

0.02

25.45

control 4

24-48 and 48-72

0.06

0.02

27.69

control 5

24-48 and 48-72

0.06

0.02

24.70

control 6

24-48 and 48-72

0.06

0.02

29.89

mean

 

0.06

0.02

25.28

Test parameters and summary of the results: 

Parameter

Name

Dimension

Value

N0

Inoculum

Cells/mL

1 x 104Approx

N72

 Mean number of cells of the control at the end of the test .

Cells/mL

1.51 x 106

m

growth rate (control)

h-1

0.06

-

increase factor of cell growth over 72 h

-

151

NOEC

no observed effect concentration (Based on Growth Rate)

mg/L

0.06

LOEC

Lowest observed effect concentration (Based on Growth Rate)

mg/L

0.19

 

Value (mg/L)

95% confidence limits

EbC10

 

0.062

0.006

0.0985

EbC20

 

0.084

0.016

0.121

EbC50

 

0.148

0.088

0.217

EbC80

 

0.263

0.188

0.978

ErC10

 

0.134

0.022

0.224

ErC20

 

0.192

0.054

0.294

ErC50

 

0.386

0.236

0.618

ErC80

 

0.774

0.512

2.58

The following quality criteria have been met in the present study:

• The cell density of the controls increased at least a factor 16 within 72 hours.

• The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures should not exceed 7%.

• The EC50 values of the reference compound, potassium dichromate, should be in the range of 0.25-2.0 mg/L.

• The mean coefficient of variation for section-by-section specific growth rates in the control did not exceed 35 %.

Conclusion

After correction for background noise the stock concentration and the highest 3 concentrations were found to be within ± 20 % of their nominal concentrations. Analytical recovery in the remaining test concentrations decreased as the concentrations decreased to a level below 80% of the nominal concentrations. Taking into consideration the stock analysis and the results obtained from the highest concentrations it is clear that the correct nominal amount of test substance was added to each replicate. However due to the characteristics of this substance, particularly its tendency to adhere to organic matter greater than 80% recovery at progressively lower concentrations was not possible. Quality criteria for the analytical apparatus were met and the apparatus was considered to be working correctly during analysis. A validated glass extraction method demonstrated that 42.1% of the test chemical was bound to the glass wear. Measurement of the remaining dissolved fraction at 0.19 mg/L demonstrates that 24.5% of the test chemical was present in solution. Due to the test substance not being significantly biodegradable during the test period and considering that 42.1% of the test chemical was found to be bound to the glass the remainder substance can be assumed to be bound to suspended solids or algae. The test organisms were therefore exposed to the test chemical in keeping with the bulk approach as far as was possible. The adsorption to glass was higher than expected. Applying this as a correction factor to calculate exposure to available fraction of the test chemical in the other concentrations is not considered an appropriate way to display this data as the level of absorption to glass would vary dependant on the test concentration and no data on the kinetics of the binding is known. However this does demonstrate that this test chemical binds significantly to negatively charged surfaces as well as algae and suspended matter. It can be concluded that under environmental conditions such binding would also occur, very likely to an even  greater extent. It is therefore considered appropriate to continue to apply the bulk approach to this chemical. Study director concludes that all possible measures were taken to recover the test  substance, and that the substance was exposed to the test material in keeping with the bulk approach as far as was possible despite full analytical recovery not being achieved. Nominal values were therefore used for endpoint calculations. Loss of the test substance to organic material and negatively charged surfaces is acceptable and would occur to an even greater extent environmentally.

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 72 h ErC10, ErC50, NOEC and LOEC for inhibition based on growth rate were calculated to be 0.13, 0.39, 0.06 and 0.19 mg/L, respectively.

