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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:
2002-01-02 - 2002-01-04 (experimental phase biological part)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals (1984) No 202 "Daphnia sp, Acute Immobilisation Test and Reproduction Test"
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
Method C.2 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.10, 0.056, 0.032, 0.018, 0.010, 0.0056, 0.0032, 0.0018, 0.0010 and 0 mg/L (nominal)
- Sampling method: The concentration and stability of the test material in centrifuged and uncentrifuged test samples were verified by chemical analysis at 0 and 48 hours. Water samples were taken from the solvent control (replicates R1 – R2 pooled) and all the test groups (replicates R1 – R2 pooled) at 0 and 48 hours for quantitative analysis. Two samples of the solvent control (replicates R1 – R2 pooled) and all the test groups (replicates R1 – R2 pooled) were taken at each occasion. One sample was analysed uncentrifuged and one sample after centrifugation (40000 G for approximately 30 minutes).
- Sample storage conditions before analysis: Further samples (in duplicate) were taken and stored frozen (approximately -20 °C) for further analysis if necessary.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:A defined amount of test material (100 mg) was dissolved in dimethylformamide and the volume adjusted to 100 mL to give a 100 mg/100 mL solvent stock solution from which serial dilutions were made to give further solvent stock solutions having the same spacing factor than used to define the nominal test concentration. An aliquot (50 μL) of each of these solvent stock solutions was separately dispersed in reconstituted water
and the volume adjusted to 500 mL to give the nominal test concentrations of 0.0010, 0.0018, 0.0032, 0.0056, 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L. Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
- Controls: Control group daphnids without test material and solvent control group
- Chemical name of vehicle: dimethylformamide (100 µL/L)
- Evidence of undissolved material: None, the preparations were all observed to be clear, colourless solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna Straus
- Stage and instar at study initiation: 1st instar
- Source: in-house laboratory cultures
- Method of breeding: Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at 21 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a suspension of algae (Chlorella sp.). Culture conditions ensured that reproduction was by parthenogenesis.
- Feeding during test : The daphnids received no food during exposure.

ACCLIMATION
- Acclimation period: Not necessary, as the reconstituted water used to maintain the stock animals was the same as that used for both the range-finding and definitive tests.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
No post exposure observation period is described.
Hardness:
250 mg/L CaCO3
Test temperature:
21 °C (temperature controlled room)
pH:
start: 7.9
end: 8.0
Dissolved oxygen:
test start: 8.4 mg/L (Air Saturation Value, ASV: 94 %)
test end: 8.1 - 8.2 mg/L (Air Saturation Value, ASV: 91 - 92 %)
Salinity:
No details available
Nominal and measured concentrations:
nominal: 0.0010, 0.0018, 0.0032, 0.0056, 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L
time-weighted mean measured: 0.000490, 0.000766, 0.00171, 0.00326, 0.00628, 0.00966, 0.0208, 0.0328 and 0.0597 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass jars
- Type (delete if not applicable): The test vessels were covered to reduce evaporation.
- Volume of solution: 250 mL
- Aeration: The test vessels were not aerated.
- Renewal rate of test solution (frequency/flow rate): The test solutions were not renewed during the exposure period.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water
Stock solutions:
a) CaCl2.2H2O: 11.76 g/L
b) MgSO4.7H2O: 4.93 g/L
c) NaHCO3: 2.59 g/L
d) KCl: 0.23 g/L
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 μS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
- Culture medium different from test medium: The reconstituted water used for both the range-finding and definitive tests was the same as that
used to maintain the stock animals.

