Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-03-10 to 2010-09-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

and EU Method C.2

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentration levels and the control were analytically verified by LC-MS/MS in the fresh media (0 hours) and old media (48 hours).
- Sampling method: For the old media, separate replicates without daphnids were prepared and incubated under test conditions until analysis. The samples were stabilized directly after sampling with methanol containing 4% formic acid (5mL sample + 5 mL methanol containing 4% formic acid).
- 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 (10 mg/L test item was weighed out) was freshly prepared with dilution water. The stock solution was treated with ultrasound for 15 min and stirred with approx. 1100 rpm for 2 hours at room temperature.
- Eluate: Natural river water
- Differential loading: 0.250 - 0.433 - 0.750 - 1.30 - 2.25 - 3.90 mg/L
- Controls: Four replicates of dilution water without test item were tested under the same conditions as the test groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In 2-3 L glass vessels with appr. 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µExm-2 x s-1
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Hardness:

Dilution water: 96.1 (mg CaCO3/L)

Test temperature:

21.6 °C

pH:

Water Quality Parameters of the Fresh Media (0 hours)
(measured in one additionally prepared separate replicate per concentration level and control)
Nominal Test Item Concentration
[mg/L] pH-Value


3.90 7.59
2.25 7.61
1.30 7.67
0.750 7.68
0.433 7.83
0.250 7.69
Control 7.33

Water Quality Parameters of the Old Media (48 hours)
(measured in all replicates per concentration level and control)
Nominal
Test Item Concentration
[mg/L] pH-Values
Replicates
1 2 3 4
3.90 7.92 7.90 7.86 7.88
2.25 7.83 7.80 7.82 7.85
1.30 7.72 7.78 7.75 7.77
0.750 7.68 7.66 7.66 7.66
0.433 7.67 7.69 7.70 7.64
0.250 7.76 7.76 7.71 7.67
Control 7.97 7.91 7.81 7.71

Dissolved oxygen:

Water Quality Parameters of the Fresh Media (0 hours)
(measured in one additionally prepared separate replicate per concentration level and control)
Nominal Test Item Concentration
[mg/L] Dissolved O2-Concentration

[mg/L]
3.90 8.39
2.25 8.67
1.30 8.72
0.750 8.78
0.433 8.77
0.250 8.76
Control 7.88

Water Quality Parameters of the Old Media (48 hours)
(measured in all replicates per concentration level and control)
Nominal
Test Item Concentration
[mg/L] Dissolved O2 -Concentration [mg/L]
Replicates
1 2 3 4
3.90 8.42 8.29 8.35 8.21
2.25 7.94 8.05 7.86 8.08
1.30 7.84 7.63 7.62 7.79
0.750 6.41 7.05 7.15 7.15
0.433 6.40 6.88 6.93 6.97
0.250 7.71 7.37 6.94 7.24
Control 8.42 8.37 8.07 8.16

Salinity:

Freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): One application at test start, static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural river water of the river Böhme was used.
River: Böhme
Location Fischendorfer Straße,
D-29683 Bad Fallingbostel, Germany,
(52° 53’ 51.81 N, 9° 45’ 41.44 E)
Sampling Date 2010-05-19
Weather on Day of Sampling cloudy, ca. 14 °C
Colour brownish, clear
pH 7.63
Conductivity [µS/cm] 462
Dissolved Oxygen [mg O2/L] 10.11
DOC [mg C/L] 5.9
TOC [mg C/L] 5.9
Ammonium-N [mg N/L] 0.065
Nitrate-N [mg N/L] 1.48
Total Nitrogen [mg N/L] 2.53
o-Phosphate-p [mg P/L] 0.076
Total Phosphate [mg P/L] 0.115
Suspended Matter [mg/L] 17.6
Total Hardness [mg CO3/L] 114
Storage conditions 6 ± 2 °C



OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility was assessed in all treatment levels after 24 h and 48 h. An animal was considered to be immobile, if it was not able to swim during a 15 second observation period following gentle agitation of the test vessel.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (1.29-1.49)

Details on results:

- Behavioural abnormalities: None
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: 1.87 mg/L (CI 1.74 - 2.00 mg/L)

Reported statistics and error estimates:

EC10 and EC50-values after 24 and 48 hours were calculated by sigmoidal dose-response regression. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism. The concentration effect relationships are shown graphically.

