Reaction products of stearic acid with 2-aminoethanol, maleic anhydride and sodium sulphite

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:

From November 24th, 2017 to April 12th, 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The test item is a complex substance composed of various organic constituents having substantially different chemical structures. As the potential ecotoxicity of the test item cannot be assigned to a specific constituent, no appropriate analytical method for an accurate analysis could be developed and the biological endpoints were therefore expressed based on nominal test concentrations (loading rates).

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

Analytical confirmation of the test concentrations were not performed due to technical reasons. As the test item is a complex substance composed of various organic constituents, no appropriate analytical method for an accurate analysis could be developed.

Vehicle:

no

Details on test solutions:

The test solutions were separately prepared at nominal loading rates by directly mixing the corresponding amounts of the test item in test medium. The test solutions were stirred for 18.5 hours (overnight) at ambient temperature in the dark using a magnetic stirrer. Thereafter the test solutions were left to settle for at least 35 minutes. In all stock solutions particles were visible. The stock solutions were filtered using a glass fibre filter with an average retention capacity of 0.4 pm (MN GF-5). The stock solutions were filtered in an ascending order of the test item concentrations (loading rates). The filter was exchanged before filtration of the 06 stock solution. The initial > 0.1 L of each filtrate of the corresponding test concentrations were discarded. The filtrates prepared thereafter were used as test solutions. Therefore, the final filtrates of the C1-03 stock solutions were transferred in 500 mL measuring flasks filled up to the calibration mark, whereas the 04, 05 and 06 test solutions were measured off using a measuring cylinder and transferred in a 500 mL volumetric flask. The remaining stock solution volumes not used (filtered) were discarded. A Tyndall effect was not observed in the filtrates. The individual filtrates were directly used for the test.

The test solutions were prepared once at the beginning of the test. After temperature adaptation of the test solutions, 0.849, 0.424 or 0.255 mL of the algal pre-culture (Raphidocelis subcapitata) were added to each of the volumetric flasks containing 1000, 500 or 300 mL of the test solutions to achieve an algal cell concentration of approximately 0.3x 104 cells/mL respectively. These manipulations were performed under a laminar flow box. The test solutions were homogenised by manual shaking 15 times overhead, before adding 100±5 mL of the test media to each test vessel to ensure a homogeneous distribution of the algal cells. The test vessels were placed onto a shaker under light and temperature controlled conditions.

The medium was sterilised by sterile filtration (pore size 0.2 pm) before use. All glassware and materials used for testing purposes were sterilised for at least 3 hours at 150 °C using a heating furnace or autoclaved for 20 minutes at 120°C, except preparation flask (volumetric flask) used for preparation of test solution.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: Raphidocelis subcapitata (former name: Pseudokirchneriella subcapitata) (SAG 61.81).
- Source (laboratory, culture collection): The organisms were originally supplied by Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany.

ACCLIMATION
- Acclimation period: yes (to adapt the algae to the test conditions, a pre-culture was inoculated by a liquid algal culture and incubated under test conditions).
- Culturing media and conditions (same as test):
o pH-value of the algal medium: 8.2;
o Culture medium: mod. OECD medium (OECD 201, EN ISO 8692);
o Volume of liquid stock culture per pre-culture vessel: 100±5 mL;
o Pre-culture vessels: 300 mL Erlenmeyer flasks;
o Number of replicates: 2;
o Light: Permanent illumination (24/0 h light/dark);
o Light intensity: 60–120 µE m-2s-1;
o Shaker: 100±5 oscillations/min;
o Temperature in the test room: 21–24°C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.8-22.2°C

pH:

7.7-7.9

Nominal and measured concentrations:

Nominal concentrations (loading rates): 0.0 (control), 0.960, 3.36, 11.8, 41.1, 144.0 and 504.0 mg test item/L, equivalent to 0.0 (control), 0.242, 0.846, 2.96, 10.4, 36.3 and 127.0 mg active ingredients/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass Erlenmeyer flasks (300 mL) covered by air-permeable lids.
- Volume of test solution per test vessel: 100±5 mL.
- Renewal rate of test solution (frequency/flow rate): none (static system).
- Age of the pre-culture: 4 days.
- Number of cells per mL in the pre-culture before inoculating the test solutions: 353.5 x 10000 cells/mL.
- Number of cells per mL test solution at the beginning of the test: 0.3 x 10000 cells/mL.
- Control end cells density: 101.68 x10000 cells/mL.
- No. of vessels per concentration (replicates): 3.
- No. of vessels per control (replicates): 6.