OTHER TEST CONDITIONS
- Adjustment of pH: not specified
- Photoperiod: photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 0.00010, 0.0010, 0.010 and 0.10 mg/L
- Results used to determine the conditions for the definitive study: 0 % immobilisation at nominal 0.00010 and 0.0010 mg/L, 30 % immobilisation at 0.010 mg/L and 100 % immobilisation at 0.10 mg/L
Reference substance (positive control):
not required
Remarks:
according to OECD 202
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.007 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % CL: 0.0057 - 0.0079 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.003 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.004 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % CL: 0.0033 - 0.0047 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.002 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Mortality of control: no
- Immobilisation of control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No, the test material preparations were all observed to be clear, colourless solutions throughout the duration of the study.
Reported statistics and error estimates:
The EC50 value and associated confidence limits at 48 hours were calculated by the maximumlikelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown.
Validity criteria fulfilled:
yes
Conclusions:
The study was conducted under GLP according to OECD Guideline 202 and EU Method C.2 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards daphnia.
In a static test neonates (< 24 h) of the freshwater invertebrate Daphnia magna was exposed to the test substance at the concentrations of nominal 0.0010, 0.0018, 0.0032, 0.0056, 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L for a period of 48 hours under defined conditions. Based on the obtained results, the 48 h EC50 for immobilisation was determined to be nominal 0.0067 mg/L, corresponding to 0.0040 mg/L based on time weighted mean measured concentrations. Hence, no acute toxicity against aquatic invertebrates is recorded at levels up to the water solubility of < 0.53 µg/L (Holzaht-Grimme, 2019).
Executive summary:

The acute toxicity of bis-(2,6 -diisopropylphenyl)carbodiimide to aquatic invertebrates was tested according to OECD guideline 202 and EU Method C.2 in a static freshwater test with Daphnia magna as test organisms, which is a freshwater invertebrate representative of a wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems. The study was conducted under certificated GLP compliance. In-house laboratory cultures of the test animals were used, whereby young daphnids, less than 24 h old were used for testing. These daphnids were not individually identified and received no food. The test concentration to be used in the definitive test was determined by a preliminary range-finding test. Based on the range-finding study, the definitive test was conducted with test concentrations of nominal 0.0010, 0.0018, 0.0032, 0.0056, 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L (each in 100 µL DMF/L), a solvent control (100 µL DMF/L) and a control, corresponding to time-weighted mean measured test concentrations of 0.000490, 0.000766, 0.00171, 0.00326, 0.00628, 0.00966, 0.0208, 0.0328 and 0.0597 mg/L. For both purposes the test solutions were prepared using a preliminary solution in dimethylformamide to achieve stable and reproducible concentrations of the test material in the test medium. Duplicate test vessels were used for each test and control group. No aeration was performed. No evidence of undissolved substance was observed, since all preparations were clear, colourless solutions. This solutions were not renewed during the exposure period. The vessels were covered to reduce evaporation and maintained at 21.0 °C in a temperature controlled room with a photoperiod of 16 h light and 8 h darkness with 20 min dawn and dusk transition periods for a test duration of 48 hours. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure.High Performance Liquid Chromatography (HPLC) was the analytical method of choice.

Based on the obtained results, the 48 h EC50 value was determined to be nominal 0.0067 mg/L with 95 % Confidence limits of 0.0057 - 0.0079 mg/L, corresponding to 0.0040 mg/L with 95 % Confidence intervalls of 0.0033 - 0.0047 mg/L based on time weighted mean measured concentrations. The corresponding 48 h No Observed Effect Concentration (NOEC) is nominal 0.0032 mg/L, corresponding to 0.0017 mg/L based on time weighted mean measured concentrations.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2004-04-20 - 2004-04-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline Study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Qualifier:
according to guideline
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
GLP compliance:
no
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No surrogate or analogue material.
Analytical monitoring:
no
Details on sampling:
No details available.
Vehicle:
no
Details on test solutions:
Since the test item is poorly water soluble, water-accomodated fractions (WAF) of the concentrations to be tested were prepared. The nominal load was shaked with the appropriate amount of dilution water for 24 h, followed by membrane filtration (pore diameter: 0.45 µm).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain: Stamm Berlin
- Source: Umweltbundesamt Berlin
- Age at study initiation (mean and range, SD): 0 - 24 hours
- Method of breeding: 5 adults per L, transfer into fresh medium twice a week, temperature: 20 +/- 2 °C, lighting cycle: 16 h/8 h (light/dark)
- Food type: green algae
Test type:
not specified
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
No post exposure observation period is described.
Hardness:
No details available.
Test temperature:
20 +/- 1 °C (deviation within one test max. +/- 1 °C)
pH:
No details available.
Dissolved oxygen:
No details available.
Salinity:
No details available.
Nominal and measured concentrations:
0 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL-beakers (DIN, HF)
- No. of vessels per concentration: Each concentration was tested fourfold.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic dilution water; following DIN 38412 part 40 (EN ISO 6341)
- Ca2+: 28.2 mg/L
- Mg2+: 4.9 mg/L
- K+: 3.0 mg/L
- Na+: 11.3 mg/L

