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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:
14 - 21 Feb 2019
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
Deviations:
yes
Remarks:
Please see section "Any other information on material and methods incl tables".
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
adopted 30. May 2008
Deviations:
yes
Remarks:
Please see section "Any other information on material and methods incl tables".
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations at the beginning and end of the test and at medium renewal (every 24 h) in the new and old test solutions.
- Sample preparation:
Low Concentrations, 22 – 4.6 mg/L, and control: To 100 mL solution 6 g NaCl was added, then, the solution was extracted two times with the solvent di-chloromethane (2 * 4 mL), the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloro-methane after addition of 100 µL ISTD stock solution (100 mg/L) and the solution was measured via GC/MSD. Tenfold enrichment was achieved during extraction procedure.
High Concentrations, 46 - 100 mg/L:
To 10 mL solution 2 g NaCl was added, then, the solution was be extracted two times with the solvent dichloromethane (2 * 4 mL), the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 100 µL ISTD stock solution (100 mg/L) and the solution was measured via GC/MSD.
- Storage: All samples were analysed directly after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: To homogenize the test item, it was melted at 40 °C before use. A WAF was preparded: 100 mg/L (real load 102.7 mg/L at the beginning and 101.3 mg/L after 24 h), adding the corresponding amount of dilution water (M4 Medium) and shaking vigorously for 23 h and 10 min (at the beginning) and 23.5 h (at medium renewal). The resulting solution was filtrated through 0.45 µm PTFE filters. The lower treatments were prepared by dilution of this solution (WAF) with dilution water.
- Differential loading: No
- Controls: Yes, medium was used
- 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: Water flea, STRAUS
- Strain/clone: Berlin
- Source: Umweltbundesamt Berlin
- Age and sex at test start: Females 0 - 24 h
- Feeding during test: no feeding during the test

ACCLIMATION
- Acclimation conditions (same as test or not): M4-Medium (recipe of ELENDT) was used for husbandry and deionised water with an enrichment of certain minerals was used in the test.
- Type and amount of food: Green algae (Desmodesmus subspicatus)
- Feeding frequency: twice a week

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: 18.5 h before the start of the test, the adult animals were separated from the young. 0.5 h before test start, the adults were caught with the help of a glass tube, and the newborn daphnia (age < 24h) were sieved from the medium and immediately placed into a beaker containing dilution water. After the settling-in 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
Test temperature:
22.2 – 23.6 °C
pH:
7.7 - 8.0 (Control)
7.7 - 8.1 (Test item concentrations)
Dissolved oxygen:
8.4 - 8.7 (Control)
7.8 - 8.9 (Test Item concentrations)
Nominal and measured concentrations:
4.6; 10; 22; 46 and 100 mg/L (nominal)
-, -, 0.34; 1.64 and 1.79 mg/L (Time Weighted Mean)
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: glass beakers, nominal volume 50 mL, tall shape, 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: yes, non-GLP range-finder was performed but no details given on range- finder
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.11 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: None
- Mortality of control: 0%
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
The EC50 (24 h) value was determined as 2.05 mg/L. The value was within the requested range for the EC50 (24 h) between 0.6 - 2.1 mg/L of potassium dichromate K2Cr2O7 as specified in the OECD guideline.
Reported statistics and error estimates:
The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.3.0.
Test design: Hypothesis testing (NOEC) and regression (LCx).

Biological Results

Table 1: Biological Results Test Item based on the time-weighted mean of the measured concentrations

Parameter

Value

95%-confidence interval

24h EC50

1.45 mg/L

n. d.

48h EC50

1.11 mg/L

n. d.

48h NOEC

0.34 mg/L

--

48h LOEC

1.64 mg/L

--

n. d.: not determined either due to mathematical reasons or value is beyond the tested concentrations by more than factor 1000

Table 2: Immobility

Nominal Concentration in mg/L

Measured Concentration based on TWA in µg/L

Immobility 24 hours

Immobility 48 hours

absolute

in %

absolute

in %

Blank control

--

0

0

0

0

0

0

0

0

0

0

4.6

--

0

0

0

0

0

0

0

0

0

0

10

--

0

0

0

0

0

0

0

0

0

0

22

0.34

0

0

0

0

0

0

0

0

0

0

46

1.64

2

0

4

0

30

5

3

5

3

80

100

1.79

5

5

5

5

100

5

5

5

5

100

Analytical Results:

In the highest concentrations 100 mg/L all daphnia were dead after 24 hours. Therefore, no new solution was prepared for this concentration.