Executive summary:

A study was conducted to determine the  fresh water algal growth inhibition of the test substance, C12-18 DAQ (99.6% active), according to the OECD Guideline 201, in compliance with GLP. The toxicity of the test chemical to an exponentially growing culture of Pseudokirchnerella subcapitata was determined over an exposure period of 72 h with nominal concentrations of 0.02, 0.06, 0.19, 0.625 and 2.0 mg/L. The test was carried out according to the bulk approach using enriched natural surface water with a low Dissolved Organic Carbon (DOC) and Total Suspended Solids (TSS) content, allowing a more environmentally realistic determination of the effects of the test chemical to be made. Nominal concentrations were used for calculation of the effect levels. For the highest 3 concentrations a statistically significant effect in comparison to the control was found. The 72 h ErC10, ErC50, NOEC and LOEC for inhibition based on growth rate were calculated to be 0.13, 0.39, 0.06 and 0.19 mg/L, respectively. The 72 h EbC10, EbC50, NOEC and LOEC for inhibition based on biomass were calculated to be 0.062, 0.148, 0.06 and 0.19 mg/L, respectively. The study fulfilled the validity criteria: (a) The average increase of the absorbance in the control over 72 h by a factor of 66 (b) The coefficient of variation of average specific growth rates during the whole  test period in the replicate control cultures did not exceed 7% (c) The mean coefficient of variation for section-by-section specific growth rates in the control did not exceed 35%. Under the study conditions, the 72 h ErC50, ErC10, NOEC and LOEC for inhibition based on growth rate were calculated to be 0.39, 0.13, 0.06 and 0.19 mg/L, respectively (Kean, 2010).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From March 24, 1998 to March 27, 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to guideline
Guideline:
other: ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
Sampling not performed
Vehicle:
no
Details on test solutions:
A stock solution of the test substance was prepared by dissolving 1.0 g of the test substance with about 80 mL of deionised water. After stirring the volume was made up to a final volume of 1000 mL. The test solutions were prepared by addition of the required amounts of stock solution to the test vessels to obtain the following final nominal test concentration: 0.04, 0.09, 0.020, 0.043, 0.096 and 0.210 mg/mL. The extinction in each erlenmeyer was measured after 0, 24, 48 and 72 hours. Algal medium was used a blank in the spectrophotometer.
Test organisms (species):
Phaeodactylum tricornutum
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, The Ferry house, Cumbria, Ambleside, United Kingdom
- Age of inoculum (at test initiation):
- Method of cultivation: After purchasing this strain was cultured and maintained according to Standard Operation Procedure E 3. Cultures on sloped agar tubes were stored at 4°C until required.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No data
Hardness:
Not measured
Test temperature:
19.6- 20.4 ⁰C
pH:
7.9- 8.4
Dissolved oxygen:
Not measured
Salinity:
Not measured
Nominal and measured concentrations:
Nominal concentrations: 0.004, 0.009, 0.020, 0.043, 0.096 and 0.210 mg/L

Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL erlenmeyers flasks
- Type (delete if not applicable): Closed-closed with cotton wool stoppers
- Material, size, headspace, fill volume: 100 mL erlenmeyers containing 40 mL of medium

GROWTH MEDIUM
The initial stock culture was inoculated with Phaeodactylum tricornutum from a sloped agar tube and checked for purity by microscopic means. This algal stock culture (40 mL) of Phaeodactylum tricornutum was regularly transferred to fresh medium to act as inoculum for testing. The extinction of an exponentially growing stock culture was measured. The cell density was determined using a calibration curve. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1 x 10^4 cell/mL in the test medium

OTHER TEST CONDITIONS
The culturing apparatus was a temperature-controlled illuminated orbital incubator in which the temperature was kept at 20 ± 1 °C and a continuous uniform illumination was provided in the spectral range of 400 to 700 nm by using 30 W fluorescent lamps of the type ‘universal white’ (colour temperature of approx. 4000 K) at a distance of approximately 0.35 m from the algal cultures. The culture flasks were rotated continuously at 100 rev/min to prevent sedimentation of the algae.

Test principle and procedures
For the test an amount of test medium was prepared in a 3-litre vessel. This medium was sterilized by filter sterilization (0.2 µm). The inoculum was added from an exponentially growing culture. Adequate amounts of stock solution were added to the test vessels {100 ml erlenmeyers). The test vessels were filled with inoculated medium upto a total volume of 40 ml using a sterilized dispenser. In addition six control replicates were included. The extinction in each erlenmeyer was measured after 0, 24, 48 and 72 hours. Algal medium was used as a blank in the spectrophotometer.

Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
other: EbC50
Effect conc.:
ca. 0.06 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 0.06-0.08 mg/L
Remarks:
equivalent to 0.045 mg a.i./L
Key result
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
ca. 0.13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.11-0.14 mg/L
Remarks:
equivalent to 0.098 mg a.i./L
Key result
Duration:
72 h
Dose descriptor:
other: ErC10
Effect conc.:
0.05 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.04-0.06 mg/L
Remarks:
equivalent to 0.038 mg a.i./L
Key result
Duration:
72 h
Dose descriptor:
other: EbC10
Effect conc.:
0.02 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 0.01-0.02 mg/L
Remarks:
equivalent to 0.015 mg a.i./L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.009 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate / biomass
Remarks on result:
other: equivalent to 0.0068 mg a.i./L
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 0.02 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks on result:
other: equivalent to 0.015 mg a.i./L
Details on results:
The 72 h EbC50 and ErC50 of test substance were 0.06 and 0.13 mg/L respectively. The NOEC determined from the results of the test compound is 0.009 mg/L, the LOEC is 0.02 mg/L.
Results with reference substance (positive control):
- EbC50 and Erc50 values of the reference compound, potassium dichromate (5.4 and 27 mg/L, respectively, the increase of the extinction of the control over 72 h by a factor of 93 and by a maximum deviation of the pH of 0.5 unit
Reported statistics and error estimates:
- Confidence limits were computed on the basis of Fieller's theorem. All computations were performed using the Akzo computer programme 'Algal' version 3.3. made in SAS.

Table 1. Mean Extinctions in definitive inhibition test.

Concentration

0h

24h

48 h

72 h

Control

0.005

0.021

0.098

0.430

0.004

0.005

0.021

0.095

0.414

0.009

0.004

0.018

0.092

0.405

0.02

0.004

0.018

0.086

0.369

0.043

0.005

0.019

0.089

0.397

0.096

0.005

0.007

0.038

0.172

0.210

0.005

0.000

0.003

0.007

 

Table 2. Inhibition algal growth and growth rate

Conc substance (in mg/L)

% inhibition growth

% inhibition growth rate

control

-

-

0.004

3.7

0.9

0.009

6.3

0.6

0.020

14.0

3.0

0.043

8.9

1.5

0.096

63.3

17.4

0.210

n.d.

n.d.

n.d. = not determined

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 72 h EbC50, ErC50, EbC10, ErC10, NOEC and LOEC values of the test substance for Phaeodactylum tricornutum were determined to be 0.06, 0.13, 0.02, 0.05, 0.009 and 0.02 mg/L, respectively (equivalent to 0.045, 0.098, 0.015, 0.038, 0.0068 and 0.015 mg a.i./L, respectively)


Executive summary:

A study was conducted to determine the marine algal growth inhibition of the test substance, C12-18 DAQ (75% active) to Phaeodactylum tricornutum in accordance with ISO test guideline 10253 in compliance with GLP. The toxicity of test substance to exponentially growing Phaeodactylum tricornutum was determined over an exposure period of 72 h. The test was conducted in a mineral salts medium at a temperature of 20± 1°C in an illuminated orbital incubator. The pH in the test media varied from 7.9 to 8.4. Based on the results of a range finding test, a definitive test was carried out at nominal concentrations of 0.004, 0.009, 0.02, 0.043, 0.096 and 0.21 mg/L. No chemical analyses of the test concentrations were performed during the test and all concentrations mentioned are nominal concentrations of the test substance. The definitive test is valid as shown by the EbC50 and ErC50 values of the reference compound, potassium dichromate (5.4 and 27 mg/L, respectively), the increase of the extinction of the control over 72 h by a factor of 93 and by a maximum deviation of the pH of 0.5 unit. Under the study conditions, the 72 h EbC50, ErC50, EbC10, ErC10, NOEC and LOEC values of the test substance for Phaeodactylum tricornutum were determined to be 0.06, 0.13, 0.02, 0.05, 0.009 and 0.02 mg/L, respectively (equivalent to 0.045, 0.098, 0.015, 0.038, 0.0068 and 0.015 mg a.i./L, respectively) (Van Wijk, 1998).

