Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-04-27 to 2010-06-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

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 replicates.

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 hours 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 item 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)
pH-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

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.78 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.63-0.95

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)

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.

EC - values and 95 % Confidence Intervals of C20/22 ATQ trocken (0-72 h)

                                            (based on the geometric mean measured test item concentrations of C20/22ATQ

                                 trocken)¹⁾ [mg/L]

	Rate-related Inhibition
ErC10	0.927 (0.798 – 1.07)
ErC20	1.53 (1.38 – 1.68)
ErC50	3.48 (3.28 – 3.70)
	Inhibition of Yield
EyC10	0.412 ( 0.0110 – 0.757)
EyC20	0.678 (0.482 – 0.914)
EyC50	1.17 (0.955 – 1.30)

Cell Densities

	Geometric mean measured test item concentration of            C20/22 ATQ trocken^1)		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
15.4		1		4996		16961	2910	8840
		2		4996		17392	4763	6501
		3		4996		16236	3905	5645
		Mean		4996		16863	3859	6995
4.39		1		4996		17740	26897	39859
		2		4996		19896	26979	46414
		3		4996		20513	26112	42600
		Mean		4996		19383	26663	42958
1.39		1		4996		25519	117447	430852
		2		4996		27670	104868	334361
		3		4996		25982	117267	421409
		Mean		4996		26390	113194	395541
0.443		1		4996		28773	172202	982629
		2		4996		25717	158800	875632
		3		4996		28127	176993	939302
		Mean		4996		27539	169332	932521
0.128		1		4996		28593	188188	1025336
		2		4996		26997	169225	946990
		3		4996		30024	170078	839834
		Mean		4996		28538	175830	937387
0.0405		1		4996		30885	203617	1040806
		2		4996		32835	191395	928157
		3		4996		27885	198725	868508
		Mean		4996		30535	197912	945824
0.0110		1		4996		27044	188064	962026
		2		4996		26861	183813	1001500
		3		4996		34301	237494	1089092
		Mean		4996		29402	203124	1017539
Control		1		4996		28626	210673	1006506
		2		4996		25375	206098	950279
		3		4996		28735	207245	1054860
		4		4996		25723	215567	1077837
		5		4996		26201	216862	1048452
		6		4996		27661	203354	1090944
		Mean		4996		27054	209967	1038146

Evaluation after 72 h

                              

Geometric mean measured test item concentration of            C20/22 ATQ trocken^1)	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
15.4	1		0.190	89.30		3844	99.63
	2		0.088	95.06		1505	99.85
	3		0.041	97.71		649	99.94
	Mean		0.106	94.02		1999	99.81
4.39	1		0.692	61.08		34863	96.63
	2		0.743	58.22		41418	95.99
	3		0.714	59.83		37604	96.36
	Mean		0.717	59.71		37962	96.33
1.39	1		1.486	16.46		425856	58.78
	2		1.401	21.21		329365	68.12
	3		1.478	16.88		416413	59.69
	Mean		1.455	18.18		390545	62.20
0.443	1		1.761	1.01		977633	5.37
	2		1.722	3.17		870636	15.73
	3		1.745	1.86		934306	9.57
	Mean		1.743	2.01		927525	10.22
0.128	1		1.775	0.21		1020340	1.24
	2		1.748	1.70		941994	8.82
	3		1.708	3.95		834838	19.19
	Mean		1.744	1.95		932391	9.75
0.0405	1		1.780	-0.07		1035810	-0.26
	2		1.742	2.08		923161	10.65
	3		1.719	3.32		863512	16.42
	Mean		1.747	1.78		940828	8.94
0.0110	1		1.753	1.41		957030	7.37
	2		1.767	0.65		996504	3.55
	3		1.795	-0.92		1084096	-4.93
	Mean		1.772	0.38		1012543	2.00
Control	1		1.769			1001510
	2		1.749			945283
	3		1.784			1049864
	4		1.791			1072841
	5		1.782			1043456
	6		1.795			1085948
	Mean		1.778			1033150

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

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	1.746	1.996	1.564	1.769	0.2169	12.3	13.6
	2	1.625	2.095	1.528	1.749	0.3029	17.3
	3	1.749	1.976	1.627	1.784	0.1768	9.91
	4	1.639	2.126	1.609	1.791	0.2901	16.2
	5	1.657	2.113	1.576	1.782	0.2898	16.3
	6	1.711	1.995	1.680	1.795	0.1735	9.66
				Mean	1.778
				± SD	0.02
				CV [%]	0.96

Validity criteria fulfilled:

yes

Conclusions:

In this study C20/22 ATQ trocken was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 3.48 (3.28 – 3.70) and 1.17 (0.955 – 1.30) mg/L, respectively.
The test effect was observed to be reversible at all tested concentrations following 72 h exposure and 3 – 7 days of growth under test conditions.
All effect levels are given based on geometric mean measured concentrations of C20/22 ATQ trocken.

