Fatty acids C16-18 (even numbered), mono, di and tri esters with sucrose

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 - 24 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen

Analytical monitoring:

no

Remarks:

Analytical measurement was performed in the preceding range-finding test indicating that it is not possible to quantify any test item in the aqueous phase. Therefore, no analytical measurements were performed during the test course of the definitive test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test solutions of 10.0, 3.16 and 1.0 mg test item/L: separately prepared as individual Water Accommodated Fractions (test item was weighed and transferred into 2 L brown glass flasks with drain ports near the bottom, flasks were filled up to a volume of 2 L with test medium and stirred slowly to formation for about 96 h at about 20°C; after 1 h of settling the first 200 mL were discarded to remove any insoluble test item), test soulutions with 0.316 and 0.1 mg/L: test solution with a nominal loading of 1.0 mg/L were diluted with dilution water. Solutions were freshly prepared before test start and before each renewal.
- Controls: yes, dilution water only

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Justification for species other than prescribed by test guideline:
- Age at study initiation: 4 - 24 h
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene, Germany
- Age of parental stock: 3 weeks
- Feeding during test : yes
- Food type: mixture of suspensions of unicellular alga Desmodesmus subspicatus
- Amount: 0.2 mg C/(Daphnia * day)

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: 3 weeks old daphnids were separated from the stock population by sieving. Batches of 30 to 50 animals were held at room temperature in ca. 1.8 L dilution water for one week and were fed daily with an algal suspension (Desmodesmus subspicatus) and ArtemioFluid (JBL). Water was changed once per week. Newborn D. magna were separated by sieving, the first generation was discarded. Individuals selected for the test were transferred with a bore Pasteur pipette a few hours after sieving to ensure applying only healthy specimens.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

250 – 350 mg CaCO3/L

Test temperature:

19.5 - 20.0 °C

pH:

7.71 – 9.42

Dissolved oxygen:

8.35 - 12.12 mg/L

Conductivity:

236 µS/cm

Nominal and measured concentrations:

Nominal loading rates: Control, 0.10, 0.316, 1.00, 3.16 and 10.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers (50 mL nominal volume)
- Type: covered with glass panes
- Fill volume: 50 mL
- Aeration: no
- Renewal rate of test solution: 3 times/week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: drinking water purified via filtration with activated charcoal and aeration
- Non purgeable organic carbon: 0.579 mg/L
- Alkalinity: 1.6 mmol/L
- Conductivity: 236 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: Oxygen concentration, pH value and temperature: checked at each media renewal in new and aged ( pooled samples) test solutions of the control and each test concentration, hardness: checked once a week at water renewal in new and aged media of the control and the highest test concentration

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: light/dark cycle of 16/8 hours
- Light intensity: ≤ 15 µE/(m2*s) or 1125 lux

EFFECT PARAMETERS MEASURED: daily: numbers of immobile daphnids, abnormalities in appearance and behaviour, newborn daphnids per beaker, abnormalities in condition (including individuals of male sex), presence of winter eggs, at test end: length of the adults without significant mortality

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: yes
- Nominal test loadings: 0.1, 1.0 and 10.0 mg test item/L
- Results used to determine the conditions for the definitive study: No significant effect on the cumulative offspring was observed for any treatment. No significant effect on immobility occurred.

Reference substance (positive control):

yes

Remarks:

performed in regular intervals with K2Cr2O7

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

mobility, age of first reproduction, length, development and intrinsic rate

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

mobility, age of first reproduction, length, development and intrinsic rate

Details on results:

- Immobilisation of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium: yes, water solubility in test medium appears to be lower than 0.3 mg test item/L.

Results with reference substance (positive control):

EC50 (24 h): 1.126 mg/L (95% CL : 0.653 – 1.942 mg/L)
Results of the latest reference study are in agreement with the stated range given in the OECD guideline (EC50 = 0.6 – 2.1 mg/L)

Reported statistics and error estimates:

LOEL was calculated by using ANOVA followed by Student-t test, Fisher`s Exact Binomial test, Dunnett’s or Williams’ test or an appropriate non-parametric test. When the test results showed a concentration-response relationship, the data was analysed
by regression to determine the EL50 including the 95% confidence interval as well as the EL10 using Probit-analysis assuming log-normal distribution of the values. The computer software ToxRat® was used for statistical evaluations.

Table 1: Survival data and percent reduction of survival compared to the control after 21 days.

 

Loading rate [mg/L]	Parental survival [number of individuals]	Reduction of survival [%] (immobility)
Control	10	0.0
0.10	10	0.0 (-)
0.316	9	10.0 (-)
1.00	10	0.0 (-)
3.16	10	0.0 (-)
10.0	10	0.0 (-)

(+) statistically significant difference between control and treatments / (-) no statistically significant difference between control and treatments

Table 2: Reproduction data after 21 days

Loading rate [mg/L]	Cumulative offspring per introduced parent Mean ± SD [Ind.]	Cumulative offspring per survived parent Mean ± SD [Ind.]	Age at first brood Mean ± SD [days]	Development rate Mean ± SD [Ind.]	Intrinsic rate Mean ± SD
Control	103.1 ± 18.9	103.1 ± 18.9	10.3 ± 0.79	0.098 ± 0.007	0.326 ± 0.022
0.10	110.7 ± 23.4	110.7 ± 23.4	10.0 ± 0.97	0.101 ± 0.010	0.330 ± 0.035
0.316	99.1 ± 36.9	105.7 ± 32.4	9.8 ± 0.95	0.103 ± 0.010	0.330 ± 0.035
1.00	112.4 ± 15.0	112.4 ± 15.0	10.3 ± 0.63	0.097 ± 0.006	0.328 ± 0.013
3.16	106.1 ± 24.7	106.1 ± 24.7	10.1 ± 0.97	0.100 ± 0.010	0.330 ± 0.027
10.0	109.9 ± 14.0	109.9 ± 14.0	10.1 ± 0.52	0.099 ± 0.005	0.327 ± 0.011

SD: standard deviation

 

 

Table 3: Validity criteria for OECD 211.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals in the control (female Daphnia) does not exceed 20% at the end of the test.	0%	yes
The mean number of living offspring produced per parent animalsurviving at the end of the test is ≥ 60 in the control.	103.1	yes

Validity criteria fulfilled:

yes

Remarks:

For further details refer to "Any other information on results incl. tables".

Description of key information

NOELR (21 d) ≥ 10 mg/L (nominal, OECD 211)

Additional information

One experimental study is available investigating the long-term toxicity of the test item to aquatic freshwater invertebrates. The study was performed according to OECD 211 (GLP). Daphnia magna was exposed to a control and five nominal loading rates of 0.10, 0.316, 1.00, 3.16 and 10.0 mg/L. The LOQ was determined to be 0.3 mg test item/L in the method validation during the range finding study. However, measurements of test item solutions showed that it is not possible to measure any test item in the aqueous phase due to the very low water solubility of the substance. Thus, the effect concentrations were based on the nominal loading rates. This procedure of evaluation of data of WAFs based on the nominal loading rates is in line with the recommendations of the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2019. Since no effect occurred for any endpoint up to and including the highest loading rate of 10.0 mg/L, no ECx were calculated. The NOELR was set to be ≥ 10.0 mg/L and the LOELR was > 10.0 mg/L for all considered endpoints.