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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-2018 till 11-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Substance is poorly soluble in water and therefore a WAF approach was followed

Analytical monitoring:

yes

Details on sampling:

All loading levels and the control were analytically verified via LC-MS/MS in the fresh media at the start of exposure and at the renewal of the test solutions (0 and 24 hours) as well as in the 24-hours old media at the renewal and at the end of the exposure (24 and 48 hours).
The samples were analysed with an LC-MS/MS method, implemented under non-GLP and documented finally in the GLP raw data.

Vehicle:

no

Details on test solutions:

The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette (see ‘Application’).

Test loadings
5 loading levels of the test item in a geometric series with a separation factor of 2 were tested as follows: 0.0625 - 0.125 - 0.250 - 0.500 - 1.00 mg/L.
The test item loadings were selected based on the results of two non-GLP preliminary range finding test. For results, see Annex II.

Water Accommodated
In view of the difficulties associated with the evaluation of aquatic Fraction 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 was 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 Fractions
Five water soluble fractions (WSF) were prepared with nominal loadings as specified below.
For the highest loading level of 1.00 mg/L, an appropriate amount of the test item was weighed out and transferred into a brown glass flask with an appropriate amount of demineralized water. The test item dispersion was shaken for 24 hours with 20 rpm at room temperature. After completion of shaking and a separation phase of 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 presaturated in order to avoid adsorption during the filtration. The first 25 mL of filtrate were 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. The following filtrate, i.e. the WSF, was used in the test and for preparation of further loading levels by dilution with dilution water (see Table 3) after addition of the mineral components as specified in Table 3. During filtration, the filter was always kept covered. This procedure was carried out one day before the start of the exposure (at day -1) and one day before the renewal of the test solutions (at day 0). With regard to the water solubility (i.e. CMC) of 80 mg/L the initial measured concentrations in the range finding test which were prepared according to the procedure described here are considered to be in equilibrium as they were distinctly lower than the CMC. Since weighing below 1 mg is inaccurate according to the test facility SOPs the WSFs 0.0625 to 0.500 mg/L were prepared by dilution of the WSF 1.00 mg/L.

Control Dilution water without test item incubated under the same conditions as the test groups

Additional Observations during the Definitive Test: All tested loading levels were colorless and visually clear throughout the exposure period.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test System and Culture

Test system Daphnia magna STRAUS, obtained from continuous laboratory cultures.

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline and is bred at the test facility.

Origin Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture I In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination, light intensity of max. 1500 lx

Culture medium Elendt M4, according to OECD 202, Annex 3 (2004), is used. The composition of the culture medium

Feeding of the culture stocks: The daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae were cultured at the test facility.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

282 mg CaCO3/L at start
275 mg CaCO3/L after 24h

Test temperature:

During the test period, the temperature in the incubator was 19.0 – 20.0 °C.

pH:

pH range 7.39 - 8.50

Dissolved oxygen:

DO2 range: 7.92 - 9.02 mg/L

Salinity:

Fresh water

Conductivity:

598 - 602 uS/cm

Nominal and measured concentrations:

Nominal test loadings: Control, 0.0625, 0.125, 0.250, 0.50, 1.0 mg/L
Geometric mean measured concentration: LOQ = 5 ug/L

Details on test conditions:

Test method: The study was performed under semi-static conditions with a renewal of the test solutions after 24 hours.
Test duration 48 hours
Test vessels Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses
Test volume 20 mL
Test temperature (target) 18 - 22 °C, constant within ± 1 °C
Illumination (target) Diffuse light, light intensity of max. 1500 lx
Photoperiod (target) 16/8 hours light/dark cycle
Feeding The daphnids were not fed during the study.
Dilution water Prior to the start of the exposure (0 hours) and the renewal of the test solutions (24 hours), the water quality parameters (i.e. pH value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured.
Test media At the start of the exposure and at the renewal (0 and 24 hours), the water quality parameters of the fresh media (i.e. pH value, dissolved oxygen concentration) were measured in one additional replicate (without daphnids) per loading level and control.
At renewal and at the end of the exposure (24 and 48 hours), the water quality parameters of the old media were measured in one appropriate replicate (containing daphnids) per loading level and control. The replicate with the highest immobilization rate per loading level and control was measured.
Temperature The incubator temperature (measurement in air with a thermo-hygrograph) was recorded throughout the period of the test.

Reference substance (positive control):

yes

Remarks:

A reference test with potassium dichromate was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium (Table 3) under static conditions with a test duration of 24 hours once per month.

