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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 October 2017 to 20 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Principles of method if other than guideline:
An acute immobilization test to Daphnia magna was carried out to determine the effects of the test item after 24 and 48 hours of exposure under static conditions in a passive dosing system in a dose-response test at and below maximum water solubility under test conditions.
GLP compliance:
yes
Remarks:
Date of Inspection: Jun 07th - Jun 10th, 2016 & Jul 13th, 2016 / Date of signature: 03.01.2017
Specific details on test material used for the study:
- Relative density: 0,94
- Water solubility: 2,91 mg/L at 20°C and pH 6 in double distilled water (Slow-stirring Flask method)
- Vapour pressure: 0,19 Pa at 25°C
- Log Kow: 5,154 +/- 0,002 (25 +/- 1°C, mean pH 5,410) (Slow-stirring method)
- Storage conditions: dry, ambient temperature
- Stability under test conditions: stable
Analytical monitoring:
yes
Details on sampling:
At the start of the exposure (0 hours), samples of the fresh media were taken from additional replicates after completion of the equilibration phase (same incubation conditions as for the test vessels) and analyzed. After 24 hours of exposure samples of the test media were taken from additional replicates and analyzed. These replicates were prepared without daphnids and incubated under test conditions until sampling. At the end of the exposure (48 hours), samples of the old media were taken from the test vessels and analyzed.
Vehicle:
no
Details on test solutions:
Passive dosing system:
Due to the low water solubility of the test item, the test item was dosed via a passive dosing system. A carrier (silicone disc) was loaded with an appropriate amount of the test item. The carrier was inserted in an appropriate amount of dilution water. After an equilibration phase, a saturated test item solution with constant concentration as near as possible at the water solubility of the test item under test conditions was achieved by the continuous diffusion of the test item into the water body. Additionally, 4 lower concentration levels with a separation factor of 1.2 were tested below the maximum water solubility.

Preparation of the test item concentrations:
Silicone discs were used as carrier for the test item in the passive dosing system. Each silicone disc was loaded with a stock solution of concentrations of the test item. Then, each silicone disc was first immersed in purified water to remove any adhering test item, dabbed with a tissue and then inserted in each test vessel, which was filled with 20 mL of the dilution water. The test vessels were covered with watch glasses. To achieve a saturated test item solution by passive dosing, the silicone discs were incubated for 24 hours under test conditions before addition of the daphnids for equilibration of the saturated test item solution. The silicone discs also remained in the test vessels during the exposure phase. This ensured a constant concentration near and below the water solubility of the test item.

Preparation of silicone discs:
Stock solutions of 1000 - 833 - 694 - 578 - 482 mg test item/L (separation factor 1.2) were prepared. The weighed test item was first diluted in a definitive amount of methanol and then filled up to volume with water to reach a mixing ratio of methanol : water 1.5 : 1. Discs of 25 mm diameter were prepared with a stamping tool out of PDMS foil (thickness 1 mm). The PDMS discs were placed into glass vessels (flask with screw cap) containing 160 mL of stock solution and immersed under constant shaking (150 rpm) on an orbital shaker for 96 hours.
Silicone discs for use in the control were treated with 160 mL methanol – water (1.5 : 1) mixture without addition of test item and incubated under the same conditions as the test item discs.

Control:
A control silicone disc immersed in dilution water without test item under the same conditions as the test groups.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna STRAUS (Clone 5)
- Justification for species other than prescribed by test guideline: Daphnia magna is the preferred species in accordance with the test guidelines and is bred at the test facility.
- Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
- Age of parental stock (mean and range, SD): Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.
- Feeding during test: The daphnids will not be fed during the study.

