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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Sampling for the analytical monitoring: At the start of an exposure-renewal interval (0 hours ), samples of the fresh media (at least three aliquots) were taken after preparation of the concentration level and the solvent control and analyzed. At the end of an exposure-renewal interval (24 hours), samples of the 24-hours old media were taken directly from the test vessels.

Sampling procedure: 40 mL of the test item concentration, the solvent control and the control were given in a 59 mL headspace vial, covered with aluminium tops with PTFE seals.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
One limit concentration of the test item was prepared by dissolving the test item in DMF and adding the test item dissolved in the solvent to the dilution water. The solution was shaken manually at room temperature. The solvent concentration was 50.0 μL/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (water flea)
- Strain/clone: The test species is Daphnia magna STRAUS, obtained from continuous laboratory cultures. The daphnids of the laboratory culture originated from clone 5.
- Age at study initiation (mean and range, SD): less than 24 hours old daphnids from a healthy stock were used for the study.
- Source: Noack Laboratorien GmbH, Sarstedt, Germany (origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu),14195 Berlin, Germany)
- Age of parental stock (mean and range, SD):
- Feeding during test : Daily feeding per test vessel; Pseudokirchneriella subcapitata and Desmodesmus subspicatus suspension was provided as food, corresponding to 0.05 - 0.1 mg C per daphnia, alga species and day.

ACCLIMATION
- Acclimation period: Acclimatization was not necessary, because the composition of the dilution water is equivalent to the culture medium.
- Acclimation conditions (same as test or not): yes

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Remarks on exposure duration:
The regular test duration is 21 days. The test was prolonged for one day as the release of juveniles had not been synchronous. Therefore, the evaluation is performed based on the data of days 0-21 and 0-22.
Test temperature:
20 ± 1 °C
Nominal and measured concentrations:
Test concentration (nominal): 1 µg/L
The measured concentrations of the test item in fresh test media (0 hours) were in the range of 0.509 μg/L to 1.66 μg/l. At the end of the respective exposure intervals (24 hours), the measured concentrations in the old media were in the range of < LOQ to 0.152 μg/L. The tested concentration was above the limit of solubility. Thus, the observed decrease may be caused by separation of undissolved test material.
Details on test conditions:
Test method: Due to the volatile nature of the test item, the study will be performed in a closed system without headspace, to reduce contact with air and losses of the test item by evaporation.

TEST SYSTEM
- Test vessel: Sealed glass flasks (4.5 (10) x 9.5 (H) cm) with screw caps were used. A test volume of approximately 130 mL was provided in each test vessel.
- Type (delete if not applicable): closed (without Headspace)
- Aeration: Test vessels were not aerated during the test.
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic fresh water (Elendt M4) is used as media for culture
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Max. 1500 lx

TEST CONCENTRATIONS
- Range finding study: The test item concentration was selected based on the water solubility of the test item and based on the results o a non-GLP preliminary range findings test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.25 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
22 d
Dose descriptor:
NOEC
Effect conc.:
0.25 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Reproductive Output:
The number of juveniles in all replicates of the controls and the test group was counted and
recorded every day. Results concerning the number of living juveniles of the concentration level
and the controls for every test day are presented in Table 11 to Table 13.
The average number of living juveniles at the end of the test after 21 and 22 days per introduced
parental daphnid was 92.4 and 96.2 in the pooled control group. Thus, the validity criterion for the
reproduction of a mean of ~ 60 living offspring per surviving parent animal in the control was met.
One parental daphnid (replicate 10 from the test concentration) was excluded from the evaluation
of the reproductive output due to accidental mortality (animal was not found; e.g. erroneously not
transferred at the renewal of the test solutions). According to the guideline, the effects on
reproduction were evaluated based on the reproduction per survived parent animal as no significant
trend in mortality was detected.
No significant reduction of the reproductive output of introduced or survived parent animals in
comparison to the control groups was observed at the geometric mean measured test item
concentration of 0.250 μg/L. For effect values, please refer to Table 1. Further details are given in
Table 3 and Figure 1 and 2.

Adult Mortality
According to the guideline, the Cochran-Armitage test was performed to detect a significant trend
in mortality. Results show no significant trend in mortality and are presented in Table 4 below.
Accidental mortality as defined in the OECD test guideline was observed in replicate 10 of the test
concentration (animal was not found; e.g. erroneously not transferred at the renewal of the test
solutions), which was excluded from analysis entirely.

First Appearance of living Juveniles
The first appearance of living juveniles was observed on days 8 to 10 in all introduced parental
daphnids of the controls and the geometric mean measured concentration 0.250 μg/L. For details,
see Table 7.

