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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-02-19 to 2018-05-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted: 23 March 2006, Annex 5 corrected: 28 July 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item: Sodium-Isostearoyl-Lactylate (CAS 66988-04-3)
Trade name: Pationic® ISL
Chemical name: Sodium-Isostearoyl-2-Lactylate
CAS number: 66988-04-3
EINECS number: 266-533-8
Lot/Batch number: 1731200025
Molecular formula: C21H39NaO4
Purity: 100% test item
Water solubility: No information provided
Chemical stability (water/light): Stable under recommended storage conditions (SDS)
State of matter and appearance: Clear viscous, light yellow liquid (TDS)
Storage conditions: Store in original container; keep original container closed and store in a well-ventilated area at 13–29 °C.
Origin of the test item: Caravan Ingredients, Lenexa KS 66215, USA
Date of receipt: January 10, 2018
Expiry date: November 10, 2018
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control, 41.0, 51.2, 64.0, 80.0, 100 mg test item/L (nominal loading rate, spacing factor of 1.25)
- Sampling method: Two samples of 5 mL were taken from the test solutions and the control at the beginning of the test prior to addition of the algae. At the end of the test, the samples were taken directly from representative replicates per test concentration and the controls. The 5 mL samples were added to 5 mL methanol + 2 mM ammonium acetate immediately after sampling for stabilisation of the sample. At sampling time point, two 5 mL samples were taken from the test vessel and stabilized by addition of 5 mL methanol, resulting in an initial diultion factor of 2. A 25 uL aliquot of each sample was diluted with 975 uL of methanol + 2 mM ammoniumacetate: test medium (1:1).
- Sample storage conditions before analysis: Stored at < -20°C for up to 14 days. One set of the samples was kept as retain samples.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The highest test concentration was prepared with a loading of 100 mg test item/L following the recommendation of the OECD guidance No. 23 for difficult substances. 210.1 mg test item was transferred into a 2 L sterile glass flask. Sterile growth medium (2.1 L) and an acetone washed, sterilised star shaped stirring bar were added to the bottle and stirred vigorously for 3.5 hours at room temperature to achieve maximum solubility of the test item. To separate insoluble test item from the test solution and to sterilise the test solution, the test item preparation was filtered using a 0.22 µm polyethylensulfone filter under sterile conditions. The filtrate was used as the highest test concentration and was subsequently diluted with sterile growth medium to obtain the other test concentrations.
- Controls: Growth medium without test substance
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata, Chlorophycea, Chlorophyta (other name: Pseudokirchneriella subcapitata, formerly known as Selenastrum capricornutum).
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Experimental Phycology and Culture Collection of Algae at the University of Goettingen (EPSAG), Nikolausberger Weg 18, 37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The stock cultures were maintained fulfilling the criteria of the OECD 201 guideline. Prior to testing, a pre-culture was established in standard OECD growth medium to obtain exponentially-growing algae for the test. The culture duration of the pre-culture was 3 days.

ACCLIMATION
- Acclimation period: None
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: Not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
Observations recorded at 24, 48, and 72 hours.
Hardness:
Not reported
Test temperature:
22.5 °C
pH:
7.92 (control at test start), 7.87–7.91 (test media at test start)
8.75 (control at test termination), 8.19–8.41 (test media at test termination)
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
Control, 41.0, 51.2, 64.0, 80.0, 100 mg test item/L (nominal);
6.01, 4.92, 7.39, 11.1, 14.4 (measured, geom. mean)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical glass flasks
- Type: Closed, covered with air-permeable silicone-sponge caps
- Material, size, headspace, fill volume: 100 mL test solution
- Aeration: No
- Illumination: The culture vessels were continuously illuminated with a light intensity, it ranged from 89.02 to 96.41 µE m-2 s-1
- No. of organisms per vessel: Cell density 1.715E+06 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8
- Positive control: 3,5-dichlorophenol non-GLP tests twice a year

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The sterilised synthetic growth medium according to OECD 201 was used as growth medium. All stock solutions and the medium were prepared with purified water processed using an ELGA „PURELAB Ultra“. The pH of the medium was obtained at equilibrium between the carbonate system of the medium and the partial pressure of CO2 in atmospheric air.
Growth medium according to OECD 201, final concentrations (all in mg/L):
NaHCO3 - 50
NH4Cl - 15
KH2PO4 - 1.6
MgSO4 x 7 H2O - 15
MgCl2 x 6 H2O - 12
CaCl2 x 2 H2O - 18
FeCl3 x 6 H2O - 0.064
H3BO3 - 0.185
MnCl2 x 4 H2O - 0.415
ZnCl2 - 0.003
CoCl2 x 6 H2O - 0.0015
CuCl2 x 2 H2O - 0.00001
Na2MoO4 x 2 H2O - 0.007
Na2EDTA x 2 H2O - 0.1
pH, at test start: about 8.0
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: Not specified
- Photoperiod: Continuous light
- Light intensity and quality: 89.02 to 96.41 µE m-2 s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (CASY TT, OMNI Life Science GmbH & Co. KG, Bremen, Germany)
- Interval of observations: the cell density was determined daily during the 3-day growth test.