Immobilization Rates in the Definitive Test

                     (n = 20, divided into 4 replicates with 5 daphnids each)

C20/22 ATQ trocken	IMMOBILIZATION [%]
Nominal Test Item Concentration [mg/L]	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
3.90	100	100	100	100	100	100	100	100	100	100
2.25	20	20	0	20	15	100	100	80	100	95
1.30	20	0	0	0	5	60	40	40	20	40
0.750	0	0	0	0	0	0	0	0	0	0
0.433^1)	0	0	0	0	0	0	20	0	0	5
0.250^1)	0	0	0	0	0	0	0	0	20	5
Control	0	0	0	0	0	0	0	0	0	0

Mass Balance of C20/22 ATQ trocken (C20 fraction) in Aqueous Phase and at Glass Walls of Stock and Test Solutions at 0 and 48 hours

Nominal concentration		Aqueous Phase (Volume: 1000 mL^1) and  20 mL^2))				Adsorbed to Glass		Aqueous Phase (0 hours) corrected versus
Test item [mg/L]	C20-fraction [mg/L]	Fresh Media (0 hours)		Old Media (48 hours)		after Exposure of 48 hours		Adsorbed to Glass (48 hours)
		Meas. conc. [mg/L]	RR [%]	Meas. conc. [mg/L]	RR [%]	Meas. conc. [mg/L]	RR [%]	Calc. conc. [mg/L]	RR [%]
10.0	1.37	1.47	107	Not determined		0.0458^3^)	3	-	-
3.90	0.534	0.559	105	0.160	30	0.138	26	0.421	79
2.25	0.308	0.340	110	0.0910	30	0.0655	21	0.275	89
1.30	0.178	0.186	104	0.0451	25	0.0297	17	0.156	88
0.750	0.103	0.113	110	0.0273	27	0.0226	22	0.0904	88
0.433	0.0593	0.0632	107	0.0149	25	0.0127	21	0.0505	85
0.250	0.0343	0.0359	105	0.00845	25	0.00659	19	0.0293	85
Control		LOQ		LOQ		LOQ		LOQ

Mass Balance of C20/22 ATQ trocken (C22 fraction) in Aqueous Phase and at Glass Walls of Stock and Test Solutions at 0 and 48 hours

Nominal concentration		Aqueous Phase (Volume: 1000 mL^1) and 20 mL^2) )				Adsorbed to Glass		Aqueous Phase (0 hours) corrected versus
Test item [mg/L]	C22-fraction [mg/L]	Fresh Media (0 hours)		Old Media (48 hours)		after Exposure of 48 hours		Adsorbed to Glass (48 hours)
		Meas. conc. [mg/L]	RR [%]	Meas. conc. [mg/L]	RR [%]	Meas. conc. [mg/L]	RR [%]	Calc. conc. [mg/L]	RR [%]
10.0	8.09	8.98	111	Not determined		0.150^3^)	2	-	-
3.90	3.16	3.46	109	0.866	27	0.578	18	2.88	91
2.25	1.82	2.14	118	0.479	26	0.265	15	1.88	103
1.30	1.05	1.20	114	0.285	27	0.122	12	1.08	103
0.750	0.607	0.697	115	0.165	27	0.130	21	0.567	93
0.433	0.350	0.393	112	0.104	30	0.0858	25	0.307	88
0.250	0.202	0.219	108	0.0607	30	0.0332	16	0.186	92
Control		LOQ		LOQ		LOQ		LOQ

Validity criteria fulfilled:

yes

Conclusions:

In this study C20/22 ATQ trocken was found to be toxic to Daphnia magna after 48 h at nominal concentrations of 1.30 mg/L (LOEC48 h) and higher. The NOEC after 48 hours was 0.750 mg/L. Based on the nominal concentrations of the test item the 48-hour EC50 for Daphnia magna was calculated to be 1.39 mg/L (95 % confidence limits: 1.29 – 1.49 mg/L).
For the assessment of the endpoints the nominal concentrations of the test item were used, because the calculated exposure concentrations demonstrate that the daphnids were fully exposed to the test item concentrations (dissolved in water and sorbed also called bulk). Due to the properties of the test item nominal concentrations have to be used instead of measured ones. This so called Bulk Approach is described by ECETOC (2003).