Description of key information

Three algae studies are available for C12-18 DAQ: Two Klimisch 1 studies performed under freshwater conditions and one Klimisch 2 study under marine conditions.  


DAQ C12-18 is a multi-component mixture (UVCB) of cationic surface-active constituents with different water solubilities. The fate of cationic surfactants in general deviates from standard chemicals. These substances are therefore considered as difficult substances for which the results of standard guideline studies are very difficult to interpret when considering them in a standard way. The two available freshwater algae tests were therefore performed following two different approaches. Both studies are considered to be of equal quality but are performed for different purposes. One test which is focused on determining the intrinsic toxicity of C12-18 DAQ (for C&L purposes) is performed according to the Water Accommodated Fraction (WAF) approach as described in OECD guidance No. 23. The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration. The other test which is suited to derive a realistic risk ratio for the aquatic compartment is performed according to the PECaquatic bulk/PNECaquatic bulk approach as described in ECETOC Technical Report is used in the environmental risk assessment to cope with the earlier mentioned lack of realistic PEC estimation and problems observed with the reproducibility of the specific chemical analyses.   


For the test performed using the WAF approach, the dose-response results were calculated based on nominal Test Loadings and on Time Weighted Average (TWA) measured concentrations. The ErL10/ErC10 for reproduction after 72 hours is 28.7/2.91 µg a.i./L. The ErL50/ErC50 after 72 hours is 55.64s.0/3.49 µg a.i./L. The TWA results are presented here despite the fact that per the definition of the WAF, all terms related to concentration level should be given as loading rates because partly dissolved compounds and mixtures cannot be related to concentrations. For the test performed according to the Bulk approach, the dose-response results are calculated based on nominal test concentrations corrected for active ingredient and sorption to glassware. The 72h ErC10, ErC50, NOEC and LOEC for inhibition based on growth rate were calculated to be 0.075, 0.226, 0.035 and 0.11 mg/L, respectively.    


Therefore, for freshwater, based on the WAF approach, the lower 72 h ErC50 = 55.64 µg a.i./L and ErC10 = 28.68 µg a.i./L (loading concentration) for effects on growth rate determined in the recently conducted algae study has been considered for C&L purposes. While based on the Bulk approach, the ErC10 = 0.0753 mg a.i./L based on effects on growth rate has been considered for the risk assessment. ​

Key value for chemical safety assessment

EC50 for freshwater algae:
55.64 µg/L
EC50 for marine water algae:
98 µg/L
EC10 or NOEC for freshwater algae:
28.68 µg/L
EC10 or NOEC for marine water algae:
38 µg/L

Additional information

Three algae studies are available for C12 -18 DAQ: Two studies performed under freshwater conditions and one under marine conditions.

Freshwater: 

Study 1:A study was conducted to determine the fresh water algal growth inhibition of the test substance, C12-18 DAQ (95.7% active), according to the OECD Guideline 201 and EU method C.3, in compliance with GLP. The aim of the study was the determination of the effects on growth rate and yield of the algaeover a period of 72 hours. The study was conducted withPseudokirchneriella subcapitataunder static conditions with an initial cell density of 4886 cells/mL. The test substance is a UVCB substance with constituents of different water solubility and therefore in agreement with OECD guidance 23 the Water Accommodated Fractions (WAF) were prepared. Six WAFs were prepared 24 ± 1 hour prior to the start of exposure with nominal loading rates of the test substance in the range of 0.632 to 200 µg/L set up in a geometric series with a factor of √10: 0.632 - 2.00 - 6.32 - 20.0 - 63.2 - 200 µg/L. For the preparation of the loading rates, the test substance was melted at 70°C and resuspended in methanol (2 g/L). The test substance with methanol was applied onto a curved glass slide and the methanol was evaporated. The glass slide with the test substance was inserted in a glass flask with an appropriate amount of dilution water. A slow stirring procedure was applied for 24 ± 1 hour at room temperature. The magnetic stirrer bar was placed with a fish-clip® system a few centimetres above the bottom of the flask to prevent direct contact with the test substance on the bottom. After a separation phase of 1 hour, the aqueous phase of the WAF was removed by siphoning (from the approximate middle of the glass flask). The WAFs were checked via laser beam (Tyndall effect) for undissolved test substance (formation of an emulsion). The Tyndall effect was negative. The resulting WAF were used in the test. The test vessels were pre-saturated with the WAFs for at least 12 hours. Three replicates were tested for each test substance 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 substance C12-18 DAQ was determined at the start of the exposure (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) of all tested concentration levels and the control via LC-MS/MS with algae. Additionally, the loading rate of 6.32 µg/L was analytically verified without algae determined at the start of the exposure (0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) via LC-MS/MS. The time-weighted average concentration of the test substance 0.101 – 0.159 – 0.488 – 1.74 – 3.71 – 3.97 μg/L were calculated despite the fact that a WAF approach is used. The test substance is a UVCB substance with constituents of different water solubility which means that per the definition of the WAF approach, the dose-response can only be based on loading rates because partly dissolved compounds and mixtures cannot be related to concentrations.  

