Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine

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:

from 2018-11-26 to 2018-11-30 at the test facility

Reliability:

1 (reliable without restriction)

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 (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The limit loading and the control were analytically verified via LC-MS/MS in the fresh media at the start of exposure and at renewal (0 and 72 hours) and in the 24-hours old media after 24 and 96 hours. The analytical method validation was done according SANCO/3029/99 rev.4 (2000) which is consistent with the ECHA guidance on information requirements (Version 1, July 2013).

Vehicle:

no

Details on test solutions:

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

Test loading One loading level of nominal 0.300 mg/L was tested as a threshold loading in a limit test. Taking into account the aquatic toxicity data of the test item, the test was performed without a range-finding test. The threshold loading (TL) was based on an algae toxicity data of the test item: ErL50 (0-72 h) = 0.276 (0.116-0.669) mg/L and a daphnia toxicity test of the test item:
EL50(0-48 h) = 0.934 (0.574 – > 1.00) mg/L (both: nominal loadings, SCHEERBAUM 2018).
Per definition of the WSF, all terms related to the concentration level were given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations.

Water Accommodated FractionI n view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000) is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture, an UVCB substance and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

Preparation of the Water Soluble Fraction One water soluble fraction (WSF) was prepared with the nominal loading of 0.300 mg/L as specified below.
For test start and each renewal day two brown glass flasks (each filled with 2 L dilution water) were prepared to achieve a sufficient volume for testing. Appropriate amounts of the test item were weighed out on glass slides and transferred into the glass flasks. The test item dispersions were shaken for 24 hours with 20 rpm at room temperature. After completion of shaking and a separation phase of at least 1 hour of standing at room temperature, the WAF was removed by siphoning from the approximate center of the water body. The WAF was filtrated to get the WSF.  
The filter (membrane filter 0.45 µm, RC, MACHEREY-NAGEL) was saturated in order to avoid adsorption during the filtration. The first 25 mL of filtrate was discarded. The filtration was interrupted for ca. 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item.
Thereafter, the filtration was continued. The next 25 mL were discarded. During filtration, the filter was always kept covered. The following filtrate, i.e. the WSF, was used in the limit test. This procedure was carried out one day before the start of the exposure (at day -1) and on days of 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: 2.36 cm
- Weight at study end (mean and range, SD): Average body weight: 0.09 g
- Method of breeding:Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (7 - 750 lux, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. 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. Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within the last 7 days before the start of the exposure were used in the test. No mortality was observed during this time. No disease treatments were administered throughout holding and testing.
- 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.

- 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:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total Hardness at day 0: 74 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 results 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 (dimensions: 11.5/13/20 cm, depth of water: approx. 17 cm) and covered with glass plates.
Test volume: About 2.5 L per vessel

- Aeration: No aeration was provided
- Renewal rate of test solution (frequency/flow rate): A semi-static test with daily renewal of the test media 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: 0.252 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Tap water of local origin was used for holding and testing. 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
Acidity: 0.10 mmol/L (recent measurement 2017-01-10)
Alcalinity: 0.80 mmol/L (recent measurement 2017-01-10)
Conductivity: 162 µS/cm (recent measurement 2018-10-17)

- 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:

LL0

Effect conc.:

0.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: See tables below
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: Not observed
- Any observations (e.g. precipitation): The test solution was visible clear throughout the period of exposure.

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

LLx-values and statistical evaluation
The LL0 after 96 h was derived from the raw data of the limit loading. LL50-values do not have to be calculated in a limit test.

Software
The data for the tables in this report were computer generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.

The following software was used for calculations: Excel, MICROSOFT CORPORATION

Sublethal observations / clinical signs:

Preliminary Test (non-GLP)

 

A preliminary test with the nominal loading of 0.5 mg/L was carried out under test conditions without fish. The prepared WSF was sampled at the start of the test and after 24 hours incubation under test conditions.

 

 

MeasuredConcentrations ofN-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%)during thePreliminary Test

Sampling date	Fresh media, 0 hours	Old media, 24 hours
Nominal loading of the test item [mg/L]	N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%)
	Meas. conc. [µg/L]	Meas. conc. [µg/L]
0.5	8.92	< LCL

Meas. conc.    = measured concentration of the test item, dilution and enrichment factors taken into account

LCL        = lowest calibration level (0.5 µg/L of the test item)

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

(n = 7, number of fish)

 

Nominal loading level of the test item (WSF) [mg/L]		Effect *		Number of fish effected at observation time [hours]
			2		24		48		72		96
0.300		(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 loading level of the test item (WSF) [mg/L]	Cumulative mortality at observation time [hours]
	2	24	48	72	96
0.300	0	0	0	0	0
Control	0	0	0	0	0

Measured Exposure Concentrations during the Definitive Test

 

 

The concentrations of the test itemN-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%)were analytically verified via LC-MS/MS from freshly prepared media after 0 and 72 hours and from the corresponding 24 hours aged test media after 24 and 96 hours in the limit tested concentration and the control.

