Amines, N-(C16-18 and C18-unsatd. alkyl)-N,N',N'-trimethyltrimethylenedi-

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:

2017-07-07 to 2017-07-20, with the definitive exposure phase from 2017-07-10 to 2017-07-14, at the test facility

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

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The limit concentration and the control were analytically verified via LC-MS/MS from freshly prepared media after 0 and 72 h and from corresponding aged test media after 24 and 96 h. Since analytical measurements of the algae and daphnia study showed no relevant adsorption to glassware (values ≤ LOQ), no adsorption of the test item onto the test vessels was determined.

Vehicle:

no

Details on test solutions:

Test method
A semi-static test with daily renewal of the test media was performed.

Test concentration
16 µg/L was tested as a threshold concentration 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) 16.1 µg/L (nominal test item concentration) and a daphnia reproduction test (EC50 (48 h) > 100 µg/L (nominal test item concentration)), both unpublished.

Preparation of the test medium
A stock solution of 10.0 mg/L (total volume 1000 mL) was freshly prepared in dilution water. The dispersion was treated with ultrasound for 30 minutes at 40 °C and used to prepare the threshold concentration of 16 µg/L. After transferring the stock solution into the test vessel the test media was mixed with an ultraturrax (1 min, at least 8500 rpm).This procedure was repeated daily for preparation of the stock solution and the threshold concentration for the daily water renewal.



Control
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: All fish used in the test were gained at NOACK LABORATORIEN from a single brood stock. (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)
- Length at study end (length definition, mean, range and SD): Average body length: 2.77 cm
- Weight at study end (mean and range, SD): Average body weight: 0.166 g
- Method of breeding:Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (0.1 - 10 µmol photons • m-2 • s-1, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. No disease treatments were administered throughout holding and testing. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started.
- Feeding during test: No feeding during test


ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within these days before the study starts were used in the test. A sufficient number of fish was acclimatized to dilution water for 7 days prior to start of the exposure.

- 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: Sera Vipan; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week.
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered throughout holding and testing.

Test type:

semi-static

Water media type:

other: River Water

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total Hardness at day 0: 299 mg CaCO3/L

Test temperature:

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

pH:

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

Dissolved oxygen:

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

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: Glass aquaria
- Type (delete if not applicable):
- Material, size, headspace, fill volume:
Glass aquaria of 3 L were used, filled with at least 2 L of natural river water (dimensions: 11.5/13/20 cm, depth of water: at least 13.5 cm), and covered with glass plates
Pre-saturation of the test vessels
A coating phase (saturation of the test vessels) was carried out. The test vessels were pre-treated with the appropriate test solutions for 72 to 96 hours under test conditions. Before the start of the exposure, the test vessels were emptied and refilled with freshly prepared test solutions. Preparation of the test concentration for coating was carried out with dechlorinated tap water.

- Aeration: Gentle aeration was provided
- Renewal rate of test solution (frequency/flow rate): A static test procedure with one application at the start of the study was performed.
- Control: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates
- Reference item: No reference item is recommended for this test according to the guideline.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.465 g fish per L test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerate for at least 24 h to remove chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
Alkalinity: 0.60 mmol/L (measured quarterly)
Acidity: 0.10 mmol/L (measured quarterly)
Conductivity: 138 µS/cm (measured quarterly)

A sufficient number of fish was acclimatized to dilution water (Natural river water) for 7 days prior to start of the exposure.

Natural river water (Dilution water)
Water of the river Innerste was used as its properties in terms of organic matter content and suspended solids are considered typical for European rivers and are close to those in the EU risk assessment methods. The water parameters and the geographical site are given in the table below.

Water Parameters of the River Water
River Innerste
Location Amtshof K514, km 26,
31157 Ruthe-Sarstedt, Germany
Latitude:52.25°, Longitude: 9.83°
Sampling Date 2016-08-09
Weather on Day
of Sampling Cloudy; 18°C
Colour Yellowish to brownish, clear
pH 7.89
Conductivity [µS/cm] 1123
Dissolved Oxygen [mg O2/L] 6.97
DOC [mg C/L] 2.71
TOC [mg C/L] 3.33
Ammonium-N [mg NH4-N/L] < 0.04
Nitrate-N [mg NO3-N/L] 2.1
o-Phosphate-p [mg P/L] < 0.05
Total Phosphate [mg P/L] 0.05
Suspended Matter [mg/L] 12.8
Total Hardness [mg CO3/L] 272
Storage conditions 15 ± 2°C
NPOC = non purgeable organic carbon

Storage conditions
The natural river water was stored at 15 ± 2 °C.
About 24 hours prior to experimental starting (pre-saturation of test vessels) the natural river water was stored at test temperature and gently aerated.

- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH-value, temperature and oxygen saturation were measured in all test vessels at the start and end of the
exposure and every 24 hours. Total hardness was determined at the start of the exposure out of the control. During the test the water temperature was recorded continuously (once per hour) with a data logger. The light intensity on the surface of the test aquaria was measured at the start of the exposure.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: A photoperiod of 16 h light / 8 h dark corresponding to natural daylight hours occurred during the course of the study.
- Light intensity: 7 - 750 Lux

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 2, 24, 48, 72 and 96 h.

TEST CONCENTRATIONS
- Range finding study: None

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

16 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 16 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.:

> 16 µg/L

Nominal / measured:

nominal

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):

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

Evaluation
The LC0 and LC100 after 96 h was derived from the raw data of the limit concentration. LC50-values do not have to be calculated in a limit test.

