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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
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 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13 April 2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item name: Sodium-Isostearoyl-Lactylate (CAS 66988-04-3)
Trade name: Pationic® ISL
Chemical name: Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl isooctadecanoate
CAS number: 66988-04-3
EINECS number: 266-533-8
Lot/Batch number: 1731200025
Purity: Technically pure
Molecular formula: C21H39NaO4
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
Expiry date: November 10, 2018
Analytical monitoring:
yes
Details on sampling:
The concentrations of the test item in the water phase were assessed by chemical analysis. Fresh samples were taken from the test solutions and control at the beginning of the exposure period and at medium renewal after 24 hours prior to distribution to test vessels. At medium renewal after 24 hours and at the end of the test, aged samples were taken directly from representative replicates per test concentrations and the control. Samples were added to methanol immediately after sampling for stabilization of the sample. At the end of the test and based on the outcome of the study it was decided that samples taken at test start, at medium renewal and at test end should be measured.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Due to the very low water solubility of the test item, the highest test concentration was prepared with a loading of 100 mg test/L with stirring of the stock solution for 1 hour. For preparation of test solutions at test start, 201.4 mg test item was transferred into a 1 L glass flask containing 2014 mL Cu-reduced dilution water. The solution was stirred for about 1 hour at room temperature (20°C) to achieve maximum solubility of the test item. To separate insoluble test item from the test solution, it was filtered using a 0.22 µm polyethersulfone filter. The filtrate was used as the highest test concentration and subsequently diluted with Cu-reduced dilution water to obtain the other test concentrations.
Test solutions were renewed after 24 hours. To prepare test solutions at medium renewal after 24 hours, 200.2 mg test item were weighed and transferred to 2002 mL Cu-reduced dilution water.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: not specified
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene. Specimens were bred in the laboratory of the Fraunhofer IME.
- Age of parental stock: at least 3 weeks
- Feeding during test: None
- Food type: algal suspension (Desmodesmus subspicatus) and ArtemioFluid (JBL).
- Amount: Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a few mL of medium. 30 mL of this suspension was given to 1 L of Daphnia medium.
- Frequency: The water was changed once a week.

ACCLIMATION
- Acclimation period: same as test
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
Observations at 24 and 48 hours
Hardness:
Hardness was below the recommended (not required) range (140–250 mg/L as CaCO3)
Test temperature:
18–22 °C (mean temperature 18.8 °C, with variation of less than 1 °C)
pH:
7.77–8.36
Dissolved oxygen:
7.62–9.34 mg/L
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal concentrations: 0, 41.0, 51.2, 64.0, 80.0, 100.0 mg/L
Geometric mean measured concentrations of fresh and aged test solutions: 0, 0.388, 0.518, 0.643, 0.867, 1.45 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL glass beakers
- Type: closed. The numbers of immobile animals were visually determined daily and dead specimens were removed. Immobility was determined according to the OECD guideline 202. Specimens which were not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilized (even if they were still moving their antennae). Any abnormalities in appearance and behavior were recorded.
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified drinking water (purification included filtration with activated charcoal, passage through a limestone column, and aeration
- Total organic carbon: Recorded but not reported
- Particulate matter: Not reported
- Metals: Recorded but not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Recorded but not reported
- Ca/Mg ratio: Not reported
- Conductivity: Not reported
- Salinity: Not applicable
- Culture medium different from test medium: no
- Intervals of water quality measurement: water chemistry data is recorded regularly

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light/8 hours dark
- Light intensity: 845–867 Ix (corresponding to 11.3–11.6 µE/(m² × s))

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The numbers of immobile animals were visually determined daily and dead specimens were removed. Immobility was determined according to the OECD guideline 202. Specimens which were not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilized (even if they were still moving their antennae). Any abnormalities in appearance and behavior were recorded.