Because of the poor solubility of the test item, the correlation between nominal and measured concentration was week. The measured concentration in the fresh prepared solution was in a similar range. Due to the test items properties (insufficient stability and adsorbing property), in the old solutions the measured values varied strongly. In the old solutions test item was clearly detectable only in the two highest concentrations. Because toxicity was observed only in the two highest concentrations, the time-weighted mean of the measured concentrations in the 3 highest concentrations was used for determination of the biological results. At the concentration 22 mg/L no test item was detectable in the old solutions. Therefore, the time weighted mean based on the measured initial concentration and the LOD (limit of detection). At the end of the test, the test item was clearly detectable only in the highest concentration 46 mg/L. Most likely due to a contamination, the measured concentration after 48 hours in the old solution (2.26 mg/L) was higher than the measured concentration after 24 hours in the new solution (1.87 mg/L) and a small signal was present also in the control solution. This signal was in the same range as the signals in the lower concentrated test solutions.

Analytical Results: Measured Concentrations (purity 48 % component 1)

Nominal
Concentration Test Item

Measured

Concentration (1)
 t = 0 h

Measured

Concentration
 t = 24 h old

Measured

Concentration
 t = 24 h new

Measured

Concentration
 t = 48 h

mg/L

mg/L

mg/L

mg/L

mg/L

Blank control

< LOD

 

 

< LOD

4.6

< LOD

< LOD

< LOD

< LOD

10

0.26 (2)

< LOD

0.17 (2)

< LOD

22

0.49 (2)

< LOD

0.58 (2)

< LOD

46

1.53

0.95 (4)

1.87

2.26

100

2.94

1.00

- (3)

- (3)

LOD = 0.2 mg/L (lowest calibration level)

1 calculated from measured concentration 100% component 1

2 measured concentration before applying enrichment factor 0.1 and therefore within the calibrated range

3 not measured because all daphnia were found dead after 24 hours

4 slightly outside of the calibrated range (0.96 – 9.6 mg/L), nevertheless used for evaluation because of the clear signal for the test item

 

Validity Criteria

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

Table :Validity criteria

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 0%.

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 7.8 mg/L.

Yes

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

Description of key information

EC50 (48 h): 1.11 mg/L (Daphnia magna, OECD 202, Time Weighted Average)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
1.11 mg/L

Additional information

The study on the acute toxicity of the substance to aquatic invertebrates was carried out according to OECD guideline 202 under GLP conditions including analytical measurement. In this semi-static test, Daphnia magna was exposed to five test concentrations (4.6, 10, 22, 46 and 100 mg/L based on the nominal test item concentrations and n.d, n.d., 0.34, 1.64 and 1.79 mg/L based on the Time Weighted Mean). Due to the low water solubility the test item concentrations were prepared via water-accommodated fractions: 100 mg/L (real load 102.7 mg/L at the beginning and 101.3 mg/L after 24 hours), adding the corresponding amount of dilution water (M4 Medium) and shaking vigorously for 23 h and 10 min (at the beginning) and 23.5 h (at medium renewal). The resulting solution is filtrated through 0.45 µm PTFE filters. The lower treatments were prepared by dilution of this solution (WAF) with dilution water. Analytical measurement was done by GC/MSD. The test item consists of two components, ethylene glycol monolaurate (component 1) and ethylene glycol dilaurate (component 2), but only one (component 1) is water soluble. The quantification of the test item was performed using the signal of component 1 only.Due to the test items properties (insufficient stability and adsorbing property), in the old solutions the measured values varied strongly. In the old solutions test item was clearly detectable only in the two highest concentrations. Because toxicity was observed only in the two highest concentrations, the time-weighted mean of the measured concentrations in the 3 highest concentrations was used for determination of the biological results. In the study report an EC50 (48 h) of 1.11 mg/L and the NOEC (48 h) was reported as 0.34 mg/L based on the time weighted average. 

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