 

The percent of the nominal concentrations of the test item at the start of the exposures were 1 % (0 h) and 3 % (72 h). At the end of exposures (24h and 96h), the measured concentrations were < LOQ. The geometric mean measured concentration was 0.00382 mg/L.

 

 

Measured Concentrations of the Test Item N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%)during the Definitive Test

Sampling date	0 hours Fresh medium		24 hours Old medium		72 hours Fresh medium		96 hours Old medium		Geometric mean measured concentration
Nominal concentration of the test item [mg/L]	N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%)
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	[mg/L]
0.300	0.00437^1)	1	< LOQ		0.00782	3	< LOQ		0.00382
Control	< LOQ		< LOQ		< LOQ		< LOQ		----

Meas. conc.= measured concentration of the test item, dilution factor taken into account

%                  = percent of the nominal concentration of the test item

LOQ             = Limit of Quantification (0.005 mg test item/L)

1)                  = < LOQ but > 70 % of the LOQ

Validity criteria fulfilled:

yes

Conclusions:

All effect levels are given based on the nominal loading level of the test item N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%). The WSF of the test item with a nominal loading of 0.300 mg/L was found to have no effects to zebrafish.
In conclusion, the LL0 corresponds to the nominal test item loading of 0.300 mg test item/L.

Executive summary:

The acute toxicity of N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%) 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).

 

An acute toxicity test with the WSF with the nominal loading of 0.300 mg/L was conducted under semi-static conditions with daily renewal of the test media. The test concentration was based on the results of the observed algae toxicity data: ErL50 (0 -72 h) = 0.276 (0.116 -0.669) mg/L and the observed daphnia toxicity data: EL50(0 -48 h) = 0.934 (0.574 – > 1.00) mg/L selected. Due to the complex structure of the test item, the WSF approach was used.Duration of the test was 96 hours. Seven test organisms were exposed to the WSF 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 limit concentration of the test item N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%)a nd the control were analytically verified via LC-MS/MS from freshly prepared test media after 0 and 72 h and from the corresponding 24 h aged test media after 24 and 96 h.

The measured concentrations in the fresh media (0 and 72 h) were 4.37 and 7.82 µg/L. The measured concentrations in the old media (24 and 96 h) were 1.20 and 2.09 µg/L. Per definition of the WSF, all terms related to concentration levels have to be given as loading levels because partly dissolved compounds and mixtures cannot be related to concentrations. Therefore, the LL0-value given in was based on the nominal loading level.

 

All validity criteria of the test guideline were met.The results of the study aresummarized in the table below

 

LL-Value (2 – 96 hours) of the Test Item

                  Based on the nominal test item loading [mg/L]

 

LL0= Highest test item loading with 0 % mortality after 96 h

0.300 mg/L

 

 

Description of key information

Three acute toxicity test robust summaries are included in the dossier. One with N-(Oleyl alkyl)- 1,3 -propanediamine mono-oleates, one with N-(Oleyl alkyl)- 1,3 -propanediamine di-oleates and one with

N-(octadecyl)- 1,3 -propanediamine. The test solutions from these tests were prepared differently. Where the mono-oleates was prepared from a water accommodated fraction in OECD test media, the di-oleates solutions were prepared by serial dilution of a homogeneous stock solution in natural river water (14 mg/L suspended mater and 2.8 mg/L DOC), the octadecyldiamine test solutions were prepared in a similar way as the di-oleates but in standard OECD medium. The results of these studies are therefore not directly comparable. The river water constituents are mitigating the toxicity because the risk assessment of the di-oleates is based on the Bulk approach and then this mitigation is taken into account in the exposure assessment.

The toxicity of both the mono and di oleates are expected to mainly originate from the diamine fraction and to a lesser extend from the salt(s) or free oleic acid (CAS no 112 -80 -1; LC50 fish > 100 mg/L Sigma-Aldrich) because under test conditions (pH ± 8) both the mono and di-oleate will be mainly dissociated (pKa = 7). For this reason, the Key diamine RSS is added and a summary of the available fish toxicity for diamines is presented in the additional info field.

Due to the complex structure of N-(Oleyl alkyl)- 1,3 -propanediamine mono-oleates (UVCB) and low water solubility, the WSF approach was used for ecotoxicity testing in the study from Scheerbaum (2018).

