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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:
28 - 30 Nov 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
adopted 13 Apr 2004
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
adopted 30 May 2008
Qualifier:
according to guideline
Guideline:
other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals
Version / remarks:
2nd edition, adopted 06 Jul 2018
GLP compliance:
yes (incl. QA statement)
Remarks:
Rheinland Pfalz, Landesamt für Umwelt, Mainz, Germany (15 May 2018)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations (Control, 2.2, 4.6, 10, 22 and 46 mg/L) at the beginning and end of the test and at medium renewal (every 24 h) in the new and old test solutions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution was prepared by adding 46.2 mg/L test item to the corresponding amount of dilution water and shaking vigorously for 24 h (no lighting but not completely in the dark). The resulting solution was filtered through blue-band paper filters. The lower concentrations were prepared by dilution of this saturated solution with dilution water. After shaking the solution was cloudy, after filtration the solution was clear, without undissolved particles.
- Preparation procedure: Sample preparation procedure was based on results of numerous non-GLP pre-tests.
- Differential loading: No
- Controls: Dilution water
- Evidence of undissolved material: After filtration through blue-band paper filter, the resulting solution was clear and no undissolved particles remained.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Planktonic crustacean / water flea
- Strain/clone: Berlin
- Source: Umweltbundesamt Berlin, Germany (27. Sep 2007)
- Age and sex at test start: Females 0 - 24 h
- Breeding conditions: According to OECD guideline and SOP 115 002 01 (12 Feb 2015, v12). Multiplication by parthenogenesis (genetically identical). Vessels: 2 L preserving glasses; Medium: M4 (Elendt); Food: Green alga (Desmodesmus subspicatus); Medium renewal: Twice/week; Photoperiod: 16 h light / 8 h dark; Temperature: 20 ± 2 °C
- Test medium: Dilution water
- Other: It has been shown that switching from M4-medium (husbandry) to dilution water (test) does not negatively affect test daphnia.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
22 h and 50 min before test start, the adult animals were separated from the young. 0.5 h before test start the adults were caught with a glass tube and the newborn daphnia (aged 0 - 22 h and 50 min) were sieved from the medium and immediately placed into a beaker containing dilution water.
After the settling period, 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:
48 h
Hardness:
250 mg CaCO3/L (dilution water)
2.502 mmol/L (dilution water)
Test temperature:
22.5 - 23.2 °C
pH:
0 h: 7.5 - 7.8
48 h: 7.5 - 7.8
Dissolved oxygen:
0 h: 8.8 - 9.0 mg/L
48 h: 8.2 - 9.2 mg/L
Nominal and measured concentrations:
Control, 2.2, 4.6, 10, 22, and 46 mg/L (nominal)
Control, 26.1, 63.35, 169.49, 468.62, and 1136.06 µg/L (time-weighted average of measured concentrations)
Details on test conditions:
TEST SYSTEM
- Test vessel: Open 50 mL glass beakers filled with 20 ± 5 mL test solution
- Renewal rate of test solution: 24 h
- 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: Not specified
- CaCl2*2H2O: 293.80 mg/L
- MgSO4*7H2O: 123.30 mg/L
- NaHCO3: 64.80 mg/L
- KCl: 5.80 mg/L
- Culture medium different from test medium: Yes, culture medium (M4, Elendt) different from test medium (dilution water).
- Intervals of water quality measurement: 24 h

OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h dark
- Light intensity: Neon tubes

EFFECT PARAMETERS MEASURED:
- Immobilization: After 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: In the pre-tests, a strong influence of filtration was observed due to the absorbing properties of the test item. Membrane filtration through PTFE filters (0.45 µm) resulted in complete loss of the test item. Unfiltered solutions were not an option because undissolved particles were still present in the solution after centrifugation and immobilised Daphnia were clogged with undissolved particles. Filtration through glass wool was not successful (visible fine particles remained in the aqueous phase).
- Results used to determine the conditions for the definitive study: Yes. At the loading rate of 100 mg/L and 46 mg/L 100% immobility of the daphnia was observed whereas no toxicity was observed at the low loading rates of 1 and 3 mg/L. Thus, the amount of dissolved substance depended on the loading rate. Therefore, the concentration of 46 mg/L was chosen as the highest concentration and the lower concentrations were prepared by dilution of this solution with test medium. Direct addition of low loading rates followed by filtration would lead to very low, non-measurable test item concentrations, possibly missing toxicity and insufficient for statistical evaluation. Therefore, the lower concentrations were not prepared by direct addition but by dilution of the stock solution with test medium.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7, CAS 7778-50-9)
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
15.93 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other:
Remarks:
95% confidence interval: 6.39 - 39.91 µg/L
Details on results:
- Behavioural abnormalities: None
- Mortality of control: 5%
- Any observations that might cause a difference between measured and nominal values: The test media were clear after filtration and remained so.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes, the reference study confirmed the reliability of the present study.
Reported statistics and error estimates:
Statistical analyses were carried out with the software ToxRat Professional v3.2.1.
Test design: Hypothesis testing (NOEC) and regression (LCx).
Statistical analyses: Logit and Probit analyses using linear max. likelihood regression; Qualitative Trend Analysis by Contrasts (Monotonicity of Concentration/Response), Tarone's Test Procedure; Multiple Sequentially-rejective Fisher Test After Bonferroni-Holm; Step-down Cochran-Armitage Test Procedure;

VALIDITY CRITERIA

The study fulfilled the validity criteria defined by the guideline (Table 1) and is thus considered valid and reliable.