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

0.934 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 0.574 - 1.00 mg/L

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

0.212 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CK <0.0625 - 0.499 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.09 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Quantification of diamine MW = 325.45 which corresponds with Oleyldiamine

Basis for effect:

mobility

Remarks on result:

other: CL: 4.6 - 6.29

Details on results:

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits during the study.

Results with reference substance (positive control):

Test of the Reference Item

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2018 10-16 to 2018-10-17. For results of the most recent of the monthly performed reference tests, see Table 7.

Table 7: EC50-Value (with Confidence Limits) of the Reference Item Potassium dichromate
based on nominal concentrations [mg/L], (0 - 24 hours)
Current Study Valid Range
EC50 2.09 mg/L 0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
Confidence limits 1.00 - 4.00 mg/L 0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A

Results of the Definitive Test

 

Biological Data

 

The percentage of immobility, determined in all loading levels of the test item and in the control after 24 and 48 hours is given in Table 4. The absolute numbers of immobile daphnids are presented in Table 5.

 

Table 4:   Immobilization Rates after 24 and 48 hours ofExposure in the Definitive Test

             (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal test item loadings   [mg/L]	Geometric mean measured test item concentrations [µg/L]	IMMOBILIZATION [%]
		24 hours					48 hours
		Replicates					Replicates
		1	2	3	4	MV	1	2	3	4	MV
1.00	6.29	0	40	0	40	20	20	100	20	60	50
0.500	4.03	20	0	0	0	5	60	60	0	20	35
0.250	3.44	0	0	0	0	0	0	0	0	20	5
0.125	2.94	0	0	0	20	5	0	0	0	20	5
0.0625	2.50	0	0	20	0	5	0	0	20	0	5
Control		0	0	0	0	0	0	0	0	0	0

 

Tabl e 5:   Absolute Numbers of immobile Daphnids after 24 and 48 hours ofExposure in the Definitive Test

             (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal test item loadings   [mg/L]	Geometric mean measured test item concentrations [µg/L]	Number of immobile Daphnids / Total number of Daphnids
		24 hours					48 hours
		Replicates					Replicates
		1	2	3	4	Sum	1	2	3	4	Sum
1.00	6.29	0 / 5	2 / 5	0 / 5	2 / 5	4 / 20	1 / 5	5 / 5	1 / 5	3 / 5	10 / 20
0.500	4.03	1 / 5	0 / 5	0 / 5	0 / 5	1 / 20	3 / 5	3 / 5	0 / 5	1 / 5	7 / 20
0.250	3.44	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	1 / 5	1 / 20
0.125	2.94	0 / 5	0 / 5	0 / 5	1 / 5	1 / 20	0 / 5	0 / 5	0 / 5	1 / 5	1 / 20
0.0625	2.50	0 / 5	0 / 5	1 / 5	0 / 5	1 / 20	0 / 5	0 / 5	1 / 5	0 / 5	1 / 20
Control		0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

 

The loading-effect relationships for immobilization after 24 and 48 hours of exposure are illustrated graphically in Figure 1 to Figure 2. The effect loadings (EL_{10 / 50 / 100}), based on the nominal loadings of the test item are presented in Table 1 .

The concentration-effect relationships for immobilization after 24 and 48 hours of exposure are illustrated graphically in Figure 3 to Figure 4. The effect concentrations (EC_{10 / 50 / 100}), based on the geometric mean measured concentrations of the test item are presented in Table2.

 

 

Measured Exposure Concentrations during the Definitive Test

 

The concentrations of the test item were analytically verified via LC-MS/MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) and in the 24-hours old media at the renewal and at the end of the test (24 and 48 hours) in all loading levels and the control.

The measured concentrations of the test item N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%) in the fresh media were in the range of < LOQ to 0.0338 mg/L at the start of the exposure (0 hours) and at the renewal (24 hours) focusing the quantification on oleyldiamine as representative for the diamine fraction in the product. The measured concentrations of the test item in the old media at the renewal and at the end of the test (24 and 48 hours) were all below the LOQ. The analytical results are presented in Table 6.
The geometric mean measured concentrations were 2.50 – 2.94 – 3.44 – 4.03 – 6.29 mg/L. For the measured values < 70% of the LOQ, the half of the LOQ was used for calculation of the geometric mean measured concentration.