TEST CONCENTRATIONS
Range finding study
- Test concentrations: 0, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: No toxicity observed.
Reference substance (positive control):
not required
Remarks:
according to OECD 202
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
No further details are available.
Results with reference substance (positive control):
No reference substance required.
Reported statistics and error estimates:
No statistics and error estimates reported.
Validity criteria fulfilled:
yes
Conclusions:
The study is regarded as a valid guideline study. No toxic effects were observed at the limit of water solubility under exposure conditions.
Executive summary:

The acute toxicity of bis-(2,6 -diisopropylphenyl)carbodiimide to aquatic invertebrates was tested according to OECD guideline 202, EU Method C.2 and EN ISO 6341 in a freshwater test with Daphnia magna as test organism. Due to the poor water solubility of the test item, the test was performed using water-accomodated fractions (WAF) after direct weighing of the nominal concentration of 1000 mg/L, which was concluded after the preliminary study. The WAF was prepared by shaking the nominal load with the appropriate amount of dilution water for 24 hours, followed by membrane filtration (pore diameter: 0.45 µm). Synthetic dilution water was used, prepared according to DIN 38412 part 40 (EN ISO 6341). Juvenile animals (aged between 0 and 24 hours) were used for the study. 5 animals were put in 20 mL solution. Each concentration was tested fourfold. The EC50 was found to be greater than the tested concentration of 1000 mg/L. No analytical monitoring was performed.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2009-08-13 - 2009-10-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. QA statement)
Remarks:
Ministery of environment and conservation, agriculture and consumerism of North-Rhine Westphalia
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No surrogate or analogue material was used.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal 1.0 mg/L and control
- Sampling method: Samples of the control and the test concentrations were taken for GC analysis from the freshley prepared (0 h) and aged test media (24 h) at test start and test medium renewal after 24 hours of test duration.
- Sample storage conditions before analysis: Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct weighings were prepared to produce the test concentration. To achieve this 1.1 mg of the test item were added to 1 L of dilution water on two consecutive days and treated for 1 h in an ultrasonic bath. Afterwards stirring was performed for 24 h and finally undissolved particles of the test item were removed by filtration using an aseptic filter with a pore size of 0.2 µm.
- Controls: dilution water only
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Name : Daphnia magna STRAUS, parthenogenetic females
- Source : Strain of Bundesgesundheitsamt Berlin
- Maintenance and Acclimatisation : A population of parthenogenetic females of synchronized age structure has been maintained for more than 15 years in the test facility under constant temperature conditions (20 +/- 1 °C) at a 16 : 8 hour light-dark photoperiod (illumination: < 1000 lux). The culture water (so-called 'M4 medium') was partly renewed once a week. The Daphnia were exclusively fed with unicellular green algae (Desmodesmus subspicatus) 'ad libitum'. Mortalities of parent Daphnia during the culture period were recorded daily in a semi-quantitative way. The neonates were separated from their parent Daphnia by filtration prior to the acute test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
No post exposure observation period is described.
Hardness:
14.7°dH (= 262.4 mg/L CaCO3).
Test temperature:
Temperature range: 18 - 22°C
With a temperature fluctuation of maximum +/- 1°C in each individual vessel.
pH:
Control: 7.7 - 8.0
Test substance: 7.8 - 7.9
Dissolved oxygen:
Control: 8.0 - 8.6 mg/L (91 - 96 %)
Test substance: 7.6 - 8.6 mg/L (85 - 95 %)
Salinity:
No details available.
Nominal and measured concentrations:
nominal: 0 and 1 mg/L
measured: < 0.275 (= LOQ)
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL glass beakers
- Type: closed
- Aeration: none
- Renewal rate of test solution: daily renewal, semi-static system
- No. of organisms per vessel: 10 neonates
- No. of vessels per concentration (replicates): two
- No. of vessels per control (replicates): two

OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8 h dark

TEST CONCENTRATIONS
The limit test at the limit of water solubility under test conditions was performed due to the low water solubility of the test item. It should be demonstrated that the EC50 value is greater than this concentration (1 mg/L).
Reference substance (positive control):
not required
Remarks:
according to OECD 202
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
< 0.275 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
< 0.275 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
No toxic effects against Daphnia were observed at a nominal limit test concentration of 1 mg/L which after aseptic filtration resulted in effective concentrations of < 0.275 μg/L (the limit of detection).
Results with reference substance (positive control):
No reference substance required.
Reported statistics and error estimates:
No statistical analysis required.
Validity criteria fulfilled:
yes
Conclusions:
The study is regarded as a valid guideline study with certificated GLP compliance. No toxic effects against aquatic invertebrates were observed at the limit of water solubility under exposure conditions.
Executive summary:

The acute toxicity of bis-(2,6 -diisopropylphenyl)carbodiimide to aquatic invertebrates was tested according to OECD guideline 202 and EU Method C.2 in a freshwater test with Daphnia magna STRAUS as test organisms under semi-static conditions with daily renewal of the test solutions. The study was conducted under certificated GLP compliance. Because of the low solubility of the test item, a limit test at the limit of water solubility under test conditions was performed in order to demonstrate that the EC50 is greater than this concentration. Several range-finding tests were conducted, which provided the concentration to be used in the main test. The final test concentration was chosen as 1 mg/L, whereby direct weighing was done for preparation. For the test item concentration as well as for the control 2 replicates were prepared. 10 neonates were placed in 20 mL test medium. A photoperiod of 16 h light and 8 h darkness was chosen during the exposure period. The quantification of the test item was performed by GC analysis and MS detection. No toxic effects against Daphnia were observed at a nominal limit test concentration of 1 mg/L which after aseptic filtration resulted in effective concentrations of < 0.275 μg/L (the limit of detection). Thus no toxic effects were observed under the established test conditions.

Description of key information

1) Key, Short-term toxicity to aquatic invertebrates: EC50 (48h) = 0.00 40 mg/L (time-weighted mean measured) for Daphnia magna based on mobility (static, OECD 203, GLP)

2) Supporting, Short-term toxicity to aquatic invertebrates: EC50 (48h) > 1 mg/L (nominal) for Daphnia magna based on mobility (semi-static, OECD 203, GLP)

3) Supporting, Short-term toxicity to aquatic invertebrates: EC50(48h) > 1000 mg/L (nominal) for Daphnia magna based on mobility (static, OECD 203, non-GLP)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.004 mg/L

Additional information

Regarding the acute toxicity of bis(2,6-diisopropylphenyl)carbodiimide to aquatic invertebrates a key study as well as two supporting studies are available. The Key study as well as the first supporting study are evaluated with Klimisch score 1 (reliable without restrictions), since these are well-documented guideline studies (according to OECD 202 and EU Method C.2) with Good Laboratory Practice (GLP) Compliance.