Table 1. Validity criteria for OECD 202.

Criterion from the guideline

Outcome

Validity criterion fulfilled

In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilized.

Immobilization in the control was 5%.

Yes

The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.

The lowest concentration of dissolved oxygen at the end of the test was 8.4 mg/L.

Yes

 

ANALYTICAL RESULTS

The measured test item concentrations confirmed the reproducibility of the preparation procedure.

Overall, the correlation between nominal and measured test item concentrations was weak due to the very poor solubility of the test item.

A decrease of the measured test item concentrations was especially observed in old solutions of the lower test concentrations (Table 2). Therefore, effect values were based on the time-weighted mean values of the measured concentrations (Table 3). For the nominal treatment of 4.6 mg/L only the measured concentrations after 24 h (new solution) and 48 h (old solution) were used for the calculation of the time-weighted mean due to an error in sampling at the beginning.

Table 2. Measured test item concentrations.

Nominal
Concentration Test Item

Measured

Concentration
 t = 0 h

Measured

Concentration
 t = 24 h old

Measured

Concentration
 t = 24 h new

Measured

Concentration
 t = 48 h old

mg/L

µg/L

µg/L

µg/L

µg/L

Blank control

n. d.

--

--

--

2.2

58.68

6.52

48.90

9.66

4.6

155.55

35.94

107.27

33.45

10

*139.08

159.44

203.50

140.24

22

436.51

454.97

525.50

380.01

46

1037.48

1043.93

1293.11

1005.13

 n. d. = not detectable

*implausible low measured concentration at the beginning whereas the measured value in the old solution after 24 h was in the expected ranged. This was most likely caused by a sampling error or an error during sample preparation.

Table 3. Time-weighted Mean values

Nominal Concentration Test Item

Time-weighted mean value of measured concentrations

mg/L

µg/L

Blank control

--

2.2

23.97

4.6

72.49

10

169.91

22

447.25

46

1091.89

 

BIOLOGICAL RESULTS

The tested concentrations caused 65 to 100% immobilization. Clear toxicity was also observed in the lowest tested concentration (Table 4). Therefore, the NOEC could only be indicated as a range. Nevertheless, the 48 h EC50 value could be determined by extrapolation due to a clear dose-response relationship. A summary of effect values is provided in Table 5.

Table 4. Immobility after 24 and 48 h.

Nominal Concentration in mg/L

Immobility 24 hours

Immobility 48 hours

absolute

in %

absolute

in %

Blank control

0

0

0

1

5

0

0

0

1

5

2.2

2

1

3

3

45

3

3

4

3

65

4.6

2

2

2

2

40

3

5

4

4

80

10

3

4

1

4

60

4

4

5

5

90

22

2

5

3

2

60

5

5

5

5

100

46

5

4

4

4

85

5

5

5

5

100

Table 5. Summary of effect values (based on Time-Weighted Mean of measured concentrations).

Parameter

Value

95%-confidence interval

24h EC50

*77.90 µg/L

27.47 – 220.89 µg/L

48h EC50

**15.93 µg/L

6.39 - 39.91 µg/L

48h NOEC

< 23.97 µg/L

--

48h LOEC

≤ 23.97 µg/L

--

*Due to the lacking concentration/response the shown 24h EC50 could not be valid.Nonetheless, the user decided to show the 24 h EC50, confidence limits and graph, see page 4 in annex

**extrapolated value


 

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.

Description of key information

EC50 (48h) = 15.93 µg/L for Daphnia magna (meas. TWA, OECD 202)

Key value for chemical safety assessment

Additional information

One study investigating the short-term toxicity of Reaction mass of N-(1-oxooctadecyl)sarcosine and N-hexadecanoyl-N-methylglycine (EC 947-850-7) to aquatic invertebrates is available.

The study was carried out according to OECD guideline 202 and GLP including analytical measurement. Daphnia magna was exposed to the following nominal concentrations 2.2, 4.6, 10, 22, and 46 mg/L under semi-static conditions. Analytical measurements verified the test item concentration to be 0.0261, 0.06335, 0.16949, 0.46862, and 1.13606 mg/L (time-weighted mean of measured concentrations). Because of the very poor solubility of the test item, the correlation between nominal and measured con-centration was weak. Therefore, the determination of the biological results was based on the time weighted mean of the measured concentration. The tested concentrations caused 65 to 100% immobilisation. Clear toxicity was also observed in the lowest tested concentration. Nevertheless, the 48 h EC50 value could be determined by extrapolation due to a clear dose-response relationship. The test revealed an EC50 (48h) of 15.93 µg/L (meas. TWA).