Stillborn Juveniles and Aborted Eggs
Stillborn juveniles or aborted eggs were observed in the concentration level but not in the control
and solvent control during the exposure period of 22 days. For details, see Table 11 to Table 15.
Results with reference substance (positive control):
The percentage of immobility for the reference item potassium dichromate was determined after 24 hours from 2018-11-14 to 2018-11-15. For results of the most recent of the monthly performed reference tests was EC50: 2.13 mg/L (valide range 0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5 and 0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A).
Reported statistics and error estimates:
Statistical Analysis after 21 and 22 days:
The STUDENT-t test for Homogeneous Variances revealed that there is no statistically significant difference between control and solvent. Thus, according to OECD 211 it is required to use the values of the pooled control for evaluation.
The Cochran-Armitage trend-test revealed that no significant trend in mortality was observed. Thus, according to OECD 211 it is required to report the NOEC/EC for the cumulative offspring per survivor provided these values are lower than in the cumulative offspring per introduced parent.

Effect values

Endpoints for reproduction and mortality after a 21 days of the test item [µg/L]

Endpoints for reproduction and mortality after a 21 days of the test item [µg/L]

LOECReproduction

   > 0.250

> 0.250

NOECReproduction

   0.250

  0.250

LC20Adult mortality

> 0.250

> 0.250

LC50Adult mortality

> 0.250

> 0.250

LC100Adult mortality

> 0.250

> 0.250

LOECAdult mortality

> 0.250

> 0.250

NOECAdult mortality

> 0.250

> 0.250

Validity criteria fulfilled:
yes
Conclusions:
According to the guideline, the effects on reproductive output of the daphnids were evaluated based on the reproduction per survived parent animal, due to no significant trend in mortality (CochranArmitage trend test; p/a = 0.05). A summary of all endpoints based on the geometric mean measured concentration of the test item Laurylacrylat 12. The overall effect threshold for effects of the test item under the test conditions was 0.250 μg/L (NOEC) and > 0.250 μg/L (LOEC) based on the geometric mean measured concentrations of the test item Laurylacrylat 12.

Description of key information

In a chronic toxicity test to Daphnia magna STRAUSS of the test substance Laurylacrylate 12, the overall effect threshold for effects of the test item under the test conditions was 0.250 μg/L (NOEC) and > 0.250 μg/L (LOEC) based on the geometric mean measured concentrations of the test item.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.25 µg/L

Additional information

A semi-static Daphnia magna reproduction with the test item Laurylacrylat 12 was conducted at the test facility according to OECD guideline 211. Test species was Daphnia magna STRAUS. Ten daphnids, held individually, were used perconcentration, solvent control and control. Since the test item is volatile, the study was carried out in a closed system without headspace (sealed glass flasks filled up to the top with the test solutions) under semi-static conditions with a daily renewal of the test solutions. One limit concentration (1 μg/L) of the test item was prepared by dissolving the test item in DMF and adding the test item dissolved in the solvent to the dilution water. The solvent concentration was 50.0 μL/L. The geometric mean measured test item concentration is 0.250 μg/L. The measured concentrations of the test item in fresh test media (0 hours) were in the range of 0.509 μg/L to 1.66 μg/l. At the end of the respective exposure intervals (24 hours), the measured concentrations in the old media were in the range of < LOQ to 0.152 μg/L. The tested concentration was above the limit of solubility. Thus, the observed decrease may be caused by separation of undissolved test material. The regular test duration is 21 days. The test was prolonged for one day as the release of juveniles had not been synchronous. Therefore, the evaluation is performed based on the data of days 0-21 and 0-22. The Cochran-Armitage trend-test revealed that no significant trend in mortality was observed. The overall effect threshold for effects of the test item under the test conditions was 0.250 μg/L (NOEC) and > 0.250 μg/L (LOEC) based on the geometric mean measured concentrations of the test item Laurylacrylat 12.

In an supporting study with a read across substane 2-Propenoic acid, C12-14-alkyl esters (mixture of CAS no. 2156 -97 -0 and 21643 -42 -5), a semi-static Daphnia magna reproduction test was performed in closed test vessels according to OECD Guideline 211 (2008) and EU Method C.20 (2008). At this method Daphnia magna, aged lower or equal 24 h at the beginning of the test, are exposed to a range of a read across substance under defined conditions over a period of 21 days. In the definitive limit test the nominal concentration was 100 mg/L. The read across substance is poorly water soluble and volatile; therefore a saturated stock solution was prepared using a liquid-liquid saturator technique. Concentration control analysis was not performed since the analytical detection limit was above the water solubility of the test substance. However, all reasonable efforts were taken to produce a saturated solution of the test substance in test media, following the guidance in OECD 23 (2000).

The following overall effect concentrations (mg/L) were obtained based on nominal concentrations: LOEC > 100 mg/L NOEC ≥ 100 mg/L No significant effect on mortality (21 d EC0 ≥ 100 mg/L) and reproduction or any adverse effects on additional endpoints (i.e. growth, immobile young, time to first brood, or aborted eggs) were observed in any of the test treatments.