Table: NOEL, LOEL and ELx-values and 95% Confidence Intervals of C12-18 DAQ (0 - 72 hours) based on the nominal test substance loadings and time-weighted average (TWA) concentrations [µg/L]    

 

Inhibition of growth rate (µg/L) (nominal)

Inhibition of growth rate (µg/L) (TWA measured)

NOEL

20.0

1.74

LOEL

63.2

3.71

ErL10

30.0(21.5–43.0)

3.04(1.82–3.69)

ErL50

58.2(51.0–61.1)

3.65(2.21–3.96)

 

 

Inhibition of Yield

(µg/L) (nominal)

Inhibition of Yield

(µg/L) ((TWA measured)

NOEL

<0.632

<0.101

LOEL

≤0.632

≤0.101

EyL10

n.d.

n.d.

EyL50

31.6(25.5–42.7)

2.53(2.12–3.20)

 

Under the study conditions, the test substance was found to inhibit the growth of the freshwater green algaePseudokirchneriella subcapitataafter 72 hours with the following effect values (based on the nominal test substance loadings and TWA concentration of the test substance): 

The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were determined to be 58.2 μg/L (55.64 μg a.i./L) and 31.6 μg/L (30.2 μg a.i./L) based on the nominal loading rates of the test substance and 3.65 μg/L (3.49 μg a.i./L) and 2.53 μg/L (2.42 μg a.i./L) based on the time-weighted mean measured test item concentrations respectively. The NOEL for both inhibition of growth rate and yield after 72 hours were determined to be 20 μg/L (19.12 μg a.i./L) and <0.632 μg/L (<0.604 μg a.i./L) based on the nominal loading rates of the test substance and 1.74 μg/L (1.66 μg a.i./L) and <0.101 μg/L (<0.096 μg a.i./L) based on the time-weighted mean measured test item concentrations respectively (Scheerbaum, 2021). 

Study 2:A study was conducted to determine the fresh water algal growth inhibition of the test substance, C12-18 DAQ (99.6% active), according to the OECD Guideline 201, in compliance with GLP. The toxicity of the test chemical to an exponentially growing culture ofPseudokirchnerella subcapitatawas determined over an exposure period of 72 h with nominal concentrations of 0.02, 0.06, 0.19, 0.625 and 2.0 mg/L. The test was carried out according to the bulk approach using enriched natural surface water with a low Dissolved Organic Carbon (DOC) and Total Suspended Solids (TSS) content, allowing a more environmentally realistic determination of the effects of the test chemical to be made. Nominal concentrations were used for calculation of the effect levels. For the highest 3 concentrations a statistically significant effect in comparison to the control was found. The 72 h ErC10, ErC50, NOEC and LOEC for inhibition based on growth rate were calculated to be 0.13, 0.39, 0.06 and 0.19 mg/L, respectively. The 72 h EbC10, EbC50, NOEC and LOEC for inhibition based on biomass were calculated to be 0.062, 0.148, 0.06 and 0.19 mg/L, respectively. The study fulfilled the validity criteria: (a) The average increase of the absorbance in the control over 72 h by a factor of 66 (b) The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures did not exceed 7% (c) The mean coefficient of variation for section-by-section specific growth rates in the control did not exceed 35%. Under the study conditions, the 72 h ErC10, ErC50, NOEC and LOEC for inhibition based on growth rate were calculated to be 0.13, 0.39, 0.06 and 0.19 mg/L, respectively (Kean, 2010).    

