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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

key study

Study period:

From March 03, 2009 to March 19, 2009

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 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 test substance 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:

semi-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 substance concentration # [mg/L] Temperature [°C]
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 substance 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 substance 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)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.651 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: mortality, physical or behavioural abnormalities

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

ca. 1.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: mortality, physical or behavioural abnormalities

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:

Under the study conditions, the 96 h LC50 of the test substance was determined to be 3.5 mg/L (geometric mean measured).

Executive summary:

A study was conducted to determine the short-term toxicity potential of the read across substance, C20-22 TMAC (active: 97.8%), to fish according to OECD Guideline 203, in compliance with GLP. Seven Danio rerio fish were exposed to 1, 1.78, 3.16, 5.62 and 10 mg/L nomimal test concentrations (corresponding to the geometric mean measured concentrations of 0.651, 1.11, 2.57, 4.70 and 8.22 mg/L) for 96 h under semi-static conditions. The test media was renewed after 48 h. Fish were considered dead if there was no visible movement (gill cover movement), reaction upon touching of caudal peduncle. Records were also kept of visible abnormalities (e.g., loss of equilibrium, swimming behaviour, respiratory function, pigmentation etc.). 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 substance 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 read across substance were found to be between 63 – 107% (fraction C20) and 74 – 101% (fraction C22) 27 – 85% (fraction C20) and 39 – 80% (fraction C22) in fresh and old media, respectively. The total recovery rates for read across substance 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 read across substance 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 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 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 read across substance can sorb partially reduces the difficulties encountered in tests with synthetic water e.g. preventing that the read across substance settles onto surfaces. The sorbed fraction of the read across 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 present the results. The read across substance was found to be toxic to zebrafish after 96 h at a concentration of 1.10 mg/L (LOEC) and higher. Since after 72 and 96 h only 0 and 100 % mortality was found, no evaluation regression analysis was carried out. Hence the LC50 was calculated as 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) and the NOEC after 96 h were determined to be 2.6, 4.7 and 0.651 mg/L (geometric mean measured). Under the study conditions, the 96 h LC50 of the read across substance was determined to be 3.5 mg/L (geometric mean measured) (Scheerbaum, 2010). Based on the results for the read across study, similar LC50 values can be expected for the test substance, C18-22 TMAC.

Description of key information

Based on the results of the read across study, the 96 h LC50 value of the test substance, C18 -22 TMAC, for toxicity to fish is considered to be 3.5 mg/L (geometric mean measured).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

3.5 mg/L

Additional information

A study was conducted to determine the short-term toxicity potential of the read across substance, C20-22 TMAC (active: 97.8%), to fish according to OECD Guideline 203, in compliance with GLP. Seven Danio rerio fish were exposed to 1, 1.78, 3.16, 5.62 and 10 mg/L nomimal test concentrations (corresponding to the geometric mean measured concentrations of 0.651, 1.11, 2.57, 4.70 and 8.22 mg/L) for 96 h under semi-static conditions. The test media was renewed after 48 h. Fish were considered dead if there was no visible movement (gill cover movement), reaction upon touching of caudal peduncle. Records were also kept of visible abnormalities (e.g., loss of equilibrium, swimming behaviour, respiratory function, pigmentation etc.). 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 substance 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 read across substance were found to be between 63 – 107% (fraction C20) and 74 – 101% (fraction C22) 27 – 85% (fraction C20) and 39 – 80% (fraction C22) in fresh and old media, respectively. The total recovery rates for read across substance 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 read across substance 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 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 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 read across substance can sorb partially reduces the difficulties encountered in tests with synthetic water e.g. preventing that the read across substance settles onto surfaces. The sorbed fraction of the read across 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 present the results. The read across substance was found to be toxic to zebrafish after 96 h at a concentration of 1.10 mg/L (LOEC) and higher. Since after 72 and 96 h only 0 and 100 % mortality was found, no evaluation regression analysis was carried out. Hence the LC50 was calculated as 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) and the NOEC after 96 h were determined to be 2.6, 4.7 and 0.651 mg/L (geometric mean measured). Under the study conditions, the 96 h LC50 was determined to be 3.5 mg/L (geometric mean measured) (Scheerbaum, 2010). Based on the results for the read across study, similar LC50 values can be expected for the test substance, C18-22 TMAC, for short-term toxicity to fish.