 

Table 6:      Measured Concentrations of the Test Item              during the Definitive Test

Sampling date	Fresh media, 0 hours	Old media, 24 hours		Fresh media, 24 hours	Old media, 48 hours
Nominal test item loadings [mg/L]	N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%)						Geometric mean measured test item concentration [µg/L]
	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%
1.00	33.8	3.71*	11	4.98*	< LOQ^#		6.29
0.500	16.9	< LOQ^#		< LOQ^#	< LOQ^#		4.03
0.250	8.94	< LOQ^#		< LOQ^#	< LOQ^#		3.44
0.125	4.75*	< LOQ^#		< LOQ^#	< LOQ^#		2.94
0.0625	< LOQ^#	< LOQ^#		< LOQ^#	< LOQ^#		2.50
Control	< LOQ	< LOQ		< LOQ	< LOQ

Meas. conc. = measured concentration of the test item, mean value of all injections, dilution factors taken into account

LOQ              = limit of quantification of the analytical method (5 µg test item/L)

%                  = percentage of the initially measured concentrations of the test item

*                    = < LOQ, but > 70% of the LOQ and therefore taken into account for the calculation of the geometric mean

                        measured test item concentrations

^#                               = Measured values < 70% of the LOQ were taken into account with half of the LOQ (= 2.50 µg/L of the test

                        item) for the calculation of the geometric mean measured concentrations.

Validity criteria fulfilled:

yes

Remarks:

The study was performed according to OECD Guideline 202 (2004). The validity criteria were fulfilled:

Conclusions:

Based on the nominal loadings of the test item N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%), the 48 hours-EL50 for Daphnia magna was 0.934 mg/L (confidence limits: 0.574 - > 1.00 mg/L).

Based on the geometric mean measured concentrations of the test item N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%), the 48 hours-EC50 for Daphnia magna was 6.09 µg/L (confidence limits: 4.60 - > 6.29 µg/L).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%) were determined according to OECD 202 (2004).

The study was conducted under semi-static conditions over a period of 48 hours with a WSF (1.00 mg/L) of the test item and four dilution levels (nominal: 0.0625 to 0.500 mg/L) prepared out of the WSF in a geometric series with a separation factor of 2. Due to the complex structure of the test item and low water solubility, the WSF approach was used. As the tested loadings were too low for exact direct weighing, the lower concentrations were diluted from the WSF.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control.

The concentrations of the test item were analytically verified via LC-MS/MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) and in the 24-hours old media at the renewal and at the end of the test (24 and 48 hours) in all loading levels and the control. Quantification was focused on the most toxic fraction i.e. the diamine fraction in the test item quantifying the oleyldiamine (MW 324.6 Da) as representative for the diamine fraction in the test item.

The measured concentrations of the test item N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%) in the fresh media were in the range of < LOQ to 0.0338 mg/L at the start of the exposure (0 hours) and at the renewal (24 hours). The measured concentrations of the test item in the old media at the renewal and at the end of the test (24 and 48 hours) were all below the LOQ.
The geometric mean measured concentrations were 2.50 – 2.94 – 3.44 – 4.03 – 6.29 µg/L. For the measured values < 70% of the LOQ, the half of the LOQ was used for calculation of the geometric mean measured concentration.

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 in Table 1 were based on the nominal loading levels.

Additionally, the EC₁₀/₅₀/₁₀₀-values were determined based on the geometric mean measured concentrations of the test item N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%). For details, see Table2.

The validity criteria of the test guideline were fulfilled.

 

Table1:      EL₁₀-, EL₅₀- (with Confidence Limits) and EL₁₀₀-Values

                     (based on the nominal loading levels of the test item)

N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%)
Effect values		Test duration [hours]		Nominal test item loadings [mg/L]
EL10 (with confidence limits)		24		0.775 (CI: Not applicable)
		48		0.212 (Cl: < 0.0625 – 0.499)
EL50 (with confidence limits)		24		> 1.00 (Cl: Not applicable)
		48		0.934 (Cl: 0.574 – > 1.00)
EL100		24		> 1.00
		48		> 1.00

 

Table2:      EC₁₀-, EC₅₀- (with Confidence Limits) and EC₁₀₀-Values

                     (based on the geometric mean measured concentrations of the test item)

N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%)
Effect values		Test duration [hours]		Geometric mean measured test item concentrations [µg/L]
EC10 (with confidence limits)		24		5.60 (CI: Not applicable)
		48		3.09 (Cl: < 2.50 – 4.28)
EC50 (with confidence limits)		24		> 6.29 (Cl: Not applicable)
		48		6.09 (Cl: 4.60 – > 6.29)
EC100		24		> 6.29
		48		> 6.29

 

B

Description of key information

Based on the structural similarity with the diamines and the fact that the diamines are the main driver of the toxicity, read-across from the diamines category to diamines mono-oleates is considered justified.

