N,N,N-tri-C8-C10-alkyl-N-methyl-1-aminium sulfate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-01-27 to 2020-02-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The limit concentration and the control were analytically verified via LC-MS/MS in the fresh media at the start of exposure and at one renewal interval (0 and 72 hours) and in the corresponding 24-hours aged media after 24 and 96 hours.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (100 mg/L of the test item were weighed out) was freshly prepared with natural river water (Table 1, diluted by factor with dechlorinated tap water) prior to the start of the exposure (at 0 hours) and prior to each renewal of the test solutions (at 24, 48 and 72 hours). The stock solution was treated with ultrasound for 5 minutes at room temperature and mixed thoroughly by manual agitation. After transferring the stock solution into the test vessel (filled with dilution water), the test media was mixed with an ultraturrax (1 min, at least 17000 rpm). This procedure was repeated daily for the preparation of the stock solution and the water renewal of the threshold concentration.
- Controls: dilution water (natural river water without test item)

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Umweltbundesamt, Schichauweg 58, 12307 Berlin, Germany
- Average body length at the test end: 1.7 cm, Average body weight at the test end: 0.05 g
- Maintenance of the brood fish: at 23 ± 2 °C and diffuse light (540 – 1000 lux, photoperiod of 12 to 16 h light / 12 to 8 h dark).
The water was changed at least once per week. The dissolved oxygen concentration was more than 80% of the air saturation value.

ACCLIMATION
- Acclimation period: At least 7 days
- Acclimation conditions (same as test or not): same as test conditions
- Type and amount of food during acclimation: Sera Vipan; SERA GMBH, 52518 Heinsberg, Germany, Amount: 4% of the fish body weight/feeding day
- Feeding frequency during acclimation: 3 times/week
- Health during acclimation (any mortality observed): < 5% mortality

FEEDING DURING TEST
- Food type: No feeding during test (+24 h before the test start)

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

40-250 mg/CaCO3/L

Test temperature:

23 ± 2 °C

pH:

6.0-8.5

Dissolved oxygen:

> 80%

Conductivity:

150 µS/cm

Nominal and measured concentrations:

Nominal loading rate of the test item was 31.3 [µg/L]
The geometric mean measured concentration of the test item was 17.7 µg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria of 3 L were used (dimensions: 11.5/13/20 cm) and covered with glass plates.
- Type (delete if not applicable): closed
- Aeration: Gentle aeration was provided
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 7
- 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 of local origin was used. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
- Total organic carbon: 18 mg/L
- Alkalinity: 0.60 mmol/L
- Culture medium different from test medium: No
- Intervals of water quality measurement: Quaterly

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/ 8 h dark corresponding to natural daylight hours
- Light intensity: 7-750 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations were made after 3, 24, 48, 72 and 96 h. Fish were considered dead if there was no visible movement (e.g. gill covvers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalties (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). For measurements of the size and weight the control fish were used at the end of the exposure.

TEST CONCENTRATIONS
- Range finding study: Not necessary due to prior acute toxicity studies, e.g. to invertebrates and algae.
- Test concentrations: 32 mg/L

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

> 31.3 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 31.3 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

> 17.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 17.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: not observed
- Observations on body length and weight: no abnormalities observed
- Mortality of control: 0%

Sublethal observations / clinical signs:

Observations of Sub Lethal Effects and Normal Behavior in the Test Vessels

             (n = 7, number of fish)

 

Geometric mean measured concentration [µg/L]	Effect *	Number of fish affected at observation time [hours]
		day 0		day 1	day 2		day 3		day 4
		2	4	24	48	52	71	76	93.5	96
17.7	(1)	7	7	7	7	7	7	7	7	7
Control	(1)	7	7	7	7	7	7	7	7	7

         *) The numbers in brackets correspond to the following observations: 

                   (1)    = Normal behavior

Cumulative Mortality[%]in the Test Vessels

Geometric mean measured concentration [µg/L]	Cumulative mortality[%] at observation time [hours]
	day 0		day 1	day 2		day 3		day 4
	2	4	24	48	52	71	76	93.5	96
17.7	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0

