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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:
2017-06-12 to 2017-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: at test start after 24 and 48 hours
- Sample storage conditions before analysis: 4 ± 4°C
- Fortified samples: 50mg of the test item were dissolved in 50mL acetonitrile to obtain a stock solution of approx. 1g test item/L. Different dilutions were carried out and exact values are documented in the raw data of the study.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Stock solution: 400 mg of test item/L in 2000 mL test water, mixed manually;
- Dilutions of stock solution: 1:2, 1:4, 1:8, 1:16 prepared for testing
- Controls: test water without addition of the test item
- Preparation of test solutions: pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: straus, clone 5
- Source: available in study report
- Age at start of experiment: 6.25 to 22.75 hours old
- Feeding during test: no feeding during test
- Feeding of culture (before test): all working days with green algae

ACCLIMATION
- Acclimation period: 6 hours
- Acclimation conditions (same as test or not): under test conditions
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L as CaCO3
Test temperature:
20.7 to 21.0 °C in the freshly prepared media
20.4 to 20.9°C in the aged test media
pH:
6.1 to 6.2 in the freshly prepared media
6.1 to 6.2 in the aged test media
Dissolved oxygen:
8.4 to 9.0 mg/L in the freshly prepared media
8.5 to 8.7 mg/L in the aged test media
Conductivity:
< 5 μS/cm
Nominal and measured concentrations:
Nominal concentrations: stock solution, 1:2, 1:4, 1:8, 1:16 (dilutions of stock solution)
Measured concentrations (geometric mean measured): 0.744, 0.325, 0.145, 0.0650 and 0.0426 mg test item/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beaker, 110 mL capacity
- Type: closed
- Material, size, headspace, fill volume: 110 mL filled with as much as test medium possble
- No. of organisms per treatment group: 20 daphnids
- No. of treatment groups: 6 (5 dose rates of the test item and a control)
- Replicates: 2 per treatment group

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, test water sterile filtered
- Alkalinity: 0.9 mmol/L
- Conductivity: < 5 μS/cm
- Culture medium different from test medium: same as Elendt M4

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: 340 to 580 lux

EFFECT PARAMETERS MEASURED: Immobilisation

RANGE-FINDING STUDY
- Test concentrations: test item dissolved in Elendt medium (adjusted to pH6). A water accommodated fraction of the highest test concentration (200 mg/L) was prepared at test start and on day 1. Dilutions of 1:10, 1:100, 1:1000 were prepared from this stock solution.
- Sampling points : at test start (0h) and test end (24h).
- Results used to determine the conditions for the definitive study: nominal concentration of 200 mg test item/L caused an immobilisation rate of 90 % after 48h. The lower concentrations showed no immobility of the daphnids. Based on these results, the main test concentrations were chosen: water accommodated fraction of 200 mg test item/L as highest test concentration with a dilution series of 1:2, 1:4, 1:8 and 1:16.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate tested at least twice a year
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.298 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.174 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.145 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Observations in control group: no immobilisation, no signs of disease or stress.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24h) = 1.48 mg test item/L, indicating that the sensitivity of the daphnids was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).
Reported statistics and error estimates:
The 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated by probit analysis.
The NOEC and LOEC after 24 and 48 hours were determined directly from the raw data.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Summary of Biological results:

Geometric mean measured concentration (mg test item/L) % of immobilised daphnids after
24 hours 48 hours
control 0 0
0.0426 0 0
0.0650 0 0
0.145 0 0
0.325 0 14
0.744 0 19

Results for the determination of the test item in the test samples:

Sample description
(mg test item/L)
Age (h)
0 (fresh) 24 (aged) 24 (fresh) 48 (aged)
Control        
Rep 1 < LOD < LOD < LOD < LOD
Rep 2 < LOD < LOD < LOD < LOD
E.F. 2.5
Concentration calculated-mean  n.a. n.a. n.a. n.a.
1:16 dilution of stock solution        
Rep 1 < LOQ < LOQ 0.167 < LOQ
Rep 2 < LOQ < LOQ 0.183 0.194
E.F. 2.5      
Concentration calculated-mean  0.025 0.025 0.07 0.0515
1:8 dilution of stock solution        
Rep 1 0.130 < LOQ 0.330 0.186
Rep 2 0.144 0.128 0.360 < LOQ
E.F. 2.5
Concentration calculated-mean 0.055 0.038 0.138 0.05
1: 4 dilution of stock solution        
Rep 1 0.266 0.257 0.657 0.349
Rep 2 0.268 0.200 0.656 0.337
E.F. 2.5
Concentration calculated-mean 0.107 0.092 0.263 0.138
1:2 dilution of stock solution        
Rep 1 0.630 0.405 1.405 0.893
Rep 2 0.643 0.406 1.410 0.884
E.F. 2.5 
Concentration calculated-mean 0.255 0.163 0.563 0.356
stock solution        
Rep 1 1.440 0.947 3.036 2.211
Rep 2 1.457 0.843 3.125 2.118
E.F. 2.5
Concentration calculated-mean 0.580 0.358 1.232 0.866
Validity criteria fulfilled:
yes
Conclusions:
The 48-hour EC50 for Daphnia magna is of 0.298 mg/L.
Executive summary:

The short-term toxicity to Daphnia magna was determined according to OECD 202 guidelines and EU method C.2. The study was conducted under semi-static conditions. The daphnids were exposed over a period of 48 hours to 5 concentrations of the test item (nominal concentrations were the stock solution and its dilutions: 1:2, 1:4, 1:8, 1:16).

The validity criteria were fulfilled.

The EC50 value was determined to be 0.298 mg/L and the NOEC is 0.145 mg/L.

Description of key information

 EC50 (48 h): 0.298 mg/L based on immobilisation of D. magna (OECD 202)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.298 mg/L

Additional information

One experimental study is available investigating the short-term effects of the substance to aquatic invertebrates. The study was performed according to the OECD guideline 2002 and GLP using Daphnia magna as test organism. 5 dilution levels of 1:1, 1:2, 1:4, 1:8 and 1:16 were tested under semi-static conditions, corresponding to geometric mean measured concentrations of 0.744, 0.325, 0.145, 0.0650, 0.0426 mg/L. Test concentrations were verified by GC-FID-analysis at the beginning and end of the exposure intervals. Immobilisation of Daphnia magna was observed after 48 h resulting in an EC50 (48 h) of 0.298 mg/L based on the geometric mean of measured concentrations.