ACCLIMATION
Acclimatization is not necessary, because the dilution water is equivalent to the culture medium (Elendt M4, according to OECD 202 Annex 3 (2004)).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
205 mg CaCO3/L
Test temperature:
20.2°C (so well comprised between 18 - 22 °C, constant within ± 1 °C)
pH:
Comprised between 7.38 to 8.01 (not exceed 1.5 units)
Dissolved oxygen:
The dissolved oxygen concentration between 6.62 to 9.95 mg/L in all test concentrations and the control (so > 3 mg/L).
Salinity:
Not applicable
Conductivity:
616 µS/cm
Nominal and measured concentrations:
Nominal concentrations: 1000 - 833 - 694 - 578 - 482 mg test item/L
Measured concentrations: The measured concentrations of the test item in fresh media were in the range of 0.286 to 0.807 mg/L at the start of the exposure (0 hours). Then, after 24 hours, these were in the range of 0.250 to 0.668 mg/L and between 0.348 to 0.570 mg/L after 48 hours (end of the exposure). Since the calculated geometric mean measured concentrations were 82 to 116% of the initially measured concentration, the results can be expressed as initial measured concentrations. See table 6.1.3/2 in "Any other information on results incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (4 (ID) x 7 (H) cm) with a nominal capacity of 50 mL, were used and covered with watch glasses during the exposure phase to reduce volatilization of the test item. A test volume of 20 mL was provided in each test vessel. A mesh made from inert glass material (glass fiber filter) was used as separation device to prevent the daphnids from direct contact to the silicone disc.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable
- Application: 20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. Then, one silicone disc was inserted into the base of each test vessel and incubated for 24 hours under test conditions for equilibration. Before addition of the daphnids, the silicone disc was covered by insertion of a glass fiber filter (MN GF-5, MACHEREY-NAGEL) to prevent the daphnids from coming into direct contact with the silicone discs while still allowing the test item from the silicone disc to diffuse across the entire aqueous phase. Thereafter, the daphnids were transferred to the test vessel by pipette together with a small amount of the dilution water that they were kept in.

TEST MEDIUM / WATER PARAMETERS
Elendt M4, according to OECD 202, Annex 3 (2004) is used.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobilization was determined in all groups after 24 and 48 hours. An animal was considered immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other adverse effects did not appear.

VEHICLE CONTROL PERFORMED: no

PRELIMINARY RANGE-FINDING TESTS (NON-GLP)
See "Attached background material ".
Reference substance (positive control):
yes
Remarks:
Potassium dichromate p.a. (SIGMA)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.682 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: 0.627 - 0.753 mg/L
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 0.807 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: maximum achievable test item concentration under test conditions
Duration:
48 h
Dose descriptor:
other: Highest tested concentration without observed effect
Effect conc.:
0.424 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
See Table 6.1.3/1 in "Any other information on results incl. tables".
Test solutions were clear throughout the exposure. No further additional observations were noticed.
Results with reference substance (positive control):
- Results with reference substance valid? yes
0-24h-EC50 = 1.89 mg/L (95% CL: 1.00-4.00 mg/L)
Reported statistics and error estimates:
None

Table 6.1.3/1: Immobilization rates after 24 and 48 hours of exposure in the definitive test

Initial measured concentration
[mg/L]

IMMOBILIZATION [%]

24 hours

48 hours

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

0.807

60

80

80

60

70

80

100

100

80

90

0.646

0

0

20

40

15

0

40

60

60

40

0.412

0

0

20

0

5

0

20

20

0

10

0.424

0

0

0

0

0

0

0

0

0

0

0.286

0

0

0

0

0

0

0

0

0

0

Control

0

0

0

0

0

0

0

0

0

0

Table 6.1.3/2: Measured conentrations of the test item during the definitive test

Sampling:

0 hours

24 hours

48 hours

 

Geometric mean measured test item concentration
[mg/L]

%

Nominal loading concentration
of the test item
[mg/L]

Test item

Meas. conc.

[mg/L]

Meas. conc.

[mg/L]

Meas. conc.

[mg/L]

1000

0.807

0.668

0.531

0.659

82

833

0.646

0.513

0.570

0.574

89

694

0.412

0.526

0.504

0.478

116

578

0.424

0.390

0.381

0.398

94

482

0.286

0.250

0.348

0.292

102

Control

< LOQ

< LOQ

< LOQ

< LOQ

Meas. conc.  = measured concentration of the test item, enrichment and dilution factor taken into account

% = percentage of the initial concentration of the test item

LOQ  = limit of quantification of the analytical method (0.05 mg test item/L)

Validity criteria fulfilled:
yes
Conclusions:
Based on the initial measured concentration of the test item, the 48 hour EC50 for Daphnia magna was 0.682 mg/L (95% confidence limits: 0.627 – 0.753 mg/L) in a passive dosing system near the maximum water solubility of the test item under test conditions.
Executive summary:

