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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:
From 14 November 2015 to 08 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other 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
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23
Version / remarks:
(2000)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Not completely soluble in test medium at 100 mg/L
Analytical monitoring:
yes
Details on sampling:
- Concentrations sampled: all test concentrations (and the control), at t=0 and t=48h
- Volume: 2.0 mL from the approximate centre of the test vessels
- At the end of the exposure period, the replicates were pooled at each concentration before sampling
- Sample storage conditions before analysis: in a freezer
Vehicle:
no
Details on test solutions:
Preparation of test solutions started with a loading rate of 100 mg/L. A magnetic stirring period of two days was applied to ensure reaching maximum dissolution in test medium. The resulting dispersion was left to settle for one hour (combined limit/range-finding) or 1¾ hour (final test) after which the clear and colourless Water Soluble Fraction (WSF) was collected. Lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. The final test solutions were all clear and colourless.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
- Source: In-house laboratory culture with a known history.
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Characteristics: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg CaCO3/L
Test temperature:
19 - 20°C
pH:
7.8 - 7.9
Dissolved oxygen:
8.6 - 9.5 mg/L
Nominal and measured concentrations:
- 10, 18, 32, 56 and 100% of a WSF prepared at a loading rate of 100 mg/L
- 8.4, 16, 26, 45 and 82 mg/L (initial measured concentrations)
- During the exposure period the concentrations remained constant (98-100% of initially measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass
- Aeration: No aeration of the test solutions
- Feeding: No feeding
- No. of organisms per vessel: 5 per vessel containing 80 mL of test solution
- No. of vessels per concentration (replicates): 4
- Introduction of daphnids: Within 36 minutes after preparation of the test solutions
- No. of vessels per control (replicates): 4

TEST MEDIUM
Adjusted ISO medium. The following chemicals (analytical grade) were dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, The Netherlands):
CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCl: 4.2 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: Not applicable
- Photoperiod: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED
The 48h-EC50 value was calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations (initially measured) using the maximum likelihood estimation method (ToxRat Professional v 3.0.0).

COMBINED LIMIT/RANGE-FINDING TEST
- Test concentrations: 0.1, 1.0, 10 and 100% of a WSF prepared at a loading rate of 100 mg/L
-- At 100% of the WSF and control: Twenty daphnids (four replicates, 5 daphnids per vessel)
-- At 0.1, 1.0 and 10% of the WSF: Ten daphnids (two replicates, 5 daphnids per vessel)
- Results used to determine the conditions for the definitive study:
-- All daphnids exposed to the undiluted WSF were immobilised within 24 hours
-- No immobility was observed in any of the WSF dilutions during the test period
-- Samples taken from 10 and 100% of the WSF were analysed. The actual concentrations were 7.2 and 71 mg/L at the start of the test, respectively. The measured concentrations remained stable during the 48-hour test period (96-100% relative to initial). The expected EC50 was therefore between 7 and 70 mg/L (e.g. between 10 and 100% of a WSF).
Reference substance (positive control):
yes
Remarks:
(sensitivity check using potassium dichromate)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
19 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95%-confidence interval: 16-22 mg/L
Details on results:
- At t=24h, daphnids were immobilized at 82 (100%), 45 (45%) and 26 mg/L (10%)
- At t=48h, daphnids were immobilized at 82 (100%), 45 (100%), 26 (85%) and 16 mg/L (30%)
Results with reference substance (positive control):
In the latest sensitivity check, performed in the period 13 - 16 December 2015, an 48h-EC50 of 0.38 mg/L was determined. The sensitivity of the daphnia was within the range determined with the historical data collected at the testing laboratory.
Validity criteria fulfilled:
yes
Conclusions:
In a study performed in accordance with OECD 202 (2004) and according to GLP principles, an 48h-EC50 of 19 mg/L was determined for the substance, based on initially measured concentrations.
Executive summary:

In a study performed in accordance with OECD 202 (2004) and according to GLP principles, the acute toxicity of the substance to daphnids was investigated. As the substance was not completely soluble in test medium at 100 mg/L, preparation of test solutions started with a loading rate of 100 mg/L. A magnetic stirring period of two days was applied to ensure reaching maximum dissolution in test medium. The resulting dispersion was left to settle for 1¾ hour after which the clear and colorless Water Soluble Fraction (WSF) was collected. Lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. A final test was performed based on the results of a preceding combined limit/range-finding test. Twenty daphnids per group (four replicates, five daphnids per replicate) were exposed to an untreated control and to test groups representing 10, 18, 32, 56 and 100% of a WSF prepared at a loading rate of 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test, from all test concentrations. Analysis of the samples taken at the start showed measured concentrations of 8.4, 16, 26, 45 and 82 mg/L. During the exposure period the concentrations remained constant (98-100% of initially measured). All results were therefore expressed based on initially measured concentrations. The study met the acceptability criteria. Under the conditions of the present study, the 48h-EC50 was 19 mg/L based on initially measured concentrations (95% confidence interval between 16 and 22 mg/L).

Description of key information

In a study performed in accordance with OECD 202 (2004) and according to GLP principles, an 48h-EC50 of 19 mg/L was determined for the substance, based on initially measured concentrations.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
19 mg/L

Additional information

In a study performed in accordance with OECD 202 (2004) and according to GLP principles, the acute toxicity of the substance to daphnids was investigated. As the substance was not completely soluble in test medium at 100 mg/L, preparation of test solutions started with a loading rate of 100 mg/L. A magnetic stirring period of two days was applied to ensure reaching maximum dissolution in test medium. The resulting dispersion was left to settle for 1¾ hour after which the clear and colorless Water Soluble Fraction (WSF) was collected. Lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. A final test was performed based on the results of a preceding combined limit/range-finding test. Twenty daphnids per group (four replicates, five daphnids perreplicate) were exposed to an untreated control and to test groups representing 10, 18, 32, 56 and 100% of a WSF prepared at a loading rate of 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test, from all test concentrations. Analysis of the samples taken at the start showed measured concentrations of 8.4, 16, 26, 45 and 82 mg/L. During the exposure period the concentrations remained constant (98-100% of initially measured). All results were therefore expressed based on initially measured concentrations. The study met the acceptability criteria. Under the conditions of the present study, the 48h-EC50 was 19 mg/L based on initially measured concentrations (95% confidence interval between 16 and 22 mg/L).