Quaternary ammonium compounds, C18-22 (even numbered) alkyltrimethyl, chlorides; Docosyltrimethylammonium chloride; C18-22 TMAC

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From April 27, 2009 to June 07, 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The fractions C20 and C22 of C20/22 ATQ trocken were analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end).
- Sampling method: Samples at start and end of the test were taken directly from the test replicates.
- Sample storage conditions before analysis: All samples were stored at room temperature before preparation and before analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 40 mg/L was freshly prepared with test medium and treated with ultrasound for 30 min.
- Eluate: Natural river water
- Differential loading: 0.020 - 0.063 - 0.20 - 0.63 - 2.00 - 6.32 - 20.0 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicate

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

After 72 h of exposure, 0.5 – 1 mL aliquots of the test medium (i.e. samples of exposed algal cells) from the nominal concentrations of 2.00 – 20.0 mg/L (geometric mean measured 1.39 – 15.4 mg/L) and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 3 - 7 days under test conditions. The test substance effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 15.4 mg/L (highest geometric mean measured test concentration). Please refer to the respective table below.

Hardness:

Not measured

Test temperature:

Mean: 23 °C, Min: 22 °C, Max: 24 °C
Adsorption on Glass - Test:
Mean: 21.5 °C, Min: 21 °C, Max: 22 °C

pH:

Geometric mean measured test item concentration of C20/22 ATQ trocken1) H-value [mg/L]
Start; 0 h End; 72 h
15.4 7.98 7.87
4.39 7.97 7.84
1.39 7.95 7.91
0.443 7.93 7.92
0.128 7.91 7.89
0.0405 7.88 7.89
0.0110 7.84 7.89
Control 7.91 7.87

Adsorption on Glass-Test: Nominal test item concentration pH-value [mg/L]

Start; 0 h End; 72 h
6.32 7.88 7.96
2.00 7.88 7.93
0.630 7.89 7.90
Control 7.91 7.87

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- 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: 4996 cells/mL
- Control end cells density: Mean 1038146 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
Water of the river Innerste was used. This river is located in
Sarstedt / Ruthe. Water parameters are given below. Additionally 50 % of the components of the dilution water (total application volume 6.5 mL/L) acc. to the guideline were added to enable a sufficient growth of algae
Water Parameters of the River Water

River Innerste
Location D-31157 Sarstedt / Ruthe, Ruther Straße, Innerste
Sampling Date 2009-02-12
Weather on Day of Sampling dry, sunny, ca. 1 °C
Colour Slightly, turbid, yellowish
pH 8.08
Conductivity [µS/cm] 887
DOC [mg C/L] 2.8
TOC [mg C/L] 2.9
Ammonium-N [mg N/L] 0.163
Nitrate-N [mg N/L] 6.10
o-Phosphate-P [mg P/L] 0.103
Total Phosphate [mg P/L] 0.152
Suspended Matter [mg/L] 24
Total Hardness [mg CO3/L] 338




OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0-72 h)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
10 45 89
1 0 0
0.1 -1 -3
0.01 -1 -4

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.927 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.798 – 1.07

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 3.28-3.70

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 0.955 – 1.30

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.412 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 0.0110 – 0.757

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: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes

- EC50:
Rate-related inhibition: 0.695 (CI 0.657 - 0.729) mg/L
Yield Inhibition: 0.320 (CI 0.294 - 0.358) mg/L

Reported statistics and error estimates:

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.

Validity criteria fulfilled:

yes

Conclusions:

Under study conditions, 72 h ErC50, ECy50, ECr10 and ECy10 values for the test substance with freshwater green algae, were determined to be3.48mg/L,1.17mg/L,0.927mg/L and0.412mg/L, respectively (geometric mean measured).

Executive summary:

A study was conducted to determine the acute toxicity study of the read across substance, C20-22 TMAC (active: 97.8%), to freshwater green algae (Desmodesmus subspicatus), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the read across substance were employed. Three replicate algal cultures, with a nominal cell density of approximately 2 - 5 x 103cells/mL, were exposed to read across substance at nominal concentrations of 0.020, 0.063, 0.20, 0.63, 2.00, 6.32 and 20.0 mg/L,corresponding to geometric mean measured concentrations of 0.0110, 0.0405, 0.128, 0.443, 1.39, 4.39 and15.4 mg/L medium for 72 h under static conditions. The concentrations of read across substance were analysed at all concentration levels after 0 and 72 h via LC-MS/MS analysis. The measured initial concentrations of read across substance were in good accordance with the nominal concentrations. The recovery rates in the fresh media (0 h) of the fractions C20 and C22 of read across substance were determined to be in the range of 82 - 92% and 70 – 77% of the nominal values, respectively. In 72 h old media, the C20 and C22 fractions had decreased, but were still detectable, except for in the lowest read across substance concentration. The recoveries in the old media (72 h) of the fractions C20 and C22 of read across substance decreased to values in the range of below the limit of quantification (LOQ) to 78% and LOQ to 65% of the nominal values, respectively. Adsorption of read across substance to the test vessels after 72 h of exposure were determined exemplarily at the dosage levels 0.0630 to 6.32 mg/L. The absorbed amount of the C20- and the C22-fraction was in the range of 11 to 46 % of the applied amount. This work was done in a second series. The recoveries in the aqueous phase were comparable to the first results of the definitive test. The read across substance has a low water solubility and sorbs to organic and inorganic materials by different mechanisms. The sorption processes are mostly non-linear, means are concentration dependent. Due to these properties the read across substance is difficult to test in synthetic water (e.g. sorption to the test organisms and glass walls of the test vessels) and results from such tests depend from the test settings applied. Using natural river water which contains particulate as well as dissolved organic carbon to which the read across substance can sorb partially reduces the difficulties encountered in tests with synthetic water (e.g. preventing that the test substance settles onto surfaces). The sorbed fraction of the test substance is difficult to extract from the test system which normally leads to low analytical recoveries. Due to the short exposure period these low recoveries cannot be associated to biodegradation. This means the read across substance is present in the test system and therefore available for exposure (dissolved in water and sorbed also called bulk). This so called Bulk Approach is described by ECETOC (2003). Due to the properties of the read across substance geometric mean measured concentrations have been used to report the results. The read across substance was found to inhibit the growth of the freshwater green algae after 72 h and the ErC50, ErC10 values for growth rate and EyC50 and EyC10 values for cell particle density were determined to be at 3.48, 0.927 and 1.17 and 0.412 mg/L respectively. The test effect was observed to be reversible at all tested concentrations following 72 h exposure and 3-7 d of growth under test conditions. Under study conditions, 72 h ErC50, ECy50, ECr10 and ECy10 values of the read across substance for toxicity to freshwater green algae, were determined to be 3.48, 1.17, 0.927 and 0.412 mg/L, respectively (geometric mean measured) (Scheerbaum, 2010). Based on the results of the read across study, similar 72 h EC50 and EC10 values can be expected for the test substance, C18-22 TMAC.