VEHICLE CONTROL PERFORMED: No
Reference substance (positive control):
yes
Remarks:
K2Cr2O7 to check the sensitivity of the test clone checked twice a year in a non-GLP test
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.581 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % CI: 0.485–0.567 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.798 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % CI: 0.727–0.899 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.518 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
0.643 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Neither significant signs of disease nor symptoms of stress like discolouration or abnormal behaviour were observed in any replicate up to and including the highest test item concentration. However, test organism in the three highest test concentrations were less active than organisms in the controls.
Results with reference substance (positive control):
24-h EC50, based on immobilisation: 1.435 mg/L (95 % CI: 1.23–1.75 mg/L)
Reported statistics and error estimates:
Effect values are given as NOEC, LOEC, and ECx (where possible).
Since the deviation from the nominal or measured initial concentration was greater than ± 20 %, the evaluation of the concentration-effect-relationships and the calculation of effect concentrations was based on the mean measured concentrations (geometric mean).
Where the test results showed around 50 % immobilisation, they were statistically analysed to determine an EC50 and EC10 value together with 95 % confidence intervals using Probit-analysis assuming log-normal distribution of the values.
The NOEC and LOEC values were determined using appropriate statistical methods (e.g. Fisher's Exact Binomial Test for quantal response). All statistical calculations were performed using the computer programme ToxRat®.

Analytical results:

The geometric mean measured concentrations of the test item in freshly prepared test solutions at test start and aged test solutions at 24 hours were 0.413, 0.522, 0.636, 0.875 and 1.56 mg/L, corresponding to 0.99–1.56 % of nominal. The geometric mean concentrations of test item in freshly prepared test solutions at 24 hours and in aged test solutions at test end (48 h) were 0.363, 0.513, 0.649, 0.859 and 1.34 mg/L, corresponding to 0.89–1.34 % of nominal. The resulting overall geometric mean measured concentrations of fresh and aged test solutions were 0.388, 0.518, 0.643, 0.867 and 1.45 mg/L (0.95–1.45 % of nominal).

Since the measured concentrations were not in a range between 80 and 120 % of nominal, test concentrations at test start and test end, it was decided to perform the evaluation of the concentration-effect-relationships and the calculations of effect concentrations based on the geometric mean measured test concentrations.

Biological results:

Table 1. The mean values for immobilisation per treatment level

* Statistically significant difference between controls and treatments

 Mean measured concentration based on geometric mean (mg/L)  Total introduced  Mobile  Immobile  % Immobility
 Control  20  20  0  0.0
 0.388  20  20  0  0.0
 0.518  20  19  1  5.0
 0.643  20  16  4  20.0*
 0.867  20  8  12  60.0*
 1.45  20  0  20  100.0*
Validity criteria fulfilled:
yes
Remarks:
This Daphnia magna acute immobilization test fulfils the validity criteria of the guideline: mortality observed in controls over the test period ≤ 10 %, dissolved oxygen conc at the end of the aging period (48 h) > 3 mg/L in control and test vessels.
Conclusions:
The test item sodium-isostearoyl-lactylate showed toxic effects on Daphnia magna in a 48-h immobilization test according to OECD test guideline 202. The EC50 was calculated to be 0.798 mg/L based on geometric mean measured concentrations. The NOEC was determined to 0.518 mg/L and the LOEC was set to 0.643 mg/L.
Executive summary:

The 48-h-acute toxicity of sodium isostearoyl lactylate (ISL) to Daphnia magna was studied under semi-static conditions according to OECD test guideline 202.  Daphnids were exposed to control, and test chemical at nominal concentrations of 41.0, 52.2, 64.0, 8 0.0, 100 mg/L. Mortality, immobilization and sublethal effects were observed daily. The 48 -h-EC50 based on immobilisation was calculated to be 0.798 mg/L based on geometric mean measured concentrations. The 48-h-NOEC based on immobilisation was determined to 0.518 mg/L and the 48-h-LOEC was set to 0.643 mg/L.

The initial concentration and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to geometric mean measured concentrations.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

Description of key information

The 48-h semi-static acute toxicity test with sodium isostearoyl lactylate (ISL) in Daphnia magna was conducted according to OECD test guideline 202. The EC50 (mobility) was calculated to be 0.798 mg/L (95% CL = 0.727–0.899 mg/L) based on geometric mean measured concentrations. The NOEC was determined to 0.518 mg/L and the LOEC was set to 0.643 mg/L.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

Following the Regulation (EC) 1272/2008, the EC50 of sodium isostearoyl lactylate needs to be classified as aquatic acute 1. As the substance is readily biodegradable, classification for chronic toxicity is not warranted.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.798 mg/L

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