TEST MEDIUM / WATER PARAMETERS
- The growth medium was OECD medium as described in the test guideline, modified to optimise growth of Raphidocelis subcapitata. The OECD growth medium was prepared with a 10-fold concentrated iron (Fe)-EDTA complex compared to the original recipe according to guideline OECD 201. The growth rates of the algae in pre-culture were not sufficient in original OECD medium but satisfactory in the modified medium, which is fully described below. Additionally, the test medium was supplemented with sodium bicarbonate (in addition to the normal content; 250 mg/L NaHCO3) (Mayer et al. (2000), ISO 14442 (2006)). Before use, the pH of the medium was adjusted to 8.1±0.2. The algal medium was sterilised (filtered, pore size 0.2 pm) before use. The vessels used were sterilised using a drying oven for three hours at 150°C.
NaHCO3 50.0 mg/L
NH4Cl 15.0 mg/L
MgCl2 × 6 H2O 12.0 mg/L
CaCl2 × 2 H2O 18.0 mg/L
MgSO4 × 7 H2O 15.0 mg/L
KH2PO4 1.6 mg/L
FeCl3 × 6 H2O 0.640 mg/L
Na2EDTA × 2 H2O 1.000 mg/L
H3BO3 0.1850 mg/L
MnCl2 × 4 H2O 0.4150 mg/L
ZnCl2 0.0030 mg/L
CoCl2 × 6 H2O 0.0015 mg/L
Na2MoO4 × 2 H2O 0.0070 mg/L
CuCl2 × 2 H2O 0.00001 mg/L
Justification for modification of growth medium: The steady and sufficient supply of trace elements (e.g. iron) is essential for algal growth. In this context, the introduction of chelating agents (e.g. EDTA) is very valuable, since by using these compounds, trace element cations easily form reversible complexes, whose splitting steadily supplies the algae with the amounts required for growth. There is no indication of interaction between this medium and the test item.
- Intervals of water quality measurement: After temperature adaptation of the test solutions, the pH was measured and recorded for each test concentration and the control. Temperature was recorded once per hour throughout the test in a separate test vessel placed on the shaker containing approximately 100±5 mL deionised water. At the end of the test, the pH was measured and recorded in the pooled replicates per test concentration and control.

OTHER TEST CONDITIONS
- Sterile test conditions: yes.
- Adjustment of pH: no.
- Photoperiod: 24h light / 0h dark.
- Light intensity and quality: 82.0–87.2 µE m–2s–1 (mean 83.7 µE m–2s–1).
- Shaker: 100±5 oscillations/min (the test vessels were placed randomly on a horizontal shaker, so that each test vessel was rotated around its own axis).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: at start of the test, the cell numbers were determined microscopically using a counting chamber (Thoma chamber). Occurrence of morphological deviations of cells was checked. After 24, 48 and 72 hours, the cell numbers were determined by measuring the fluorescence intensity in 4 samples of 200 µL of test solution per replicate using a fluorometer (Multiple Reader Tecan ULTRA). The results [RFU, relative fluorescence units] were converted into biomass concentration [cells/mL] using a calibration curve, which was generated by the data analysis software TIBCO Statistica (TIBCO Software Inc. 2017). A dilution series of the pre-culture was used to determine the calibration line by comparison of cell numbers (counted using a microscope) and fluorescence intensity (measured using a fluorometer) in consideration of background fluorescence of the blank algal medium.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.5.
- Range finding study: yes (results not available in the study report).

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr207)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 504 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: statistically evaluated value = 708 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 126.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: statistically evaluated value = 177.7 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.52 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% cl = 2.13-7.86 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.13 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% cl = 0.53-1.97 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.96 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.24 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Results with reference substance (positive control):

A reference test using potassium dichromate (K2Cr207) as reference item were performed in October 2017 (ECT study number: IA01710). Standard: growth rate ErC50 (0-72 h): 0.952 mg/L (0.916 - 0.990 mg/L; 95%-CL). Based on an international ring test (ISO (2004). Water Quality - Freshwater algal growth inhibition test with unicellular green algae. European Standard EN ISO 8692, October 2004) mentioned in OECD guideline 201, the ErC50 (72h)-value for potassium dichromate obtained from different laboratories was 1.19 mg/L with a standard deviation of 0.27 mg/L. The ErC50 value for the toxic reference item, potassium dichromate, was determined as 0.952 mg/L. This value is within the range of the international ring test (ISO (2004)). Therefore, the results of this reference test are acceptable and the test conditions are reliable.