Per definition of the WSF, all terms related to concentration levels have to be given as loading levels because partly dissolved compounds and mixtures cannot be related to concentrations. Therefore, the EL10/50/100-values given were based on the nominal loading levels. The study from Scheerbaum resulted in an LL0 = 0.3 mg/L and LL50>0.3 mg/L. These values are used as Key values for the chemical safety assessment.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.3 mg/L

Additional information

The acute toxicity of N-(Oleyl alkyl)- 1,3 -propanediamine mono-oleates towards Danio rerio in standard lab water was determined according to OECD guideline 203 under GLP. A semi-static test with daily renewal of the test media was conducted with one loading level of nominal 0.30 mg/L as a threshold loading in a limit test. Taking into account the aquatic toxicity data of the test item, the test was performed without a range-finding test. The threshold loading (TL) is based on the observed algae toxicity data: E_rL_{50 (0 -72 h)}= 0.276 (0.116 -0.669) mg/L and observed daphnia toxicity data: EL_{50 (0 - 48 h)}= 0.934 (0.574 – > 1.00) mg/L. Duration of the test was 96 hours and no lethal or sub-lethal effects were observed at the loading level of nominal 0.30 mg/L (LL50>0.3 mg/L).

The toxicity of N-(Oleyl alkyl)- 1,3 -propanediamine di-oleates (N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate]) towards Danio rerio in natural river water was determined according to OECD guideline 203 under GLP. In this test, similar as in the study with N-(Oleyl alkyl)- 1,3 -propanediamine mono oleates, semi-static conditions with daily renewal of the test media was conducted with the nominal test item concentrations of 0.0855 – 0.188 – 0.413 – 0.909 – 2.00 mg/L (factor 2.2), corresponding to mean measured concentrations of 0.0625 – 0.140 – 0.290 – 0.668 – 1.56 mg/L. Duration of the test was 96 hours. The 96h-LC50 based on nominal concentrations was found to be 1.34 mg/L.

The toxicity of N-(octadecyl)- 1,3 -propanediamine towards Danio rerio in reconstituted lab water was determined according to OECD guideline 203 under GLP. In this static fish test the organisms were exposed to nominal test item concentrations of 0, 0.1, 0.22, 0.5 and 1.0 mg/L. The exposure concentrations were not monitored (1990, Kl 2). Duration of the test was 96 hours. The 96h-LC50 based on nominal concentrations was found to be 0.148 mg/L.

Summary of diamines toxicity to fish

Seven acute toxicity tests with fish are available for various alkyl-1,3-diamines with C12 -18 chains (Diamines; all reliability 2). The tests reveal a comparable toxicity for the diamines with alkyl chain lengths ranging from C12 to C18, independent of the alkyl chain length.

The tests with diamines were considered to be valid with restrictions for various reasons. The test concentrations were not analytically verified because a reliable specific method of analysis was not available at the time when the test was performed. Further concerns related to the poor solubility of the test substances and partial sorption onto the walls of test vessels.

The available acute fish toxicity data are all determined with the same species, endpoint, test duration, life stage and testing conditions and they have te same reliability rating. As the results are not more than order of magnitude apart (96h LC50 range 0.08 - 0.35 mg/L),

they can be harmonised by a geometric mean according to REACH Guidance document Chapter R.7b.

The geometric mean of all 7 tests has been calculated resulting in LC50 of 0.150 mg/L. It can be concluded that fish are less sensitive to the alkyl-1,3 -diamines than daphnia and algae in acute toxicity tests based on the geometric mean of all available acute test data. Therefore it can be concluded as well that fish toxicity is not a critical endpoint for the PNEC derivation, Classification and PBT assessment. The experimental value of 0.148 mg/L is selected as key value for the CSR.

Acute diamine toxicity to fish

CAS no	Species/test	Result	Reference	Remarks
90640-43-0	Danio rerio	0.1<96h-LC50 < 0.35 mg/L	Clariant 1985	Reliability 2, GLP
68603-64-5	Danio rerio	96h-LC50 = 0.148 mg/L	Clariant 1990	Reliability 2, GLP
7173-62-8	Danio rerio	96h-LC50 = 0.32 mg/L	Clariant 1991	Reliability 2, GLP
7173-62-8	Danio rerio	96h-LC50 = 0.16 mg/L	CECA 1997	Reliability 2, GLP
61791-55-7	Danio rerio	96h-LC50 = 0.1 mg/L	Akzo Nobel 1990	Reliability 2, GLP
61791-63-7	Danio rerio	96h-LC50 = 0.08 mg/L	Akzo Nobel 1993	Reliability 2, GLP
61791-63-7	Danio rerio	96h-LC50 = 0.15 mg/L	Clariant 1990	Reliability 2, GLP