The measured concentrations of C20/22 ATQ trocken (C20 and C22 fraction) at test start were in the range of 70 - 92% and after 72h in the range of < LOQ – 78 % of the nominal values. The recovery of the C20 fraction was a slightly higher than that of the C22 fraction.
The measured concentrations of the aqueous phases of the adsorption test which was carried out separately confirmed these values.
Adsorption to the test vessels after 72 h of exposure were determined exemplarily at the dosage levels 0.630 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 amount of adsorbed C20/C22 was higher at low nominal concentrations and lower at higher nominal concentrations.
All effect values are based on the geometric mean measured concentrations of the test item C20/22 ATQ trocken (C20 and C22 fraction).

Executive summary:

The toxicity of C20/22 ATQ trocken (batch number DEG4047322)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from 2009-04-27 to 2010-06-07 with the definitive exposure phase from 2009-06-23 to 2009-06-26. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h. The test was carried out in natural river water with a DOC of 2.8 mg/L.

The study was conducted under static conditions with an initial cell density of approximately 2 – 5 x 10³ cells/mL. Seven concentration levels were tested in a geometrical series with a dilution factor of √10, nominal: 0.020 - 0.063 - 0.20 - 0.63 - 2.00 - 6.32 - 20.0 mg/L, corresponding to geometric mean measured concentrations of 0.0110 - 0.0405 - 0.128 - 0.443 - 1.39 - 4.39 - 15.4 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of C20/22 ATQ trocken wereanalysed at all concentration levels after 0 and   72 h via LC-MS/MS analysis. The measured initial concentrations of C20/22 ATQ trocken were in good accordance with the nominal concentrations. The recovery rates in the fresh media (0 h) of the fractions C20 and C22 of C20/22 ATQ trocken were determined to be in the range of 82 to 92 % and 70 – 77 % of the nominal values, respectively. In 72h old media, the C20 and C22 fractions had decreased, but were still detectable, except for in the lowest test item concentration. The recoveries in the old media (72 h) of the fractions C20 and C22 of C20/22 ATQ trocken 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 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 analytical results are presented in Table 13 to Table 17.

The test item 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 test item 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 test item can sorb partially reduces the difficulties encountered in tests with synthetic water (e.g. preventing that the test item settles onto surfaces). The sorbed fraction of the test item 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 test 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).

However, all effect values given are based on geometric mean measured concentrations of the test item C20/22 ATQ trocken

EC - Values and 95 % Confidence Intervals of C20/22 ATQ trocken (0-72 h)

                                            (based on the geometric mean measured test item concentrations of C20/22ATQ

                                 trocken)¹⁾ [mg/L]

	Rate-related Inhibition
ErC10	0.927 (0.798 – 1.07)
ErC20	1.53 (1.38 – 1.68)
ErC50	3.48 (3.28 – 3.70)
	Inhibition of Yield
EyC10	0.412 ( 0.0110 – 0.757)
EyC20	0.678 (0.482 – 0.914)
EyC50	1.17 (0.955 – 1.30)

After 72 h algae were transferred from the nominal concentrations of 2.00 – 20.0 mg/L (geometric mean measured 1.39 – 15.4 mg/L) and control to fresh untreated medium and allowed to grow for further 3 – 7 days under test conditions. The test item effect was observed to be reversible at this concentration. Therefore, there is potential for recovery following exposure up to 15.4 mg/L (highest geometric mean measured test concentration)

Description of key information

In this study C20/22 ATQ trocken was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 3.48 (3.28 – 3.70) and 1.17 (0.955 – 1.30) mg/L, respectively. 
The test effect was observed to be reversible at all tested concentrations following 72 h exposure and 3 – 7 days of growth under test conditions.
All effect levels are given based on geometric mean measured concentrations of C20/22 ATQ trocken.
The measured concentrations of C20/22 ATQ trocken (C20 and C22 fraction) at test start were in the range of 70 - 92% and after 72h in the range of < LOQ – 78 % of the nominal values. The recovery of the C20 fraction was a slightly higher than that of the C22 fraction. 
The measured concentrations of the aqueous phases of the adsorption test which was carried out separately confirmed these values.
Adsorption to the test vessels after 72 h of exposure were determined exemplarily at the dosage levels 0.630 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 amount of adsorbed C20/C22 was higher at low nominal concentrations and lower at higher nominal concentrations. 
All effect values are based on the geometric mean measured concentrations of the test item C20/22 ATQ trocken (C20 and C22 fraction).

Key value for chemical safety assessment

EC50 for freshwater algae:

3.48 mg/L

EC10 or NOEC for freshwater algae:

0.78 mg/L