4-(octadecylamino)-4-oxoisocrotonic acid

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 - 27 Nov 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011

Deviations:

yes

Remarks:

See section "Any other information on material and methods incl tables"

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

adopted 07. December 2015

Deviations:

yes

Remarks:

See section "Any other information on material and methods incl tables"

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz, Mainz, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and all test item solutions
- Sampling method: Samples were taken at test start and test end
- Sample storage conditions before analysis: Samples were frozen

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Differential loading: A stock solution was prepared by mixing the nominal load of 100.0 mg/L with the corresponding amount of algal medium and shaking vigorously for 23 h. The resulting solution was filtrated through 0.45 μm cellulose filters. The lower treatments were prepared by dilution of this saturated solution with nutrient medium.
- Controls: Yes, algae medium only

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: SAG 86.81
- Source (laboratory, culture collection): Received from MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen; Jan 2016)
- Age of inoculum (at test initiation): 5 days (pre-culture)
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

ACCLIMATION
- Acclimation period: 5 days (for the pre-culture)
- Culturing media and conditions (same as test or not): Not the same, stock culture was kept on a solid agar. For the pre-culture the same medium (OECD 201 medium) was used.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.2 – 23.2 °C

pH:

7.8 - 9.1 (Control)
7.8 - 10.1 (Test item concentrations)

Nominal and measured concentrations:

Nominal: Control, 1, 3.2, 10, 32 and 100 mg/L
Measured: measured concentrations are listed in table 5 - 6 in section "Any other information on results incl tables".

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (covered with perforated plastic foil acting as a stopper)
- Material, size, headspace, fill volume: Glass flasks total volume 65 mL, 46 mL test solution, 15 mL headspace
- Initial cells density: 2400 cells/mL
- Control end cells density: 815737 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium was used according to the OECD guideline 201, all solutions were sterilized before use.
- Culture medium different from test medium: Pre-culture and test were performed with the same medium
- Intervals of water quality measurement: pH was measured at test start and test end

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continous
- Light intensity and quality: 5000 lux

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Cell numbers were determined with an electronic particle counter every 24 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: The concentrations to be tested are based on non-GLP pre-tests.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 56.58 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 56.58 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

18.11 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No, algae cells appeared in a normal shape through the whole test duration and for all test item concentrations and the control.
Others: the lowest test item concentration of 1 mg/L was excluded from the evaluation based on turbidity.

Results with reference substance (positive control):

72h ErC50: 0.79 mg/L (95% confidence interval 0.79 - 0.79 mg/L)

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1. statistical calculation using ToxRat® Professional 3.2.1 (based on nominal concentrations) and statistical calculation using ToxRat® Professional 3.2.1 (based on measured concentrations).

Table 1: Biological Results of the Test Item (based on measured concentrations)

Parameter	Value	95% confidence interval
NOEC (Growth Rate) 72 h	18.11 μg/L	--
NOEC (Yield) 72 h	18.11 μg/L	--
LOEC (Growth Rate) 72 h	56.58 μg/L	--
LOEC (Yield) 72 h	56.58 μg/L	--
72h ErC10	> 56.58 μg/L	--
72h EyC10	15.16 μg/L	2.51 – 91.75 μg/L
72h ErC50	> 56.58 μg/L	--
72h EyC50	> 56.58 μg/L	--

 

Table 2: Biological Results of the Test Item (based on nominal concentrations)

Parameter	Value	95% confidence interval
NOEC (Growth Rate) 72 h	32.00 mg/L	--
NOEC (Yield) 72 h	32.00 mg/L	--
LOEC (Growth Rate) 72 h	100.00 mg/L	--
LOEC (Yield) 72 h	100.00 mg/L	--
72h ErC10	> 100 mg/L	--
72h EyC10	26.79 mg/L	4.43 – 162.18 mg/L
72h ErC50	> 100 mg/L	--
72h EyC50	> 100 mg/L	--

 

Table 3: Inhibition Values

Nominal concentration in mg/L	% Inhibition
	Growth Rate (0-72h)	Yield (0-72h)
Blank control	0	0
1*	11.85	49.74
3.2	-1.7	-10.0
10	-0.9	-5.1
32	2.6	14.5
100	8.2	37.3

Negative inhibition values indicate a stimulation of algal growth compared to the blank control. Treatment 1 mg/L showed turbidity and is therefore not included in the evaluation.