In the key study (Wetton and Mullee, 2002) the acute toxicity of bis-(2,6 -diisopropylphenyl)carbodiimide to aquatic invertebrates was tested according to OECD guideline 202 and EU Method C.2 in a static freshwater test withDaphnia magnaas test organisms, which is a freshwater invertebrate representative of a wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems. The study was conducted under certificated GLP compliance. In-house laboratory cultures of the test animals were used, whereby young daphnids, less than 24 h old were used for testing. These daphnids were not individually identified and received no food. The test concentration to be used in the definitive test was determined by a preliminary range-finding test. Based on the range-finding study, the definitive test was conducted with test concentrations of nominal 0.0010, 0.0018, 0.0032, 0.0056, 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L (each in 100 µL DMF/L), a solvent control (100 µL DMF/L) and a control, corresponding to time-weighted mean measured test concentrations of 0.000490, 0.000766, 0.00171, 0.00326, 0.00628, 0.00966, 0.0208, 0.0328 and 0.0597 mg/L. Due to the poor water solubility of the test item the test solutions for both purposes were prepared using a preliminary solution in dimethylformamide (DMF) to achieve stable and reproducible concentrations of the test material in the test medium. Duplicate test vessels were used for each test and control group. No aeration was performed. No evidence of undissolved substance was observed, since all preparations were clear, colourless solutions. This solutions were not renewed during the exposure period. The vessels were covered to reduce evaporation and maintained at 21.0 °C in a temperature controlled room with a photoperiod of 16 h light and 8 h darkness with 20 min dawn and dusk transition periods for a test duration of 48 hours. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure.High Performance Liquid Chromatography (HPLC) was the analytical method of choice.

Based on the obtained results, the 48 h EC50value was determined to be nominal 0.0067 mg/L with 95 % Confidence limits of 0.0057 - 0.0079 mg/L, corresponding to 0.0040 mg/L with 95 % Confidence intervalls of 0.0033 - 0.0047 mg/L based on time weighted mean measured concentrations. The corresponding 48 h No Observed Effect Concentration (NOEC) is nominal 0.0032 mg/L, corresponding to 0.0017 mg/L based on time weighted mean measured concentrations.

Hence, no acute toxicity against aquatic invertebrates is recorded at levels up to the water solubility of < 0.53 µg/L (Holzaht-Grimme, 2019).

In the first supporting study Daphnia magna was used as test organisms under semi-static conditions with daily renewal of the test solutions. Because of the low solubility of the test item, a limit test at the limit of water solubility under test conditions was performed in order to demonstrate that the EC50 is greater than this concentration. Several range-finding tests were conducted, which provided the concentration to be used in the main test. The final test concentration was chosen as 1 mg/L, whereby direct weighing was done for preparation. For the test item concentration as well as for the control 2 replicates were prepared. 10 neonates were placed in 20 mL test medium. A photoperiod of 16 h light and 8 h darkness was chosen during the exposure period. The quantification of the test item was performed by GC analysis and MS detection. No toxic effects against Daphnia were observed at a nominal limit test concentration of 1 mg/L which after aseptic filtration resulted in effective concentrations of < 0.275 μg/L (the limit of detection). Accordingly, the 48 h EC50 was determined to be nominal > 1 mg/L, corresponding to < 0.275 µg/L effective concentration. Thus no toxic effects were observed at the water solubility under the established test conditions.

The second supporting study was not conducted under GLP compliance, but also according to OECD Guideline 202, EU Method C.2 and EN ISO 6341 in a freshwater test with Daphnia magna as test organism (Rudolf, 2004). Due to the poor water solubility of the test item, the test was performed using Water Accommodated Fractions (WAF) of the concentration (1000 mg/L), which was concluded after the preliminary study. The WAF was prepared by shaking the nominal load with the appropriate amount of dilution water for 24 hours, followed by membrane filtration (pore diameter: 0.45 µm). Synthetic dilution water was used, prepared according to DIN 38412 part 40 (EN ISO 6341). Juvenile animals (aged between 0 and 24 hours) were used for the study. 5 animals were put in 20 mL solution. Each concentration was tested fourfold. No analytical monitoring was performed. The EC50 was found to be greater than the tested concentration of nominal 1000 mg/L. Thus no toxic effects were observed at the limit of water solubility under exposure conditions.