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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 5, 2017 - December 13, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
Council Regulation (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Preparation of the Test Item:
A preparation with a test item concentration of 100 mg/L was freshly prepared. For that purpose, the test item will be weighed in a calibrated flask and vehicle was added. The preparation was stirred with a magnetic stirrer for 1 hour. This solution was used for the preparation of the different test item concentrations. The pH value was not adjusted.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Concentrations:
10.0, 17.8, 31.6, 56.3 and 100.0 mg/L

Sampling method:
Two replicates of each sample were deep-frozen (≤ -20°C) directly after preparation and after 48h and stored in a freezer until analysis. The stability of the test item during freezing has been demonstrated in a separate non-GLP study. One replicate was used for analysis. The second replicate was used as backup.

Test solutions

Vehicle:
yes
Details on test solutions:
Macro nutrients (mg/L)
CaCI2 •2H20 293.80
MgSO4•7H20 123.30
NaHCO3 64.80
KCI 5.80
Na2SiO3•9H20 10.00
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18

Trace elements (mg/L)
B 0.5000
Fe 0.2000
Mn 0.1000
Li, Rb and Sr 0.0500
Mo 0.0250
Br 0.0125
Cu and Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003

Macro nutrients (mg/L)
Na2EDTA • 2H20 2.50

Vitamins (µg/L):
Thiamine 75.00
B12 1.00
Biotin 0.75

- pH: 7.84
- Total hardness: 236 mg CaCO3/L
- O2-Concentration: 8.85 mg/L
- Conductivity: 666 µS/cm

Preparation of the Test Item:
A preparation with a test item concentration of 100 mg/L was freshly prepared. For that purpose, the test item will be weighed in a calibrated flask and vehicle was added. The preparation was stirred with a magnetic stirrer for 1 hour. This solution was used for the preparation of the different test item concentrations. The pH value was not adjusted.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna Straus
Origin: Daphnia magna Straus was originally obtained from IBACON GmbH (Roßdorf, Germany).
Culture conditions: The clone is bred in the laboratories of Merck KGaA (room 2068).
Parental daphnids are used for reproduction until they are about 6 weeks old. Thereafter, they are replaced by neonates.
Daphnids are kept individually in 100 mL glass vessels containing approximately 60 mL reconstituted water (ELENDT M4 medium) at a water temperature of 20 ± 2°C and a 16 hour light and 8 hour dark regime to ensure similar conditions as in the experiment. Offspring are removed from the vessels at least twice per week.
Feeding: The parental daphnids are fed ad libitum with unicellular green algae Desmodesmus subspicatus three times per week.
Age: Offspring less than 24 hours old were used for the study.
Acclimation period: same as test

Feeding during test: None

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
236 mg CaCO3/L
Test temperature:
20.4 - 20.9°C
pH:
7.12 - 7.84
Dissolved oxygen:
8.69 - 8.87 mg/L
Conductivity:
666 µS/cm
Nominal and measured concentrations:
Nominal concentrations: 10.0, 17.8, 31.6, 56.3 and 100.0 mg/L
Measured concentrations: > 80 % of nominal concentrations
Details on test conditions:
EXPOSURE:
The study was performed in an air-conditioned room. For the study 20 mL glass test tubes containing at least 20 mL either reconstituted water (control group) or test medium (test item groups) were used. Each test vessel contained five daphnids resulting in 4 mL medium per daphnid. They were not fed and the media were not aerated during the exposure.

The test was performed as a static test in open vessels.

The duration of exposure was 48 hours. During the exposure period, the mobility of the daphnids was assessed daily, i.e. after 24 and 48 hours.

NO. OF DAPHNIDS:
Control Group: 20 daphnids
10.0 mg/L: 20 daphnids
17.8 mg/L: 20 daphnids
31.6 mg/L: 20 daphnids
56.3 mg/L: 20 daphnids
100.0 mg/L: 20 daphnids

CONCENTRATION(S)
In a pre-test (non-GLP) the stability of the test item in reconstituted water for daphnids was investigated. The recovery of the test item in the test medium was >80% of the nominal concentration. The test item concentrations in reconstituted water for daphnids were stable over a period of 48 hours in open vessels and the samples could be deep-frozen until analysis (Broschard, 2017).

Therefore, the present study was performed as a static test in open vessels with the following nominal concentrations:
10.0, 17.8, 31.6, 56.3 and 100.0 mg/L

For the control, reconstituted water was used.


VEHICLE
Reconstituted water (ELENDT M4 medium) was used as vehicle.