* values calculated by Microsoft Excel®, not by ToxRat®

 

Table 4: Cell Numbers calculated

Nominal Conc.	Replicate	Cell Number/mL
[mg/L]		0 h	24 h	48 h	72 h
Blank control	1	2400	19440	161800	815900
	2	2400	21140	152620	755260
	3	2400	18120	175740	928160
	4	2400	18120	152960	860020
	5	2400	18660	160920	828940
	6	2400	16500	133900	706140
	Mean	2400	18663	156323	815737
	SD	0	1550	13827	78030
1	1	2400	14760	116060	475020
	2	2400	15680	115240	417220
	3	2400	14000	79860	341260
	Mean	2400	14813	103720	411167
	SD	0	841	20667	67085
3.2	1	2400	18360	170360	919060
	2	2400	19260	163940	874420
	3	2400	19620	176140	898040
	Mean	2400	19080	170147	897173
	SD	0	649	6103	22333
10	1	2400	17920	160840	864980
	2	2400	19980	160320	940620
	3	2400	18460	151380	766140
	Mean	2400	18787	157513	857247
	SD	0	1068	5318	87497
32	1	2400	15920	119660	675140
	2	2400	17460	162200	728120
	3	2400	14880	137700	689480
	Mean	2400	16087	139853	697580
	SD	0	1298	21352	27403
100	1	2400	14620	120080	637200
	2	2400	12400	99600	510440
	3	2400	13560	85960	390520
	Mean	2400	13527	101880	512720
	SD	0	1110	17174	123356

SD = Standard Deviation

Treatment 1 mg/L showed turbidity and is therefore not included in the evaluation

Analytical Results:

Table 5: Measured Concentrations with algae

Nominal Concentration Test Item mg/L	Measured concentration test item μg/L
	t = 0 h	t = 24 h	t = 48 h	t = 72 h
Blank control	--	--	--	<LOD
1	--	<LOD	<LOD	<LOD
3.2	--	<LOD	<LOD	<LOD
10	--	<LOD	<LOD	<LOD
32	--	10.39	3.50	<LOD
100	--	49.90*	40.02	31.90

LOD / LOQ (Limit of detection / quantification) = 1.00 μg/L (as the lowest calibration level)

Treatment 1 mg/L showed turbidity and is therefore not included in the evaluation

* 8-fold diluted instead of 2-fold like the other values

 

Table Measured Concentrations without algae

Nominal Concentration Test Item	Measured concentration test item μg/L
mg/L	t = 0 h	t = 24 h	t = 48 h	t = 72 h
Blank control	< LOD	--	--	--
1	< LOD	< LOD	< LOD	< LOD
3.2	1.24	< LOD	< LOD	< LOD
10	5.81	< LOD	< LOD	< LOD
32	24.46	9.82	5.94	3.89
100	96.93*	55.74*	45.72	41.50
100 (additional)	82.00	--	--	--

LOD / LOQ (Limit of detection / quantification) = 1.00 μg/L (as the lowest calibration level)

Treatment 1 mg/L showed turbidity and is therefore not included in the evaluation

* 8-fold diluted instead of 2-fold like the other values

Table 6: Geometric Mean of Measured Concentrations and Calculated Concentrations

Nominal Concentration Test Item	Geometric Mean	Calculated Concentrations
mg/L	μg/L	μg/L
Blank control	-	- --
1	--	0.57
3.2	--	1.81
10	--	5.66
32	--	18.11
100	56.58	56.58

Treatment 1 mg/L showed turbidity and is therefore not included in the evaluation

Table 7: Validity criteria

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.	340	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%	14%	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%.	2%	Yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

EC50 (72h) >56.58 µg/L (Desmodesmus subspicatus, OECD 201, mean measured concentrations)

EC10 (72h) >56.58 µg/L (Desmodesmus subspicatus, OECD 201, mean measured concentrations)

Additional information

One experimental study is available investigating the toxicity of 4-(octadecylamino)-4-oxoisocrotonic acid (CAS 3077-27-8) to aquatic freshwater algae. The study was conducted according to OECD 201 (GLP) using the freshwater algae Desmodesmus subspicatus (LAUS GmbH, 2018b). The test was carried out with test item concentrations of 1, 3.2, 10, 32 and 100 mg/L for 72 h. The test solution was prepared by adding appropriate amounts of test item to test medium, followed by intensive stirring and filtration. During the study the actual concentrations in the test medium was measured by HPLC/MS. The measured concentration in the freshly prepared solutions was between 1.24 μg/L and 96.93 μg/L and in the aged test solutions between 3.89 μg/L and 41.5 μg/L, respectively. Therefore the results are given based on the geometric mean of the measured concentrations. Hence the EC50 (72 h) was determined to be > 100 mg/L (nominal) and > 56.58 μg/L (geom. mean measured concentration), respectively. Statistically significant effects were determined at the highest test item concentration of 56.58 µg/L based on growth rate. Hence the NOEC was determined to be at 18.11 µg/L. Due to a low inhibition of 8% at 56.58 µg/L the EC10 was used as toxicological endpoint since effects below 10 % compared to the control are generally not considered to be ecotoxicologically relevant. According to the Guidance on Application of CLP criteria (Version 5.0, July 2017) the preference is given to EC10 to be used for classification and risk assessment. Following this strategy the EC10 and EC50 were used as toxicological endpoints. The measured water solubility for the test item was determined to be 5.9 mg/L at 25 °C, pH 5. The discrepancy between the low recovery in the test solutions and the water solubility may have been caused by the different test media, pH-values and temperature.