Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

27.03.1990-06.04.1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No analytical monitoring due to missing method, nominal concentration without checking substance recoveries, limited information on test substance

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

no

Analytical monitoring:

no

Details on sampling:

not available

Details on test solutions:

stock solution (substance in tap water) was diluted with tap water

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Brachydanio rerio (Zäbrabärbling)
- Source: Zoo-Stumpe. Hildesheim
- Length at study initiation (length definition, mean, range and SD): ca. 2.8cm
- Weight at study initiation (mean and range, SD): ca. 0.37g
- Feeding during test
- Food type: Tockenfutter, TETRA

ACCLIMATION
- Acclimation period: according to OECD-guideline

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

Dose response test

Post exposure observation period:

n.a.

Hardness:

not determined

Test temperature:

21°C

pH:

7.98 - 8.25

Dissolved oxygen:

8.0 - 8.7 mg O2/L

Salinity:

---

Nominal and measured concentrations:

Nominal:
0.05 - 0.1 - 0.25 - 0.5 - 1.0 mg/L
no measured concentration in the test system due to lack of analytical method

Details on test conditions:

TEST SYSTEM
- Test vessel: 16L
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 5L

- Type of flow-through (e.g. peristaltic or proportional diluter): static
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap-water



OTHER TEST CONDITIONS
- Adjustment of pH: 7.98 - 8.25
- Photoperiod: 12 hrs / 12 hrs
- Light intensity: ca. 10 [µE /m2 *s]


TEST CONCENTRATIONS
- 0.05 - 0.1 - 0.25 - 0.5 - 1.0 mg/L

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.05 - 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Results with reference substance (positive control):

none

Reported statistics and error estimates:

statistical method:
acc. to Spearman - Kärber in: L- Sachs, Angewandte Statistik S. 117-181, Springer Verlag 1984

Validity criteria fulfilled:

yes

Conclusions:

LC50 (96h): 0.5-1 mg/L

Executive summary:

lowest tested concentration of test substance with 100% mortality       = 1.00 mg/L

highest tested concentration of test substance without mortality            = 0.25 mg/L

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-03-09 to 2009-03-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity 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 and the control were analytically verified from freshly prepared media after 0 and 48 h and from 48 h old media after 48 and 96 h.
- Sample storage conditions before analysis:All samples were stored at room temperature before preparation and before analysis, if necessary at 6 ± 2 °C (test samples) or room temperature (adsorption samples including respective test solutions), respectively.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/L was prepared for the test item concentrations 1 - 1.78 - 3.16 mg/L and treatedw ith ultrasound for 5 minutes and stirred for 120 minutes on a magnetic stirrer. The test item concentrations 5.62 and 10 mg/L were directly weighed out.
- Eluate: Natural river water
- Differential loading: 1 - 1.78 - 3.16 - 5.62 - 10 mg/L
- Controls: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source:Aquarium am Aegi, Volgersweg 6, 30175 Hannover, Germany
- Length at study initiation (length definition, mean, range and SD): Average body length 2.16 cm
- Weight at study initiation (mean and range, SD): Average body weight 0.27 g
- Method of breeding: Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (0.1 - 10 µmol photons x m-2 x s-1, natural photoperiod). Tap water of local origin was used for holding and testing. The water was filterred on activated charcoal and aerated for at least 24 h to remove chlorine. Nominal water parameters: Total hardness: 10 - 250 mg CaCO3/L; pH-value: 6.0 - 8.5. The water is analysed biannula acc. to German tap water regulation. The water was changed at least once per week. the dissolved oxygen concentration was more than 80 % of the air saturation value.
- Feeding during test: No feeding during test



ACCLIMATION
- Acclimation period: Zebrafish with al least 12 days of acclimatisation and mortality < 5% within these days before the study starts were used.
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Stoer perlets, SERA GmbH, 52518 Heidelberg, Germany
- Feeding frequency: 3 times per week
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered thwoughout holding and testing.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Total Hardness at day 0: 313 mg/L as CaCO3; at day 2: 363 mg/L

Test temperature:

Geometric mean measured
test item concentration#
[mg/L] Temperature
[°C]
old new
Day 0 8.22 --- 22.6
4.70 --- 22.7
2.57 --- 23.2
1.11 --- 22.9
0.651 --- 22.4
Control --- 21.9
Day 1 8.22 23.4 ---
4.70 23.4 ---
2.57 23.4 ---
1.11 23.3 ---
0.651 23.3 ---
Control 22.9 ---
Day 2 8.22 23.2 23.2
4.70 23.3 23.3
2.57 23.3 23.5
1.11 23.3 23.4
0.651 23.2 23.0
Control 23.1 22.7
Day 3 8.22 22.6 ---
4.70 22.7 ---
2.57 22.7 ---
1.11 22.7 ---
0.651 22.4 ---
Control 22.1 ---
Day 4 8.22 - ---
4.70 - ---
2.57 22.2 ---
1.11 22.4 ---
0.651 22.2 ---
Control 21.7 ---

pH:

Geometric mean measured
test item concentration#
[mg/L] pH Value
old new
Day 0 8.22 --- 8.14
4.70 --- 8.20
2.57 --- 8.15
1.11 --- 8.13
0.651 --- 8.13
Control --- 8.11
Day 1 8.22 8.14 ---
4.70 8.15 ---
2.57 8.16 ---
1.11 8.11 ---
0.651 8.11 ---
Control 8.12 ---
Day 2 8.22 8.20 8.13
4.70 8.20 8.15
2.57 8.21 8.16
1.11 8.20 8.18
0.651 8.20 8.17
Control 8.16 8.10
Day 3 8.22 8.28 ---
4.70 8.24 ---
2.57 8.43 ---
1.11 8.28 ---
0.651 8.43 ---
Control 8.35 ---
Day 4 8.22 - ---
4.70 - ---
2.57 8.31 ---
1.11 8.13 ---
0.651 8.31 ---
Control 8.22 ---

Dissolved oxygen:

Geometric mean measured
test item concentration#
[mg/L] O2 -Saturation

[%]
old new
Day 0 8.22 --- 94
4.70 --- 96
2.57 --- 100
1.11 --- 100
0.651 --- 99
Control --- 100
Day 1 8.22 99 ---
4.70 98 ---
2.57 98 ---
1.11 96 ---
0.651 95 ---
Control 100 ---
Day 2 8.22 100 100
4.70 100 100
2.57 100 100
1.11 99 100
0.651 100 100
Control 100 100
Day 3 8.22 99 ---
4.70 94 ---
2.57 100 ---
1.11 98 ---
0.651 100 ---
Control 100 ---
Day 4 8.22 - ---
4.70 - ---
2.57 99 ---
1.11 90 ---
0.651 99 ---
Control 96 ---

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

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

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Type (delete if not applicable): open, loosely covered by glass tops
- Material, size, headspace, fill volume: glass, 2 L test volume per vessel
- Aeration: Gentle aeration
- Renewal rate of test solution (frequency/flow rate): One application at test start
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: less than 1 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water of the river Innerste was used. This river is located in Sarstedt / Ruthe. Water parameters are given below
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
- Adjustment of pH: No
- Photoperiod: natural
- Light intensity: 0.1 - 10 µmol photons x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 24, 48, 72 and 96 h.


TEST CONCENTRATIONS
- Range finding study: Yes
Cumulative Mortality in the Preliminary Test
(n = 5)
Nominal test item concentration [mg/L] Test Duration [hours]
24 48 72 96
10 100 - - -
1 0 0 0 0
0.10 0 0 0 0
Control 0 0 0 0

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

3.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 2.6-4.7

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

4.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Not observed
- Mortality of control: 0%
- Other adverse effects control: Not observed
- Abnormal responses: Not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not observed

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

Since after 72 and 96 h only 0 and 100 % mortality was found, no
evaluation regression analysis was carried out. Hence, the LC50 is the geometric mean value of the concentrations with 0 and 100 % mortality.
The concentration causing 0 and 100 % mortality were chosen as lower and upper confidence limit. The concentrations causing to 0 and 100 % mortality (LC0 and LC100) as well as the no effect level (NOEC) after 96 h were determined directly from the test results.