C12-18 DAQ is a multi-component mixture (UVCB) of cationic surface-active constituents with different water solubilities. The fate of cationic surfactants in general deviates from standard chemicals. These substances are therefore considered as difficult substances for which the results of standard guideline studies are very difficult to interpret when considering them in a standard way. The two available freshwater algae tests were therefore performed following two different approaches. Both studies are considered to be of equal quality but are performed for different purposes. 

One test which is focused on determining the intrinsic toxicity of C12-18 DAQ (for C&L purposes) is performed according to the Water Accommodated Fraction (WAF) approach as described in “OECD guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (No. 23 Feb. 2019). The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration. 

The other test which is suited to derive a realistic risk ratio for the aquatic compartment is performed according to the PECaquatic bulk/PNECaquatic bulk approach as described in ECETOC Technical Report “Environmental Risk Assessment of difficult substances” (TR 88, 2003). The so called “Bulk approach” is used in the environmental risk assessment to cope with the earlier mentioned lack of realistic PEC estimation and problems observed with the reproducibility of the specific chemical analyses. 

For the test performed using the WAF approach, the dose-response results were calculated based on nominal Test Loadings and on Time Weighted Average (TWA) measured concentrations. The ErL10/ErC10 for reproduction after 72 hours is 28.7/2.91 µg a.i./L. The ErL50/ErC50 after 72 hours is 56.0/3.49 µg a.i./L. The TWA results are presented here despite the fact that per definition of the WAF, all terms related to concentration level should be given as loading rates because partly dissolved compounds and mixtures cannot be related to concentrations. For the test performed according to the Bulk approach, the dose-response results are calculated based on nominal test concentrations corrected for active ingredient and sorption to glassware. The 72h ErC10, ErC50, NOEC and LOEC for inhibition based on growth rate were calculated to be 0.075, 0.226, 0.035 and 0.11 mg/L, respectively.  

The mitigation factor of the river water constituents can be calculated by taking the ratio between the bulk approach effect concentrations and the WAF approach effect concentrations based on loading: 

 ErC50 bulk/ErL50 WAF = 0.226/0.0560 = 4.0 

 ErC10 bulk/ErL10 WAF = 0.0753/0.0287 = 2.6    

Salt water:  

A study was conducted to determine the marine algal growth inhibition of the test substance, C12-18 DAQ (75% active) toPhaeodactylum tricornutumin accordance with ISO test guideline 10253 in compliance with GLP. The toxicity of test substance to exponentially growingPhaeodactylum tricornutumwas determined over an exposure period of 72 h. The test was conducted in a mineral salts medium at a temperature of 20± 1°C in an illuminated orbital incubator. The pH in the test media varied from 7.9 to 8.4. Based on the results of a range finding test, a definitive test was carried out at nominal concentrations of 0.004, 0.009, 0.02, 0.043, 0.096 and 0.21 mg/L. No chemical analyses of the test concentrations were performed during the test and all concentrations mentioned are nominal concentrations of the test substance. The definitive test is valid as shown by the EbC50 and ErC50 values of the reference compound, potassium dichromate (5.4 and 27 mg/L, respectively), the increase of the extinction of the control over 72 h by a factor of 93 and by a maximum deviation of the pH of 0.5 unit. Under the study conditions, the 72 h EbC50, ErC50, EbC10, ErC10, NOEC and LOEC values of the test substance forPhaeodactylum tricornutumwere determined to be 0.06, 0.13, 0.02, 0.05, 0.009 and 0.02 mg/L, respectively (equivalent to 0.045, 0.098, 0.015, 0.038, 0.0068 and 0.015 mg a.i./L, respectively) (Van Wijk, 1998).  

Therefore, based on the WAF approach, the lower 72 h ErC50 = 55.64 µg a.i./L and ErC10 = 28.68 µg a.i./L (loading concentration) for effects on growth rate, determined in the recently conducted algae study has been considered for C&L purposes. While based on the Bulk approach, the ErC10 = 0.0753 mg a.i./L based on effects on growth rate has been considered for the risk assessment.