Reported statistics and error estimates:

The biological results, i.e. growth rate (p) and yield (Y), were evaluated statistically. Probit and Weibull analyses using linear max. likelihood regression was used to determine of the concentration-response function. The statistical software package ToxRat 3.2 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.

A clear concentration-response relationship was observed for the biological parameters yield and growth rate during the exposure period.

Inhibition of Yield in relation to Concentration and Time

The EyC50 of Raphidocelis subcapitata was stated to be 6.99 mg test item/L (equivalent to 1.75 mg active ingredients/L) based on nominal concentrations. The NOEC based on nominal concentration appeared to be lower than 0.960 mg test item/L (equivalent to 0.242 mg active ingredients/L).

Nominal concentration [mg/L]	Mean yield [cells]	Std. Dev. [cells]	n	%Inhibition after 72 hours	Significant difference from control (Williams test; p ≤0.05)
Control (C0)	101.38	7.573	6		–
0.960 (C1)	85.85	5.106	3	15.3	Yes
3.36 (C2)	55.43	4.819	3	45.3	Yes
11.8 (C3)	39.10	1.849	3	61.4	Yes
41.1 (C4)	24.46	0.365	3	75.9	Yes
144 (C5)	22.58	1.756	3	77.7	Yes
504 (C6)	5.83	0.010	3	94.2	Yes

Mean: arithmetic mean; Std. Dev.: standard deviation; n: number of replicates.

Inhibition of Growth Rate in Relation to Concentration and Time

The ErC50 of Raphidocelis subcapitata was stated to be >504 mg test item/L (statistically evaluated value: 708 mg test item/L, equivalent to 177.7 mg active ingredients/L) based on nominal concentrations. The NOEC based on nominal concentration appeared to be lower than 0.960 mg test item/L (equivalent to 0.242 mg active ingredients/L).

Nominal concentration [mg/L]	Mean growth rate [day^-1]	Std. Dev. [day^-1]	n	%Inhibition after 72 hours	Significant difference from control (Williams test; p ≤0.05)
Control (C0)	1.9412	0.02455	6		–
0.96 (C1)	1.8863	0.02007	3	2.8	Yes
3.36 (C2)	1.7407	0.02829	3	10.3	Yes
11.8 (C3)	1.6257	0.01584	3	16.3	Yes
41.1 (C4)	1.4710	0.00493	3	24.2	Yes
144 (C5)	1.4441	0.02523	3	25.6	Yes
504 (C6)	1.0057	0.00054	3	48.2	Yes

Mean: arithmetic mean; Std.Dev.: standard deviation; n: number of replicates.

Summary of biological results based on nominal test item concentrations in mg test item/L

	Endpoints [mg test item/L]
Parameter	EC10	EC20	EC50	NOEC	LOEC
Yield (95% cl)	0.20 (0.08 – 0.39)	0.68 (0.34 – 1.11)	6.99 (5.08 – 9.41)	<0.960	≤0.960
Growth Rate (95% cl)	4.52 (2.13 – 7.86)	33.9 (22.7 – 46.4)	>504 * (n.a. – n.a.)	<0.960	≤0.960

cl: confidence limit, n.a.: not applicable.

*The statistically evaluated value was 708 mg test item/L, 95% cl = 513–1070 mg/L (extrapolated beyond the highest tested concentration).

Observations

In none of the concentration levels C0-C2, deformed and/or damaged algal cells were observed during microscopic inspection. In the C3 treatment a few cells seemed thickened. Most of the cells were thickened, enlarged and partly damaged in the C4-C6 concentration levels.

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were fulfilled as required by the study plan (mean biomass increase in the control: ≥ 16; mean CV for section-by-section specific growth rates in the control: ≤ 35%; CV of average specific growth rates in replicate controls: ≤ 7%).

Conclusions:

A clear concentration-response relationship was observed for the biological parameter yield and growth rate during the exposure period.