Software
All data were computer-generated and rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.

 

Sublethal observations / clinical signs:

The test solution was visible clear throughout the period of exposure.

All effect levels are given based on the nominal threshold concentration of the test item after 96 hours. The LC₀after 96 hours was 16 µg/L.

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

(n = 7, number of fish)

Nominal test item concentration [µg/L]	Effect *	Number of fish effected at observation time [hours]
		2	24	48	72	96
16	(1)	7	7	7	7	7
Control	(1)	7	7	7	7	7

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

               (1)          = Normal behavior

                    

 Cumulative Mortality [%] in the Test Vessels

Nominal test item concentration [µg/L]	Cumulative mortality at observation time [hours]
	2	24	48	72	96
16	0	0	0	0	0
Control	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

All effect levels are given based on the nominal threshold concentration of the test item N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine. The threshold concentration of the test item was found to have no lethal nor sub lethal effects to zebrafish.
In conclusion, the LC0 corresponds to the nominal concentration of 16 µg test item/L.

Executive summary:

The acute toxicity of the test item N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine (batch no.211401A Morris) to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (1992) and EU Council Regulation (EC) No. 440/2008/ Method C.1 (2008) from 2017-07-07 to 2017-07-20, with the definitive exposure phase from 2017-07-10 to 2017-07-14, at the test facility.

An acute toxicity test with the threshold concentration of 16 µg/L was conducted under semi-static conditions with daily renewal of the test media. Duration of the test was 96 hours. Seven test organisms were exposed to the threshold concentration and the control. 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 determination of concentrations of three components of thetest itemN-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diaminewas carried out via LC-MS/MS on a reversed phase column using external standard calibration. Evaluation was done from freshly prepared media after 0 and 72 hours and from the corresponding aged test media after 24 and 96 hours. The measured concentrations of the three components in freshly prepared media were 93 and 118 %of the nominal value. In corresponding aged test media, the measured concentrations were 10 and 68 % of the nominal value.

 

The aquatic ecotoxicity tests withN-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diaminewere performed in river water to allow a PEC_aquatic,bulk/PNEC_aquatic,bulk approach and is considered to be conservative but more environmentally realistic than the standard method. This approach is based on PEC estimations representing'total aquatic concentrations'. To characterize the risk to the aquatic compartment the PEC_aquatic,bulk is compared with the PNEC_aquatic,bulk derived from river water ecotoxicity studies (ECETOC, 2003).

 

In order to class standard laboratory toxicity study valid, it is of particular importance that – besides information on test substance, test method / conditions and test organism used – suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations are based upon measured levels.

 

For ecotoxicity tests performed using the bulk approach, however, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the concentration-effect relationship should be based on the sum of adsorbed and dissolved substance in the volume of the medium tested. One of the advantages of the bulk approach tests with these difficult substances is that in the presence of suspended matter, humic acids, the residual sorption to glassware will be negligible. The results of these bulk approach tests are therefore much easier to interpret and more realistic, and if compared to PEC_bulk clearly provide a more appropriate assessment of risks for the environment. An individual test design was applied. Natural river water was used as dilution medium . 

Following the bulk approach and considering that biodegradation as possible reason for the observed decrease is very unlikely considering the short time frame of refreshment and the fact that the observed sorption to glassware is negligible shows that the test organisms were fully exposed to the test substance during the test. It is therefore considered justified to use the nominal test concentrations to derive the effect values.

 

All validity criteria of the test guideline were met.

The results of the study are summarized in the table below

 

LC-Values (4 – 96 hours) of the Test Item

                  Based on nominal test item concentrations

 

LC100= Lowest test item concentration with 100 % mortality after 96 h	> 16 µg/L
LC50	> 16 µg/L
LC0= Highest test item concentration with 0 % mortality after 96 h	16 µg/L

 

Description of key information

A study was conducted to determine the acute toxicity of N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine to Zebra fish (Danio rerio) according to the OECD Guideline 203 and EU Method C.1, in compliance with GLP.

The test performed with the threshold concentration of 16 µg/L was conducted under semi-static conditions with daily renewal of the test media. Duration of the test was 96 hours. Seven test organisms were exposed to the threshold concentration and the control. 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 determination of concentrations of three components of the test item N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diaminewas carried out via LC-MS/MS on a reversed phase column using external standard calibration. Evaluation was done from freshly prepared media after 0 and 72 hours and from the corresponding aged test media after 24 and 96 hours. The measured concentrations of the three components in freshly prepared media were 93 and 118 % of the nominal value. In corresponding aged test media, the measured concentrations were 10 and 68 % of the nominal value.

 

The aquatic ecotoxicity tests with N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine were performed in river water to allow a PECaquatic,bulk/ PNECaquatic,bulk approach (ECETOC 2003) and is considered to be conservative but more environmentally realistic than the standard method.

The results of these bulk approach tests are much easier to interpret and more realistic, and when compared to PECbulk clearly provide a more appropriate assessment of risks for the environment.

Following the bulk approach and considering that biodegradation as possible reason for the observed decrease is very unlikely considering the short time frame of refreshment and the fact that the observed sorption to glassware is negligible shows that the test organisms were fully exposed to the test substance during the test. It is therefore considered justified to use the nominal test concentrations to derive the effect values.

 

Under the study conditions, the 96h LC50 and NOEC of the test substance was determined to be >16 and 16 µg/L, respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

16 mg/L

Additional information

NOEC = 16 µg/L; LC50>16 µg/L