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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:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
One sample of the stock solution and triplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test and at day 1 (after 24h of exposure).

For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, triplicate samples from the aged test media of all test concentrations and the control were collected at day 1 (after 24 hours of exposure) and at the end of the test by pouring together the contents of the test beakers of each treatment.

All samples were diluted by a factor of 2 with acetonitrile.

Additional samples of the control blank and the dilution solvent were taken at test start and test end (after 48 hours of exposure) without any sample treatment.

The concentrations of the test item Dichlone were analysed in one of the triplicate test media samples from all test concentrations, and in one of the triplicate control samples, from all sampling times.
Vehicle:
no
Details on test solutions:
Before test start and before the test item renewal (after 24 hours of exposure), a stock solution of 2 mg test item/L was prepared by suspending 4.02 mg test item in 2010 mL test water, each. The test item was mixed into the test water as homogeneously as possible with a mixing period of 24 hours. Non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 μm cellulose nitrate filter). Then, adequate volumes of the resulting solution were mixed into test water to obtain the desired test concentrations. The test media were prepared just before introduction of the Daphnia (= start of the test) and test medium renewal on Day 1 (after 24h of exposure).
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 1.5 to 19 hours old
Sex: Female
Origin: The Daphnia introduced in the test were taken from ibacon's in-house laboratory culture.

Breeding Conditions:
The Daphnia were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental Daphnia was performed in Elendt M4 medium. The test organisms were not first brood progeny. The Daphnia in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.

Reference Item:
For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Acclimatisation: Was not necessary, since the test was performed in the same medium as the culturing.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
1.068 mmol/L as CO3
Test temperature:
20 °C
pH:
8.2 to 8.5 in the freshly prepared media
7.7 to 8.1 in the aged test media
Dissolved oxygen:
8.2 to 9.2 mg/L in the freshly prepared media
7.9 to 8.5 mg/L in the aged test media
Salinity:
n/a
Nominal and measured concentrations:
Nominal concentrations of 500, 230, 100, 50 and 20 μg test item/L, prepared from a filtrate of nominal 2 mg test item/L, corresponding to initial mean measured concentrations of 330, 154, 53.0, 31.3 and 12.4 μg test item/L and time weighted mean measured concentrations of 281, 130, 46.7, 24.2 and 10.40 μg test item/L and a control.
Details on test conditions:
Test Environment: Controlled environment room
Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 480 to 640 lux (measured once during the test).
Recording: Test conditions were recorded with suitable instruments and documented in the raw data.
Introduction of Individuals: 20 Daphnia per control and test concentration, divided into 4 groups of 5 animals, each group in 60 mL test medium.
Exposure Time: 48 hours
Test Procedure: A semi-static test system with a test medium renewal on Day 1 (after 24 hours of exposure) was chosen to keep the concentrations of test item as constant as possible in the test media.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
39.6 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% conf.interval: 35.9-43.7
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
29.7 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% conf. interval: 25.2-33.1
Details on results:
At the start of the test and at the water renewal recoveries of the nominal test concentration varied between 52 and 68% (of the nominal concentrations of 50, 100, 230 and 500 μg test item/L). The nominal test concentrations of 20 μg test item/L and the nominal concentration of 50 μg test item/L at test start could not be measured at all as they were lower than the limit of quantification (LOQ = 9 μg test item/L). After 24 hours test duration from test start and from the water renewal, the recoveries of the nominal values varied between 31 and 54% (of the nominal concentrations of 50, 100, 230 and 500 μg test item/L). The nominal test concentration of 20 μg test item/L could not be measured at all as it was lower than LOQ, and for the nominal 50 and 100 μg test item/L concentration the recovery 24 h after the test start was lower than the LOQ, too.
Results with reference substance (positive control):
In the most recent test with the reference item potassium dichromate performed in January 2015 the EC50 after 24 hours was determined to be 1.85 mg test item/L indicating that the sensitivity of the Daphnia was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).

Description of key information

Very toxicy (acute effects) to aquatic invertebrates. 

Key value for chemical safety assessment

Additional information

The toxicity to aquatic invertebrates was assessed in a 48h study conducted according to OECD guideline 202 and under consideration of GLP. The nominal test concentrations were 500, 230, 100, 50, and 20 μg test item/L. The test concentrations were confiremd by specific analysis. The quantifications of the test item was performed using liquid chromatography with UV detection. At the start of the test and at the water renewal recoveries of the nominal test concentration varied between 52 and 68% (of the nominal concentrations of 50, 100, 230 and 500 μg test item/L). The nominal test concentrations of 20 μg test item/L and the nominal concentration of 50 μg test item/L at test start could not be measured at all as they were lower than the limit of quantification (LOQ = 9 μg test item/L). After 24 hours test duration from test start and from the water renewal, the recoveries of the nominal values varied between 31 and 54% (of the nominal concentrations of 50, 100, 230 and 500 μg test item/L). The nominal test concentration of 20 μg test item/L could not be measured at all as it was lower than LOQ, and for the nominal 50 and 100 μg test item/L concentration the recovery 24 h after the test start was lower than the LOQ, too. For those concentration groups which were below the LOQ the initial mean "measured" concentrations were calculated from arithmetic mean of initial concentrations of exposure levels > 50 μg/L and considering the recovery rate. Although this approach includes an additional element of uncertainity in an ECx approach it is regarded as suitable for this endpoint as it is supported by supporiting studies which are in the same range of toxicity. The EC50 was determined to be 39.6 μg/L..

This result is in line with several publications which are available for the compound's toxicity towards aquatic invertebrates. A study from the MInistry of the Environment, Japan revealed an EC50 of 17 μg/L. Moreover, Sanders determined an EC50 of 25 μg/L after an exposure of 48h.

In conclusion, despite the analytical difficulties in the key study the result seem to be reliable and in line with the results of the supporting studies. The compound is very toxic for aquatic invertebrates.