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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:
May 03 - Jul 18, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
At the start of the exposure and at the renewal (24 and 48 h), samples of the freshly prepared test item concentrations were taken and analyzed.
At renewal and at the end of the exposure (24 and 48 h), sampled of the 24 h old media were taken directly from the test vessels and analyzed.
Vehicle:
yes
Remarks:
dilution water
Details on test solutions:
Saturated solution
A saturated solution (100 mg test item/L were weighed out) was freshly prepared with culture medium (see Table 2) one day prior to the start of the exposure (day -1) and prior to the renewal of the test solutions (day 0 hours). The stock solution was stirred for 24 ± 1 hour at approx. 1100 rpm at room temperature. After a separation phase of 1 hour at room temperature, the test solution was removed from the middle of the flask. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item. No Tyndall-effect was detected and no undissolved test item was present during the test.

Test concentrations
The saturated solution and further 4 dilution levels of the saturated solution in a geometric series with a separation factor of 2, prepared by dilution of the saturated solution with dilution water, were tested as follows:

6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution, corresponding to the geometric mean measured concentrations: 45.6 – 85.2 – 388 – 1223 – 2836 µg/L.
The test item concentrations mentioned above were selected based on the results of a non-GLP preliminary range finding test.

Control
Dilution water without test item incubated under the same conditions as the test groups
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna STRAUS (Clone 5)
- Source: Institut für Wasser-, Boden und Lufthygiene (WaBoLu), Berlin, Germany
- Age: < 24 h

FEEDING DURING TEST
- Food type: 5 x per week ad libitum with a mix of unicellular green algae
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
0 h: 262 mg CaCO3/L
24 h: 258 mg CaCO3/L
Test temperature:
19.0 - 20.3 °C
pH:
0 h: 8.27
24 h: 7.90

Dissolved oxygen:
0 h: 9.41 mg/L
24 h: 9.06 mg/L
Conductivity:
0 h: 603 µS/cm
24 h: 619 µS/cm
Details on test conditions:
Test vessels: Glass beakers, 50 mL capacity, loosely covered with watch glasses
Test volume: 20 mL

Number of daphnids and replicates:
20 daphnids, divided into 4 replicates, each with 5 daphnids were used for each concentration level and control.

Application:
20 g test solution per replicate was weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a samll amount of dilution water (start of the exposure) or test solution (water renewal) by pipette.

Renewal of the test solutions:
The test solutions were renewed after 24 h. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
61.3 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 58.9 - 63.8
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
85.2 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 45.6 - 349
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Limit test: No
- ECx: EC50 (24 hours): 0.6 - 2.4 mg/L (according to AQS 9/2); EC50 (24 hours): 0.6 - 2.1 mg/L (according to OECD 202)
- Other: An acute immobilization test was conducted with the reference substance once per month to prove the validity of the test system.

Purpose

The purpose of this study was to determine the influence of the test item on the mobility of Daphnia magna. For this purpose, young daphnids (< 24 hours old) were exposed to various concentrations under defined conditions for 48 hours.

Study Design

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 202.  The study was conducted under semi-static conditions over a period of 48 hours with a saturated solution of the test item with a nominal loading of 100 mg test item/L and further four dilution levels (nominal: 6.25 – 12.5 – 25.0 – 50.0 - 100%) of the saturated solution prepared in a geometric series with a separation factor of 2.
Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each dilution level and the control.
The concentrations of the test item were analytically verified via LC-MS/MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) and in the old media at the renewal and at the end of the test (24 and 48 hours) in all dilution levels and in the control.

The recorded effects were the mobility of the daphnids after 24 and 48 hours. The test method of application and the test system are recommended by the test guidelines and Daphnia magna is the recommended test species.
The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Results

Biological Results

The percentage of immobility, determined in all dilution levels of the test item and in the control after 24 and 48 hours is given below:

Mean Measured Concentration
[mg test item / L]
% of immobilised daphnids after
24 hours
48 hours
2836
100
100
1223
100
100
388
100
100
85.2 50
100
45.6
0
5
Control 0
0


The effect concentrations (EC10 / 50 / 100), based on the geometric mean measured concentrations of the test material are given below:

Effect concentrations
Duration / [h]
Geometric Mean measured the test material concentrations / [µg/L]
EC10
(with confidence limits)
24
72.1 (< 45.6 - 334)
48
47.1 (< 45.6 – 49.5)
EC50
(with 95% confidence limits)
24
85.2 (45.6 - 349)
48
61.3 (58.9 – 63.8)
EC100
24
388
48
85.2


Analytical Results

The concentrations of the test item were analytically verified via LC-MS/MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) and in the old media at the renewal and at the end of the test (24 and 48 hours) in all dilution levels and in the control.

Sampling
0 h (fresh)
24 h (old)
24 h (fresh)
48 h (old)









Dilution level of the
saturated solution
[%]
Measured
conc.
[µg/L]
Measured
conc.
[µg/L]


[%]
Measured
conc.
[µg/L]
Measured
conc.
[µg/L]


[%]
Geometric mean
measured concentration
[mg/L]
100
5174
1554
30
Not determined due
to 100 % mortality
after 24 hours
2836
50
2125
704
33
1223
25
862
175
20
388
12.5
448
24.5
5
481
< LOQ
85.2
6.25
192
< LOQ
225
< LOQ
45.6
Control
< LOQ
< LOQ
< LOQ < LOQ


Conclusion

The toxic effect of the test item to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour EC10was determined to be 47.1 µg test item/L and the 48-hour EC50 value was calculated to be 61.3 µg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to geometric mean measured concentrations.

Validity criteria fulfilled:
yes
Conclusions:
The toxic effect of the test item to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour EC10was determined to be 47.1 µg test item/L and the 48-hour EC50 value was calculated to be 61.3 µg test item/L. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to geometric mean measured concentrations.

Description of key information

OECD 202: EC50 (48 h) = 61.3 µg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
61.3 µg/L

Additional information