The ErC50 of Raphidocelis subcapitata was stated to be >504 mg test item/L (statistically evaluated value: 708 mg test item/L, equivalent to 177.7 mg active ingredients/L) based on nominal concentrations. The ErC10 was evaluated to be 4.52 mg test item/L (equivalent to 1.13 mg active ingredients/L). The NOEC based on nominal concentration appeared to be lower than 0.960 mg test item/L (equivalent to 0.242 mg active ingredients/L).

The EyC50 of Raphidocelis subcapitata was stated to be 6.99 mg test item/L (equivalent to 1.75 mg active ingredients/L) based on nominal concentrations. The EyC10 was evaluated to be 0.20 mg test item/L (equivalent to 0.05 mg active ingredients/L). The NOEC based on nominal concentration appeared to be lower than 0.960 mg test item/L (equivalent to 0.242 mg active ingredients/L).

Executive summary:

The toxicity to the green alga Raphidocelis subcapitata (former name: Pseudokirchneriella subcapitata) of the REACH substance "Stearic acid, reaction product with 2-aminoethanol, maleic anhydride and sodium sulfite" (UVCB) was investigated in a GLP-compliant study performed according to OECD Guideline 201. A clear concentration-response relationship was observed for the biological parameters yield and growth rate during the exposure period.

The ErC50 of Raphidocelis subcapitata was stated to be >504 mg test item/L (statistically evaluated value: 708 mg test item/L, equivalent to 177.7 mg active ingredients/L) based on nominal concentrations. The ErC10 was evaluated to be 4.52 mg test item/L (equivalent to 1.13 mg active ingredients/L). The NOEC based on nominal concentration appeared to be lower than 0.960 mg test item/L (equivalent to 0.242 mg active ingredients/L).

The EyC50 of Raphidocelis subcapitata was stated to be 6.99 mg test item/L (equivalent to 1.75 mg active ingredients/L) based on nominal concentrations. The EyC10 was evaluated to be 0.20 mg test item/L (equivalent to 0.05 mg active ingredients/L). The NOEC based on nominal concentration appeared to be lower than 0.960 mg test item/L (equivalent to 0.242 mg active ingredients/L).

A summary of biological results based on nominal test item concentrations (mg test item/L) is reported below.

	Endpoints [mg test item/L]
Parameter	EC10	EC20	EC50	NOEC	LOEC
Yield (95% cI)	0.20 (0.08 – 0.39)	0.68 (0.34 – 1.11)	6.99 (5.08 – 9.41)	<0.960	≤0.960
Growth Rate (95% cl)	4.52 (2.13 – 7.86)	33.9 (22.7 – 46.4)	>504 * (n.a. – n.a.)	<0.960	≤0.960

cl: confidence limit, n.a.: not applicable.

*The statistically evaluated value was 708 mg test item/L, 95% cl = 513–1070 mg/L (extrapolated beyond the highest tested concentration).

Description of key information

The toxicity to the green alga Raphidocelis subcapitata (former name: Pseudokirchneriella subcapitata) of the REACH substance "Stearic acid, reaction product with 2-aminoethanol, maleic anhydride and sodium sulfite" (UVCB) was investigated in a GLP-compliant study performed according to OECD Guideline 201. A clear concentration-response relationship was observed for the biological parameters yield and growth rate during the exposure period.

The ErC50 of Raphidocelis subcapitata was stated to be >504 mg test item/L (statistically evaluated value: 708 mg test item/L, equivalent to 177.7 mg active ingredients/L) based on nominal concentrations. The ErC10 was evaluated to be 4.52 mg test item/L (equivalent to 1.13 mg active ingredients/L). The NOEC based on nominal concentration appeared to be lower than 0.960 mg test item/L (equivalent to 0.242 mg active ingredients/L).

The EyC50 of Raphidocelis subcapitata was stated to be 6.99 mg test item/L (equivalent to 1.75 mg active ingredients/L) based on nominal concentrations. The EyC10 was evaluated to be 0.20 mg test item/L (equivalent to 0.05 mg active ingredients/L). The NOEC based on nominal concentration appeared to be lower than 0.960 mg test item/L (equivalent to 0.242 mg active ingredients/L).

Key value for chemical safety assessment

EC50 for freshwater algae:

177.7 mg/L

EC10 or NOEC for freshwater algae:

1.13 mg/L

Additional information

One reliable experimental study is available and is selected as key study (ECT, 2018). It was performed according to OECD Guideline 201 and under GLP principles.