Macro nutrients (mg/L)
CaCI2 •2H20 293.80
MgSO4•7H20 123.30
NaHCO3 64.80
KCI 5.80
Na2SiO3•9H20 10.00
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18

Trace elements (mg/L)
B 0.5000
Fe 0.2000
Mn 0.1000
Li, Rb and Sr 0.0500
Mo 0.0250
Br 0.0125
Cu and Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003

Macro nutrients (mg/L)
Na2EDTA • 2H20 2.50

Vitamins (µg/L):
Thiamine 75.00
B12 1.00
Biotin 0.75

- pH: 7.84
- Total hardness: 236 mg CaCO3/L
- O2-Concentration: 8.85 mg/L
- Conductivity: 666 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: 48h


OTHER TEST CONDITIONS
- Photoperiod: 16h light / 8h dark
- Light intensity: The mean light intensities were 575 Lux and 629 Lux prior to and at the end of the exposure period, respectively.

References:

ELENDT, B.-P. Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus.
Protoplasma 154, 25-33, 1990
Reference substance (positive control):
no
Remarks:
No positive control used in this study. The accuracy and reliability of the test method is demonstrated periodically as recommended by guidelines with potassium dichromate.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
74.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: The EC50 values were calculated with the nominal concentrations.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
41.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: The EC50 values were calculated with the nominal concentrations.
Details on results:
Under the conditions of the present study, the test item showed a 48h EC50 of 41.1 mg/L.
Results with reference substance (positive control):
Potassium dichromate:
24h EC50 1.13 mg/L (0.98 – 1.34 mg/L)
48h EC50 0.74 mg/L (0.66 – 0.82 mg/L)

The test item Art. 104864 (Potassium dichromate) showed a 24h EC50 value of 1.13 mg/L which is within the range of the published data of 0.6 to 1.7 mg/L (Council Regulation (EC) No. 440/2008) and 0.6 to 2.1 mg/L (OECD Guideline No. 202)..

Any other information on results incl. tables

Objective

The objective of this study was to determine the effect of the test item on the immobilization of Daphnia magna.

Study Design

Juvenile daphnids were exposed to different test item concentrations of nominal 0.0, 10.0, 17.8, 31.6, 56.3 and 100.0 mg/L in an open static system. 20 daphnids, divided into four replicates, each of five animals were used per test item group and the control group. The daphnids were observed for immobilization after 24 and 48 hours of exposure.

Samples of the test and control medium were taken directly after preparation and at the end of exposure (48h) and stored at ≤ -20°C until further analysis of the test item concentration via HPLC with UV detection.

Results

The measured concentrations were between 93.0% and 100.1% of the nominal concentrations.

 

0h

48h
Nominal

[mg/L]

%

[mg/L]

%

0 mg/L <LOD <LOD -
10.0 mg/L 10.012 100.1 9.417 94.2 
17.8 mg/L 17.733 99.6 16.845 94.6 
31.6 mg/L  31.271 99.0 29.385 93.0 
56.3 mg/L  55.719 99.0 54.403 96.6
100.0 mg/L  99.541 99.5 98.303 98.3

As the recoveries of the test item in the aqueous solutions were >80% of the nominal concentrations during the study, the EC50 values were calculated with the nominal concentrations.

No effect on the mobility of the daphnia was observed at the two lowest concentrations of nominal 10.0 and 17.8 mg/L. A concentration dependent immobilization was observed between 31.6 and 100.0 mg/L as given in the following table.

  Number of daphnids immobilized / exposed    % of daphnids immobilized 
Nominal 24 hours 48 hours 24 hours 48 hours
0.0 mg/L  0/20  0/20  0%  0% 
10.0 mg/L 0/20  0/20  0%  0% 
17.8 mg/L  0/20  0/20  0%  0% 
31.6 mg/L  3/20  7/20  15%  35% 
56.3 mg/L 10/20  14/20  50%  70% 
100.0 mg/L 11/20  20/20  55%  100% 

For the test item the following nominal EC50 values and 95% confidence intervals were determined:

24h EC50       74.1 mg/L (57.5 - 110.9 mg/L)

48h EC50       41.1 mg/L (34.7 - 48.7 mg/L)

Conclusion

Under the conditions of the present study, the test item showed a 48h EC50 of 41.1 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study, the test item showed a 48h EC50 of 41.1 mg/L.
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 202. Under the conditions of the present study, the test item showed a 48h EC50 of 41.1 mg/L.