In the acute immobilization test with Daphnia magna, the effects of the test item were determined at the test facility according to OECD Guideline 202 with GLP statement from 2017-10-12 to 2018-04-20 with the definitive exposure phase from 2018-04-17 to 2018-04-19. Due to the low water solubility of the test item (2.91 mg/L in double distilled water) the test item was dose via a passive dosing system. The study was performed under static conditions in a dose-response test at and below maximum achievable water solubility. The maximum concentration achievable in the test solution preparation and test conditions was 0.807 mg/L. All test media were visually clear throughout the exposure period. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control. The concentrations of the test item were analytically verified via GC-MS in fresh media at the start of the exposure (0 hours), at 24 hours and the end of the exposure (48 hours) in each concentration level and the control. The measured concentrations of the test item in fresh media were in the range of 0.286 to 0.807 mg/L at the start of the exposure (0 hours), between 0.250 and 0.668 mg/L after 24 hours and between 0.348 and 0.570 mg/L after 48 hours (end of exposure). The geometric mean measured concentrations were calculated and were 82 to 116% of the initial concentrations, so within ± 20% of the initial measured test item concentration. Therefore, all effect values are given based on the initial measured concentrations of the test item.

Based on the results of this study, the 48 hour‑EC50for Daphnia magna was 0.682 mg/L(95% confidence limits: 0.627 – 0.753 mg/L).

All validity criteria were fulfilled.

 

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
From 2017-01-27 to 2017-01-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
iSafeRat® HA-QSAR toolbox

2. MODEL (incl. version number)
iSafeRat® holistic HA-QSAR v1.7

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
O=C1CCC=CCCCCCCCCCC1
The water solubility given as input was experimentally measured: 2.91 mg/L

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached QPRF

6. ADEQUACY OF THE RESULT
See attached QPRF
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
not applicable
Remarks:
(QSAR model)
Principles of method if other than guideline:
The acute toxicity to aquatic invertebrates was determined using a validated QSAR for the Mode of Action in question, based on validated data derived from 48-hour EC50 tests on daphnia, for which the concentrations of the test item had been determined by chemical analyses over the test period. The QSAR is a simple linear regression of sub-cooled liquid solubility versus 48-h EC50 for daphnia and is valid within the applicability domain defined in the QMRF.
GLP compliance:
no
Specific details on test material used for the study:
- Water solubility: 2.91 mg/L (from experimental study)
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
Not applicable
Test organisms (species):
Daphnia sp.
Details on test organisms:
Not applicable
Test type:
other: QSAR
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
none
Post exposure observation period:
Not applicable
Hardness:
The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.
Test temperature:
The temperatures varied from approximately 20 to 23 °C depending on the species used to construct the algorithm. This small difference is not expected to contribute to the variability of the EC50 values found in experimental data.
pH:
The QSAR is based on data from studies performed at acceptable pH between 6.0 - 9.0.
Dissolved oxygen:
The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%).
Salinity:
Not applicable.
Nominal and measured concentrations:
The QSAR is based on data from studies performed using measured concentrations or with acceptable stability.
Details on test conditions:
Not applicable
Reference substance (positive control):
no
Remarks:
QSAR model
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: 1.1-1.2 mg/L
Details on results:
The substance falls within the applicability domain of the model except for the descriptor domain. From a descriptor domain point of view, the substance falls within the intermediate domain where baseline toxicity cannot be experimentally measured accurately. Moreover the toxicity is likely greater than the water solubility limit. Based on a k-NN approach (with k = 3), the predicted acute toxicity (48h-EC50) to daphnid is considered as the geometric mean of the iSafeRat® prediction and the subcooled water solubility limit.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Not determinable

No additional information

Validity criteria fulfilled:
yes
Conclusions:
The substance falls within the applicability domain of the model except for the descriptor domain. From a descriptor domain point of view, the substance falls within the intermediate domain where baseline toxicity cannot be experimentally measured accurately. Moreover the toxicity is likely greater than the water solubility limit. Based on a k-NN approach (with k = 3), the predicted acute toxicity (48h-EC50) to daphnid was determined at 1.1 mg/L (95% CL: 1.1-1.2 mg/L), corresponding to the geometric mean of the iSafeRat® prediction and the subcooled water solubility limit.
Executive summary:

A QSAR prediction was performed to assess the acute toxicity of the test substance to daphnid. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modelling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 202. The criterion predicted was the EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

 

The immobility of the daphnids was determined using a validated QSAR for the Mode of Action in question, (MOA 1, non-polar narcosis). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour tests on daphnids, for which the concentrations of the test substance had been determined by chemical analyses over the test period.