Description of key information

Based on the results of the read across substance, the 72 h ErC50, ECy50, ECr10 and ECy10 values of the test substance, C18-22 TMAC, for toxicity to freshwater green algae, are considered to be 3.48 mg/L,1.17 mg/L, 0.927 mg/L and 0.412 mg/L, respectively (geometric mean measured).

Key value for chemical safety assessment

EC50 for freshwater algae:

3.48 mg/L

EC10 or NOEC for freshwater algae:

0.927 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the read across substance, C20-22 TMAC (active: 97.8%), to freshwater green algae (Desmodesmus subspicatus), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the read across substance were employed. Three replicate algal cultures, with a nominal cell density of approximately 2 - 5 x 103cells/mL, were exposed to read across substance at nominal concentrations of 0.020, 0.063, 0.20, 0.63, 2.00, 6.32 and 20.0 mg/L,corresponding to geometric mean measured concentrations of 0.0110, 0.0405, 0.128, 0.443, 1.39, 4.39 and15.4 mg/L medium for 72 h under static conditions. The concentrations of read across substance were analysed at all concentration levels after 0 and 72 h via LC-MS/MS analysis. The measured initial concentrations of read across substance were in good accordance with the nominal concentrations. The recovery rates in the fresh media (0 h) of the fractions C20 and C22 of read across substance were determined to be in the range of 82 - 92% and 70 – 77% of the nominal values, respectively. In 72 h old media, the C20 and C22 fractions had decreased, but were still detectable, except for in the lowest read across substance concentration. The recoveries in the old media (72 h) of the fractions C20 and C22 of read across substance decreased to values in the range of below the limit of quantification (LOQ) to 78% and LOQ to 65% of the nominal values, respectively. Adsorption of read across substance to the test vessels after 72 h of exposure were determined exemplarily at the dosage levels 0.0630 to 6.32 mg/L. The absorbed amount of the C20- and the C22-fraction was in the range of 11 to 46 % of the applied amount. This work was done in a second series. The recoveries in the aqueous phase were comparable to the first results of the definitive test. The read across substance has a low water solubility and sorbs to organic and inorganic materials by different mechanisms. The sorption processes are mostly non-linear, means are concentration dependent. Due to these properties the read across substance is difficult to test in synthetic water (e.g. sorption to the test organisms and glass walls of the test vessels) and results from such tests depend from the test settings applied. Using natural river water which contains particulate as well as dissolved organic carbon to which the read across substance can sorb partially reduces the difficulties encountered in tests with synthetic water (e.g. preventing that the test substance settles onto surfaces). The sorbed fraction of the test substance is difficult to extract from the test system which normally leads to low analytical recoveries. Due to the short exposure period these low recoveries cannot be associated to biodegradation. This means the read across substance is present in the test system and therefore available for exposure (dissolved in water and sorbed also called bulk). This so called Bulk Approach is described by ECETOC (2003). Due to the properties of the read across substance geometric mean measured concentrations have been used to report the results. The read across substance was found to inhibit the growth of the freshwater green algae after 72 h and the ErC50, ErC10 values for growth rate and EyC50 and EyC10 values for cell particle density were determined to be at 3.48, 0.927 and 1.17 and 0.412 mg/L respectively. The test effect was observed to be reversible at all tested concentrations following 72 h exposure and 3-7 d of growth under test conditions. Under study conditions, 72 h ErC50, ECy50, ECr10 and ECy10 values of the read across substance for toxicity to freshwater green algae, were determined to be 3.48, 1.17, 0.927 and 0.412 mg/L, respectively (geometric mean measured) (Scheerbaum, 2010).Based on the results of the read across study, similar72 h EC50 and EC10 values can be expected for the test substance,C18-22 TMAC, for toxicity to algae.