Oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Nov 2017 - 26 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

EC Number:

248-813-1

EC Name:

Oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

Cas Number:

28065-97-6

Molecular formula:

C34H69NO2 C36H67NO2 C36H69NO2 C36H71NO2 C36H73NO2

IUPAC Name:

oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test item concentrations and the control
- Sampling method: The level of TOC (total organic carbon) measured in freshly prepared test media specimens and aged (after 3 days) test media specimens, were compared against the carbon content of the test item to derive the concentrations of test item at each time point. The suitability of the carbon analyser was cross-checked with a second carbon analyser at 0.5 mg/L TOC.
- Sample storage conditions before analysis: the specimens were stored at 2 - 8 °C

Test solutions

Vehicle:

yes

Remarks:

dichloromethane

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution (100.0 mg/100 mL) was prepared with dichloromethane. Aliquots of that solution (0.125 mL, 0.250 mL, 0.500 mL, 1.000 mL, and 2.000 mL) were pipetted into screw cap glass bottles and the solvent was evaporated under a hood until no odour of solvent was detectable. Thereafter 1000 mL of ultrapure water was added and shaken for approx. 24 h. After shaking, the suspension was filtered through glass fibre filters and membrane filters. The eluates („WAFs“) were used directly without any further dilution step. By the addition of the "intermediate dilution" of mineral nutrients and the algal inoculation the final nominal concentrations (loadings) were achieved.
- Controls: Yes

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Desmodesmus subspicatus CHODAT Strain-No. 86.81
- Source: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen [SAG], Germany
- Age of inoculum (at test initiation): 72 h (pre-culture)

ACCLIMATION
- Acclimation period: 3 d
- Culturing media and conditions: same

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

pH:

8.05 - 9.54 (Control)
8.02 - 9.58 (Test Item Concentrations)

Nominal and measured concentrations:

Control, 0.1, 0.2, 0.4, 0.8 and 1.6 mg/L (nominal)
Control, 0.06, 0.04, 0.03, 0.08 and 0.10 mg/L (based on the geomertic mean concentrations)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: 50 mL glass cylinders, 50 mL volume;
- Initial cells density: 3 x 1000
- Control end cells density: 4.5 x 100000
- No. of vessels per concentration (replicates): 7
- No. of vessels per control (replicates): 7

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ultra pure water
- Intervals of water quality measurement: test start and test end

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: constant illumination
- Light intensity and quality: ≥120 µE/m²s (ca 8000 lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: photometrical measurement (spectrophotometer at 578 nm); cell numbers were calculated using a calibration curve of cell numbers versus extinction values (578 nm)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.658 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential limits

Remarks:

0.383 - 1.136 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

0.277 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential limits

Remarks:

0.178 - 0.437 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.176 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential limits

Remarks:

0.111 - 0.281 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.076 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential limits

Remarks:

0.068 - 0.085 mg/L

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

0.048 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential limits

Remarks:

0.044 - 0.052 mg/L

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.038 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential limits

Remarks:

0.035 - 0.041 mg/L

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.04 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities: no

Reported statistics and error estimates:

Estimation of the inhibition values for yield and the growth rates of the algae as well as the statistical calculations were conducted according to the Guideline using the commercial computer program ToxRatPro Version 3.2.1.

Any other information on results incl. tables

Biological Results:

Table 1: Cell Numbers (Mean Values, Main Test)

Times	Nominal Concentrations (“WAF”) [mg Test item / L]
	Control	0.1 mg/L	0.2 mg/L	0.4 mg/L	0.8 mg/L	1.6 mg/L
t0	3*10 3	3*10 3	3*10 3	3*10 3	3*10 3	3*10 3
t 24.5 h	1.9*10 4	2.0*10 4	2.1*10 4	1.0*10 4	6*10 3	5*10 3
t48.5 h	1.25*10 5	1.16*10 5	1.42*10 5	2.8*10 4	6*10 3	7*10 3
t72 h	4.54*10 5	4.51*10 5	5.22*10 5	1.28*10 5	1.5*10 4	1.3*10 4

  

Table 2: Inhibition Values

Nominal Concentration (“WAF”) of the test item [mg/L]	Growth Rates (day-1)	Inhibition of the Growth Rate after 72h [%]	Yield after 72h*	Inhibition of the Yield after 72h [%]
Control (0)	1.716	-	4.52*10 5	-
0.100	1.713	0.2	4.48*10 5	0.8
0.200	1.762	-2.6**	5.20*10 5	-15.1**
0.400	1.162	32.3	1.25*10 5	72.3
0.800	0.582	66.1	1.2*10 4	97.2
1.600	0.541	68.5	1.1*10 4	97.6

*Yield = Cell number at time t72 h – cell number at time t0; **negative value indicates an activation

Table 3: Analytical Results

Values Time t0	Nominal Concentrations
	Control	0.1 mg/L	0.2 mg/L	0.4 mg/L	0.8 mg/L	1.6 mg/L
Measured TOC (NPOC) [mg/L]	0.219	0.303	0.288	0.404	0.411	0.249
	0.179	0.172	0.163	0.185	0.288	0.229
	0.130	0.153	0.146	0.173	0.189	0.160
	0.177	0.322	0.243	0.252	0.389	0.438
	0.141	0.222	0.172	0.188	0.254	0.337
	0.152	0.182	0.130	0.175	0.314	0.281
Mean Values	0.166	0.226	0.190	0.230	0.308	0.282
Net-Control	0.000	0.060	0.024	0.063	0.141	0.116

Values Time t72h	Nominal Concentrations
	Control	0.1 mg/L	0.2 mg/L	0.4 mg/L	0.8 mg/L	1.6 mg/L
Measured TOC (NPOC) [mg/L]	0.239	0.283	0.257	0.226	0.341	0.338
	0.183	0.187	0.197	0.158	0.139	0.172
	0.125	0.178	0.116	0.156	0.120	0.178
	0.198	0.245	0.369	0.244	0.265	0.259
	0.169	0.189	0.211	0.137	0.176	0.212
	0.136	0.161	0.158	0.190	0.168	0.128
Mean Values	0.175	0.207	0.218	0.185	0.201	0.214
Net-Control	0.000	0.032	0.043	0.010	0.027	0.048

Table 4: Summary Concentrazions of the test item during incubation

Nominal Conc. [mg/L]	mg/L TOC net t0	mg/L TOC net t72h	% of t0	Mean* Concentration TOC [mg/L]	Calculated mean* Concentration of the Test Item [mg/L]	% Recovery of the Nominal Concentrations
0	0	0	-	0	0
0.1	0.06	0.03	54.1	0.04	0.06	56.2
0.2	0.02	0.04	179.1	0.03	0.04	20.7
0.4	0.06	0.01	16.1	0.03	0.03	8.2
0.8	0.14	0.03	18.8	0.06	0.08	9.8
1.6	0.12	0.05	41.5	0.075	0.10	6.0

*Geometric mean concentration

Table 5: Validity criteria for OECD 201

Criterion from the guideline	Outcome	Validity criterion Fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	172.3	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	19.9%	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	0.9%	Yes

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect was investigated by determination of the inhibition of the growth rate of the algae and the yield during the exposure period of 72 h. The EL50 for growth rate was determined to be at 0.076 mg/L based on mean measured concentration and 0.658 mg/L based on the nominal concentraions.