The substance falls within the applicability domain of the model except for the descriptor domain. From a descriptor domain point of view, the substance falls within the intermediate domain where baseline toxicity cannot be experimentally measured accurately. Moreover the toxicity is likely greater than the water solubility limit. Based on a k-NN approach (with k = 3), the predicted acute toxicity (48h-EC50) to daphnid was determined at 1.1 mg/L (95% CL: 1.1-1.2 mg/L), corresponding to the geometric mean of the iSafeRat® prediction and the subcooled water solubility limit.

Description of key information

OECD Guideline 202, GLP, passive dosing system, key study, reliability 1:

48h-EC50 (Daphnia magna) = 0.682 mg/L (95% CL: 0.627 - 0.753 mg/L), based on the intial measured concentrations.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.682 mg/L

Additional information

To assess the acute toxicity of the registered substance to aquatic invertebrates, an experimental study and a QSAR prediction are available.

The experimental study (NOACK, 2018), assessed as the key study, is an acute immobilization test with Daphnia magna, performed according to OECD Guideline 202 with GLP statement, in a static passive dosing system (due to the low water solubility of the registered substance). Five test concentrations of the registered substance were used at and below maximum achievable water solubility. The maximum achievable concentration of the registered substance in the test solution preparation and test conditions was 0.807 mg/L. All test media were visually clear throughout the exposure period. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control. The concentrations of the test item were analytically verified via GC-MS in fresh media at the start of the exposure (0 hours), at 24 hours and the end of the exposure (48 hours) in each concentration level and the control. The measured concentrations of the test item in fresh media were in the range of 0.286 to 0.807 mg/L at the start of the exposure (0 hours), between 0.250 and 0.668 mg/L after 24 hours and between 0.348 and 0.570 mg/L after 48 hours (end of exposure). The geometric mean measured concentrations were calculated and were 82 to 116% of the initial concentrations, so within ± 20% of the initial measured test item concentration. Therefore, all effect values are given based on the initial measured concentrations of the test item. Based on the results of this study, the 48 hour‑EC50 for Daphnia magna was 0.682 mg/L(95% confidence limits: 0.627 – 0.753 mg/L).

The QSAR prediction (KREATiS, 2017; Model:iSafeRat® holistic HA-QSAR v1.7), assessed as a supporting data, was performed to assess the acute toxicity of the registered substance to daphnids. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modelling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 202. The immobility of the daphnids was determined using a validated QSAR for the Mode of Action in question, (MOA 1, non-polar narcosis). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour tests on daphnids, for which the concentrations of the test substance had been determined by chemical analyses over the test period.

The substance falls within the applicability domain of the model except for the descriptor domain. From a descriptor domain point of view, the substance falls within the intermediate domain where baseline toxicity cannot be experimentally measured accurately. Moreover the toxicity may even be greater than the water solubility limit as equilibrium between the test organism and the test solution may not be reached within the experimental time frame. Based on a k-NN approach (with k = 3), the predicted acute toxicity (48h-EC50) to daphnid was determined at 1.1 mg/L (95% CL: 1.1-1.2 mg/L), corresponding to the geometric mean of the iSafeRat® prediction for the non-polar narcotic regression line (i.e. the value expected if equilibrium was reached between the test solution and the test organism) and the subcooled water solubility limit of the test substance. This QSAR result supports the key study performed on the same substance with an EC50 value in the same order of magnitude, considering the biological and experimental variabilities (factor <2) . Therefore, this QSAR is considered relevant and sufficient to assess, alone, the toxicity of the registered substance to aquatic algae, without the use of an experimental study.

In conclusion, according to the key experimental study (NOACK, 2018), the 48h-EC50 value on Daphnia magna is 0.682 mg/L (95% CL: 0.627 - 0.753 mg/L).