Sublethal observations / clinical signs:

Observations in the Test Vessels

                 (n=7)

Geometric mean measured test item concentration^# [mg/L]	Effect*	Test Duration [h]
		24	48	72	96
8.22	(E)	-	-	7/7	---
	(2.4)	7/7	7/7	-	---
	(2.6)	7/7	-	-	---
4.70	(E)	-	-	7/7	---
	(2.4)	7/7	7/7	-	---
	(2.6)	7/7	-	-	---
2.57	(1)	-	-	7/7	7/7
	(2.4)	7/7	7/7	-	-
	(2.6)	7/7	-	-	-
1.11	(1)	-	7/7	7/7	7/7
	(2.4)	7/7	-	-	-
0.651	(1)	7/7	7/7	7/7	7/7
Control	(1)	7/7	7/7	7/7	7/7

Cumulative Mortality [%] in the Test Vessels

(based on the geometric mean measured test item concentrations)

Geometric mean measured test item concentration ^# [mg/L]	Test Duration [h]
	24	48	72	96
8.22	0	0	100	100
4.70	0	0	100	100
2.57	0	0	0	0
1.11	0	0	0	0
0.651	0	0	0	0
Control	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

All effect levels are given based on the sum of the geometric mean measured concentrations of the fractions C20 and C22 of C20/22 ATQ trocken:
In this study C20/22 ATQ trocken was found to be toxic to zebrafish after 96 h at a concentration of 1.10 mg/L (LOEC) and higher.
The LC50 after 96 h was 3.48 mg/L (2.57 – 4.70 mg/L).

Executive summary:

The acute toxicity of the test item C20/22 ATQ trocken (batch no.: DEG4047322) to fish (zebrafish) was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992) from 2009 -03-09 to 2009-03-19 at Dr.U.Noack-Laboratorien, 31157 Sarstedt , Germany . A semi-static test with the nominal concentrations of 1 - 1.78 - 3.16 - 5.62 - 10 mg/L corresponding to the geometric mean measured concentrations of 0.651 – 1.11 – 2.57 – 4.70 – 8.22 mg/L and a renewal of the test media after 48 h was performed. Duration of the test was 96 h. 7 test organisms were exposed to each concentration and control. Water quality parameters pH-value, temperature and oxygen-saturation measured after 0, 24, 48, 72 and 96 h were determined to be within the acceptable limits.

The concentration of the test item and control were analytically verified via LC-MS/MS from freshly prepared media after 0 and 48 h and from 48 h old media after 48 and 96 h. The recovery rates for C20/22 ATQ trocken were found to be between 63 – 107 % for the fraction C20 of C20/22 ATQ trocken and 74 – 101 % for the fraction C22 of C20/22 ATQ trocken from the fresh media and 27 – 85 % for the fraction C20 of C20/22 ATQ trocken and 39 – 80 % for the fraction C22 of C20/22 ATQ trocken from the old media. The total recovery rates for C20/22 ATQ trocken were found to be between 71 – 102 % from the fresh media and 34 – 82 % from the 48 h old media. The measured initial concentrations of both series were in good accordance. In 48 h old media the test item concentrations had decreased but were still detectable.

Adsorption to the test vessels after 48 h of exposure was determined exemplarily at the dosage levels 1.78 to 5.72 mg/L. The absorbed amount of the C20- and the C22-fraction was in the range of 39 to 57 % 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 organism and walls of the test vessel) 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). Due to the properties of the test item geometric mean measured concentrations have to be used

LC-Values with 95 % Confidence Intervals

                      (based on the geometric mean measured test item concentrations of C20/22 ATQ trocken)¹⁾            

Test duration [h]	LC50  [mg/L]		p = 95 % [mg/L]
24	> 8.2		Not applicable
48	> 8.2		Not applicable
72	3.5		2.57 - 4.70 *
96	3.5		2.57 - 4.70 *
LC100 =		4.7 mg/L
Lowest test item concentration
with 100 % mortality after 96 h
LC0 =		2.6 mg/L
Highest test item concentration
with 0 % mortality after 96 h

Description of key information

All effect levels are given based on the sum of the geometric mean measured concentrations of the fractions C20 and C22 of C20/22 ATQ trocken:
In this study C20/22 ATQ trocken was found to be toxic to zebrafish after 96 h at a concentration of 1.10 mg/L (LOEC) and higher.
The LC50 after 96 h was 3.48 mg/L (2.57 – 4.70 mg/L).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

3.5 mg/L