Two acute daphnia toxicity test robust summaries are included in the dossier. One with N-(Oleyl alkyl)- 1,3 -propanediamine mono-oleates (Scheerbaum, 2018) and one with N-(Oleyl alkyl)- 1,3 -propanediamine (CECA, 1999).

Similar as for the fish, the test solutions from these acute daphnia tests were prepared differently. Where the mono-oleates was prepared from a water accommodated fraction in OECD test media, the oleyldiamine solutions were prepared by serial dilution of a homogeneous stock solution standard lab water. The results of these studies are therefore not directly comparable. Therefore only the results of N-(Oleyl alkyl)- 1,3 -propanediamine mono-oleates (Scheerbaum, 2018) are used for this endpoint.

Test summary:

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.

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.

Based on the nominal loadings of N-(Oleyl alkyl)- 1,3 -propanediamine mono-oleates (100%), the 48 hours-EL50 for Daphnia magna was 0.934 mg/L (confidence limits: 0.574 - > 1.00 mg/L).

These values are used as Key values for the chemical safety assessment.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.934 mg/L

Additional information

The summary of the acute daphnia test with N-(Oleyl alkyl)- 1,3 -propanediamine mono-oleate(Scheerbaum, 2018)

In the acute immobilization test with Daphnia magna (STRAUS), the effects of N-(Oleyl alkyl)- 1,3-propanediamine mono-oleates (100%) were determined according to OECD 202 (2004).

The study was conducted under semi-static conditions over a period of 48 hours with a WSF (1.00 mg/L) of the test item and four dilution levels (nominal: 0.0625 to 0.500 mg/L) prepared out of the WSF in a geometric series with a separation factor of 2. Due to the complex structure of the test item and low water solubility, the WSF approach was used. As the tested loadings were too low for exact direct weighing, the lower concentrations were diluted from the WSF.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control.

The concentrations of the test item were analytically verified via LC-MS/MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) and in the 24-hours old media at the renewal and at the end of the test (24 and 48 hours) in all loading levels and the control. Quantification was focused on the diamine fraction in the test item quantifying the oleyldiamine (MW 324.6 Da) as representative for the diamine fraction in the test item.

The measured concentrations of the test item N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%) in the fresh media were in the range of < LOQ to 0.0338 mg/L at the start of the exposure (0 hours) and at the renewal (24 hours). The measured concentrations of the test item in the old media at the renewal and at the end of the test (24 and 48 hours) were all below the LOQ.
The geometric mean measured concentrations were 2.50 – 2.94 – 3.44 – 4.03 – 6.29 µg/L. For the measured values < 70% of the LOQ, the half of the LOQ was used for calculation of the geometric mean measured concentration. Based on the geometric mean measured concentrations of the test item N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%), the 48 hours-EC50 for Daphnia magna was 6.09 µg/L (confidence limits: 4.60 - > 6.29 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 EL₁₀/₅₀/₁₀₀-values given were based on the nominal loading levels.

Based on the nominal loadings of the test item N-(Oleyl alkyl)- 1,3-propanediamine mono oleates (100%), the 48 hours-EL50 for Daphnia magna was 0.934 mg/L (confidence limits: 0.574 - > 1.00 mg/L).

Diamines short-term toxicity to aquatic invertebrates

For N-(alkyl) - 1,3 - propanediamines several acute daphnia tests were performed but for the diamines category these results are not used. Instead the result of a long term daphnia test is used. This study is similar to the fish study with diamines performed with natural river water for the intented Bulk approach.

The summary of the acute daphnia test with N-(Oleyl alkyl)- 1,3-propanediamine (CECA, 1999)

The toxicity of N-(Oleyl alkyl)- 1,3 -propanediamine to Daphnia magna has been investigated according to EEC guideline C2 and OECD 202, assessing mobility inhibition. Daphnia magna were exposed for 48h to an aqueous solution of N-(Oleyl alkyl)- 1,3-propanediamine at different nominal concentrations of 0 - 0.02 - 0.04 - 0.09 - 0.21 - 0.45 - 1.00 mg/L. EC50 (24h) and EC50 (48h) ranged from 0.013 to 0.025 mg/L (measured concentrations).