Executive summary:

In the acute immobilization test to Daphnia magna (STRAUS) with natural river water the effects of the test item C20/22 ATQ trocken (batch number: DEG4047322) were determined according to OECD 202 (2004) and Directive 92/69/EC Method C.2 (1992) from 2009-03-10 to 2010-09-06, with the definitive exposure phase between 2010-08-31 and 2010-09-02, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Straße 1, 31157 Sarstedt, Germany.

The study was conducted under static conditions with 6nominal test item concentrations ranging from 0.250 to 3.90 mg/L set up in a geometric series with a dilution factor of √3over a period of 48 hours. 20 test organisms were exposed to each test concentration and control. All concentration levels and the control were analysed via LC-MS/MS after 0 h (fresh media) and after 48 h (old media).

The recovery rates in the fresh media (0 hours) of all test concentrations were determined to be in the range of 105 to 110 % of the nominal values for the C20-fraction and 108 to 118 % of the nominal values for the C22-fraction. The recoveries in the aqueous phase of the old media (48 hours) decreased to values in the range of 25 to 30 % of the nominal values for the C20 fraction and 26 to 30 % of the nominal values for the C22-fraction.

Adsorption to the test vessels after 48 h of exposure were determined at all dosage levels. The absorbed amount of the C20- and the C22-fraction was in the range of 12 to 26 % of the applied amount.

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 at the end of the exposure phase (48 hours) cannot be associated to biodegradation. The measured extractable test item concentration in the aqueous phase the test item sorbed to DOC and suspended matter is regarded to be available for the daphnids. Therefore, the exposure concentrations at the end of the exposure phase of 48 h were calculated by correcting the initially measured concentrations versus the test item concentrations sorbed to the glassware.

For the assessment of the endpoints the nominal concentrations of the test item were used, because the calculated exposure concentrations demonstrate that the daphnids were fully exposed to the test item concentrations (dissolved in water and sorbed also called bulk). Due to the properties of the test item nominal concentrations have to be used instead of measured ones (see Table 1). This so called Bulk Approach is described by ECETOC (2003).

The water quality parameters (pH-values and dissolved oxygen concentrations), measured at 0 and 48 h, were determined to be within acceptable limits. The validity criteria of the test guideline were fulfilled.

EC₁₀-, EC₅₀- (with Confidence Interval), EC₁₀₀- Values, NOEC and LOEC

                   (based on the nominal concentrations of the test item C20/22 ATQ trocken)

	Test Duration [h]	Nominal Concentration of the Test Item C20/22 ATQ trocken [mg/L]	Confidence Interval p = 95% [mg/L]
EC10^1)	24	1.50	---
	48	0.988	---
EC50^1)	24	2.76	2.34 – 3.26
	48	1.39	1.29 – 1.49
EC100	24	3.90	---
	48	3.90	---
NOEC	48	0.750	---
LOEC	48	1.30	---

Description of key information

In this study C20/22 ATQ trocken was found to be toxic to Daphnia magna after 48 h at nominal concentrations of 1.30 mg/L (LOEC48 h) and higher. The NOEC after 48 hours was 0.750 mg/L. Based on the nominal concentrations of the test item the 48-hour EC50 for Daphnia magna  was calculated to be 1.39 mg/L (95 % confidence limits: 1.29 – 1.49 mg/L). 
For the assessment of the endpoints the nominal concentrations of the test item were used, because the calculated exposure concentrations demonstrate that the daphnids were fully exposed to the test item concentrations (dissolved in water and sorbed also called bulk). Due to the properties of the test item nominal concentrations have to be used instead of measured ones. This so called Bulk Approach is described by ECETOC (2003). 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.4 mg/L