TEST CONCENTRATIONS
Control, 41.0, 51.2, 64.0, 80.0 and 100 mg test item/L (spacing factor of 1.25)
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 14.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
10.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
other: yield
Remarks on result:
other: 95 % CI = 8.95–12.9 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
8.06 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95 % CI = 6.39–10.2 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 4.92 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
11.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
<= 4.92 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.01 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Due to the low inhibition of 7.7 and 4.9 % at 4.95 and 6.01 mg/L, the NOEC was set to this concentration since effects below 10% compared to control are generally not considered to be ecotoxicologically relevant.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 4.92 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): No abnormalities observed.
- Any stimulation of growth found in any treatment: No
Results with reference substance (positive control):
The sensitivity of the test organism is routinely checked using 3,5-dichlorophenol as primary standard following internal SOPs in a non-GLP test twice a year. The nominal growth rate ErC50 value of 2.90 mg/L (9 5% CI = 2.66–3.16 mg/L, December 2017) is in good agreement with the results of an international ring test with an ErC50 of 3.38 ± 1.30 mg/L.
Reported statistics and error estimates:
See reported effect concentrations.

Analytical results

The test item was quantified by LC-MS/MS measurements in samples of freshly prepared test solutions and controls prior to adding into the test vessels at test initiation. At the end of the test, samples were taken from two representative test replicates per treatment level and control. The level of quantification (LOQ) was determined to be 6.0 µg/L.

At the start of the exposure, the recoveries of the measured concentrations were in the range of 9.4 and 17.2 % of the loading. At the end of the test, the concentrations were between 9.7 and 14.2 % of the loading. The test item concentrations decreased by more than 20 % during the test period, except for the second and third concentration.

The test was evaluated using the geometric mean of the measured concentrations. The mean measured concentrations in increasing order were 4.92, 6.01, 7.39, 11.1 and 14.4 mg test item/L.

Effects on growth rate and yield

Growth rate

There was a slight concentration-dependent effect on the growth rate of the algae up to 16.8 % inhibition at the highest test concentration of 14.4 mg/L, resulting in an (unbounded) ErC50 of > 14.4 mg test item/L. The inhibition of 7.7 and 4.9 % compared to the control at the two lowest test concentrations was significantly different from the control, respectively. However, effects below 10 % inhibition compared to the control are generally not considered to be ecotoxicologically relevant. The statistically significant effects are a result of the low variability between the replicates. Therefore, the NOEC for growth rate was set to 6.01 mg test item/L.

Yield

There were significant, concentration-dependent effects on yield up to 60.7 % inhibition compared to the control at the highest test concentration of 14.4 mg/L during the growth test. Due to the inhibition above 50 % an EyC50 value of 10.4 mg/L could be calculated.

Other observations

Microscopic observation of the cell cultures

Date

Observations

Test start, day 0

Inoculum culture: normal appearance of intact cells

Test end, day 3

Control

Normal appearance of intact cells, almost no cell debris

4.92 mg/L

Normal appearance of cells, lower cell density than control

6.01 mg/L

7.39 mg/L

Decreasing cell density and increasing amount of cell debris with increasing test concentrations.

11.1 mg/L

14.1 mg/L

 

Validity criteria fulfilled:
yes
Remarks:
Mean growth rate of 1.839/d; Mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2, 2-3) in the control cultures was 14.2 %; mean coefficient of variation of growth rates in replicate control cultures was 1.4%.
Conclusions:
The 72-h ErC50 for growth rate of Raphidocelis subcapitata was > 14.4 mg test item/L based on geometric mean measured concentrations. The NOEC for growht rate was 6.01 mg/L (mean measured).
Executive summary:

In a 72-hour acute toxicity study, cultures of Raphidocelis subcapitata were exposed to sodium isostearoyl lactylate (ISL) at geometric mean measured concentrations of 4.92, 6.01, 7.39, 11.1 and 14.4 mg test item/L under static conditions in accordance with OECD guideline 201. The NOEC and EC50values based on growth rate were 6.01 and >14.4 mg/L, respectively.

The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any abnormalities.

This toxicity study is classified as acceptable and satisfies the guideline requirements for algae growth inhibition study according to OECD guideline 201.

Following Regulation (EC) 1272/2008, the EC50 of ISL does not fulfil any classification criteria for aquatic toxicity.

Results synopsis

Test organism: Raphidocelis subcapitata

Test type: Static

Effect levels:

72 hr ErC10: 8.06 mg/L (95 % CI = 6.39–10.2 mg/L)

72 hr ErC50: >14.4 mg/L

72 hr NOEC: 6.01 mg/L

72 hr LOEC: 11.1 mg/L

Endpoint(s) effected: growth rate

Description of key information

In a 72-hour acute toxicity study, cultures of Raphidocelis subcapitata were exposed to sodium isostearoyl lactylate (ISL) at geometric mean measured concentrations of 4.92, 6.01, 7.39, 11.1 and 14.4 mg test item/L under static conditions in accordance with OECD guideline 201. The NOEC and EC50 values based on growth rate were 6.01 and >14.4 mg/L, respectively.

The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any abnormalities.

This toxicity study is classified as acceptable and satisfies the guideline requirements for algae growth inhibition study according to OECD guideline 201.

Following Regulation (EC) 1272/2008, the EC50 of ISL does not fulfil any classification criteria for aquatic toxicity.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
8.06 mg/L

Additional information