Fatty acids, tall-oil, reaction products with 2-[(2-aminoethyl)amino]ethanol

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:

May 2016 - June 2016

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)

Specific details on test material used for the study:

batch no: 0001549819
colour: yellow-brown
Production date 17. Sep. 2014
Expiry date 17. Sep. 2016

Analytical monitoring:

yes

Details on sampling:

Samples from all treatments for analytical determinations were taken from the new solutions at the beginning and after 24 h from the old solutions. At medium renewal on day 7 and day 14 samples from the 2 highest concentrations and the control were taken from the new and the old solution. The same treatments were measured on day 20 (new solution) and at the end of the test on day 21 (old solution).

6 g NaCl was added to 100 mL of a solution in the matrix. The solution was extracted two times with the solvent ethyl acetate (10 mL, 4 mL), the organic phase was collected into a flask, after drying with Na2SO4. The solvent was evaporated by heating to approx. 40 °C, the residue was dissolved in 2 mL ethyl acetate and the solution was measured via GC/MS. 50-fold enrichment was achieved during the sample preparation.

Vehicle:

no

Details on test solutions:

The water-accommodated fraction (WAF) was prepared for the test. This was done by weighing the nominal load 10 mg/L, adding the corresponding amount of dilution water and shaking vigorously for 23 - 24 h. The resulting solution was filtrated through 0.2 μm nylon filters. The lower concentrations were prepared by dilution of this WAF with nutrient medium. This procedure is in agreement with the OECD guidance document no. 23 for the testing of mixtures which are toxic at very low loading rates.
At the beginning and at each medium renewal, a stock solution as water-accommodated fraction was prepared.
(Days: 0, 2, 5, 7, 9, 12, 14, 16, and 19. The exact loads were always 10 mg/L)

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna
Variety: STRAUS
Arrival of Strain: 27. Sep. 2007
Sex: female
Age: between 0 and 24 h
The animals which were used in the test did not belong to the first brood of the respective parent animals.

Animal Husbandry
Vessels: glass beakers, nominal volume 2 L
Medium: M4-Medium (recipe of ELENDT)
Food: green alga (Desmodesmus subspicatus)
Medium renewal: twice a week
Photo period: 16/8 h, using neon tubes
Temperature: 20 +/- 2 °C

Selection of Daphnia for the test:
19 h before the start of the test, the adult animals were separated from the young. 18.5 h later, the adults were caught with the help of a glass tube, and the newborn daphnia, aged between 0 and 18.5 h, were sieved from the medium and immediately placed into a 250 mL-beaker containing dilution water. After a settling-in period of 30 min, animals which showed no apparent damage were used for the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

267 - 356 mg CaCO3/L

Test temperature:

19.1 - 24.8 °C

pH:

7.5 - 8.0

Dissolved oxygen:

8.3 - 9.5

Nominal and measured concentrations:

The test item concentration in the test solutions and the blank control was determined using GC/MS determination. Only in the two treatments with the highest concentrations (2.2 and 1.0 mg/L nominal) the test item concentration was determined as the concentrations below 1 mg/L were lower than the limit of detection (LOD).
During validation of the analytical method stability of the test item over 3 days in test medium without test organisms and food was shown (3d-stability: 105.8 %). In almost every freshly prepared test solution the treatment with the highest concentration showed a detectable concentration of test item. During the test possibly adsorption to the test organism and/or the food occurred.
Therefore, the biological results for the treatment with the highest concentration were stated on the arithmetic mean of the measured start concentrations. The treatments with lower concentrations were calculated based on the dilution factor and the mean concentration of the highest treatment.

calculated concentrations based on the mean start value of 1.26 mg/L in the highest treatment (nominal: 2,2 mg/L):
nominal calculated (in mg/L)
2.2 1.26
1.0 0.57
0.46 0.26
0.22 0.13
0.1 0.06

Details on test conditions:

Duration: 21 days
Loading: 80 mL/animal, animals were kept individually
Replicates: 10 replicates/concentration
Test item concentrations: 0.1 / 0.22 / 0.46 / 1.0 / 2.2 mg/L nominal
Blank Control: dilution water
Photoperiod: 16/8 light/dark
Light intensity: 1000 Lux
Aeration: none
Feeding: 0.15 - 0.20 mg organic carbon/animal/working day, the amount of carbon is based on photometric measurement (linear correlation between absorption at 440 nm and measured DOC concentration).
Temperature: 19.1 - 24.8 °C

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Details on results:

Calculation of results was performed with the help of a validated software (Microsoft Excel ®). The estimation of the biological results was accomplished using the software Tox-Rat® Professional, version 3.2.0.
For the effect parameter reproduction the statistical evaluation excluded treatments with no survivors from the analysis. The remaining database leads to statistically not acceptable effective concentrations (EC). A statistically significant effect on reproduction was recorded at 0.46 mg/L, therefore the NOEC value of 0.22 mg/L was obtained.
The statistical evaluation of the parameter immobility included all tested concentrations with inhibition values between 0 and 100 %. Due to a steep concentration-response relationship the obtained lethal concentrations (LC) were not valid. A statistically significant effect on immobility was recorded at 0.46 mg/L, therefore the NOEC value of 0.22 mg/L was obtained for this parameter.

Validity criteria fulfilled:

yes

Conclusions:

Biological results based on measured / calculated concentrations
Immobility
NOEC = 0.13 mg/L
LOEC = 0.26 mg/L
Reproduction
NOEC = 0.13 mg/L
LOEC = 0.26 mg/L

Biological results based on nominal concentrations
Immobility
NOEC = 0.22 mg/L
LOEC = 0.46 mg/L
Reproduction
NOEC = 0.22 mg/L
LOEC = 0.46 mg/L

Executive summary:

The effect of test item 400112 on the reproduction ofDaphnia magnawas investigated with the OECD Guideline 211 resp. EU C.20 under GLP conditions. The water accommodated fraction of 400112 was prepared for 5 concentrations ranging from 0.1 to 2.2 mg/L. Ten daphnia were individually exposed to each concentration and a blank control for a treatment period of 21 days.

The validation of the analytical method showed a stability of the test item over 3 days in test medium without test organisms and food (3d-stability: 105.8 %). Therefore, a semistatic test design was used with medium renewal three times per week.

A significant decrease in the reproduction of the daphnia was observed at the three highest concentrations with 29 % inhibition at a concentration of 0.46 mg/L and 100 % inhibition at 1.0 and 2.2 mg/L. After 21 days, mortality of adult animals reached 100 % at 1.0, 2.2 mg/L and 40 % at 0.46 mg/L. As mortality could be assigned as test item related effect, the offspring of dead animals were not excluded from the analysis.

The test item concentration in the test solutions and the blank control was determined using GC/MS determination. Only in the two treatments with concentrations of 2.2 and 1.0 mg/L (nominal) the test item could be determined as the concentrations below 1 mg/L were lower than the limit of detection (LOD). In almost every freshly prepared test solution the treatment with the highest concentration showed a detectable concentration of test item in a similar range. Therefore, the biological results for the treatment with the highest concentration were stated on the arithmetic mean of the measured start concentrations. The treatments with lower concentrations were calculated based on the dilution factor and the mean concentration of the highest treatment. The biological results were additionally based on the nominal concentrations.

No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test is considered valid.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.13 mg/L