 

Measured Concentrations of the Test Item during the Definitive Test

Sampling date:	0 hours, Fresh medium		24 hours, Old medium		24 hours, Fresh medium		96 hours, Old medium
Nominal loading rate of the test item[µg/L]									Geometric mean measured concentration of the test item [µg/L]
	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%
31.3	23.1	74	5.37	18	29.4	94	25.1	80	17.7
QC	2.36	117	2.41	119	1.96	97	2.48	123
Control	< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc.  = measured concentration of the test item, mean value of two injections, dilution factor taken into account %               =percent of the nominal loading rateof the test item

LOQ            = limit of quantification of the analytical method (2 µg test item/L)

QC              = Quality Control (2 µg test item/L)

 

Validity criteria fulfilled:

yes

Conclusions:

The LC0 corresponds to the geometric mean measured test item concentration ≥ 17.7 µg test item/L, the LC50 was > 17.7 µg/L.

Executive summary:

The acute toxicity of the test item to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (2019) from 2020-02-10 to 2020-02-14.

The test item is a slightly yellow solid and dispersible in water. A stock solution with a nominal concentration level of 100 mg test item/L was freshly prepared with dilution water. Out of the stock solution a concentration of 31.3 µg/L was tested in a limit test. The selection of the test concentration is based on the derivation of a threshold concentration (TC) from the results of an alga toxicity test (ErC50 (0-72 h) = 99.0 µg/L (geometric mean measured test item test item concentration)) and a daphnia acute immobilization test (EC50 (0-48 h) = 31.3 µg/L (geometric mean measured test item concentration)).

The study was performed over a period of 96 hours under semi-static conditions with renewal of test solutions after every 24 hours as a worst-case exposure. Seven test organisms were exposed to the limit concentration and the control, respectively. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits. The test media was visually clear.

The concentration of the test item was analytically verified via LC-MS/MS in the fresh media at the start of the exposure and at one renewal interval (0 hours and 72 hours) and in the corresponding 24-hours aged media after 24 and 96 hours in the limit test item concentration and the control. The measured concentrations of the test item at the start of two exposure intervals after 0 and 72 hours were between 74 and 94% of the nominal values. At the end of the exposure intervals after 24 and 96 hours the measured concentrations of the test item were between 18 and 80%, the decrease over 24 hours was < 20% with one exception. All effect values were based on the geometric mean measured concentration of the test item.

All validity criteria of the test guideline were met. The test item does not show toxic effects on fish at the limit concentration of 17.7 µg/L.

Additional information

The acute toxicity of the test item to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (2019) from 2020-02-10 to 2020-02-14.

The test item is a slightly yellow solid and dispersible in water. A stock solution with a nominal concentration level of 100 mg test item/L was freshly prepared with dilution water. Out of the stock solution a concentration of 31.3 µg/L was tested in a limit test. The selection of the test concentration is based on the derivation of a threshold concentration (TC) from the results of an alga toxicity test (ErC50 (0-72 h) = 99.0 µg/L (geometric mean measured test item test item concentration)) and a daphnia acute immobilization test (EC50 (0-48 h) = 31.3 µg/L (geometric mean measured test item concentration)).

The study was performed over a period of 96 hours under semi-static conditions with renewal of test solutions after every 24 hours as a worst-case exposure. Seven test organisms were exposed to the limit concentration and the control, respectively. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits. The test media was visually clear.

The concentration of the test item was analytically verified via LC-MS/MS in the fresh media at the start of the exposure and at one renewal interval (0 hours and 72 hours) and in the corresponding 24-hours aged media after 24 and 96 hours in the limit test item concentration and the control. The measured concentrations of the test item at the start of two exposure intervals after 0 and 72 hours were between 74 and 94% of the nominal values. At the end of the exposure intervals after 24 and 96 hours the measured concentrations of the test item were between 18 and 80%, the decrease over 24 hours was < 20% with one exception. All effect values were based on the geometric mean measured concentration of the test item.

All validity criteria of the test guideline were met. The test item does not show toxic effects on fish at the limit concentration of 17.7 µg/L.