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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-11-13 - 2020-01-09 (experimental phase biological an analytical part)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
OECD Guideline for Testing of Chemicals, No. 211: "Daphnia magna Reproduction Test", adopted October 02, 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals", 2nd Ed., February 08, 2019
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 20.3, 6.44, 2.05, 0.65 and 0.206 µg test item/L (each in 100 µL DMF/L), a solvent control (100 µL DMF/L) and a control
- Sampling method: The samples were taken from the biological phase of the study. Duplicate samples from the freshly prepared test media of all test concentrations, the control, and the solvent control were taken at the test start (day 0) and at a test medium renewal period in the second and third week (days 7 and 18). One sample from the freshly prepared DMF application solutions were taken at test start (week 1), day 7 (week 2), and day 14 (week 3). 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 media renewal periods, duplicate samples of the respective aged test media and controls were sampled at days 2, 9, and 21. One of these three stability samplings was over a test medium renewal period of 72 hours (weekend), two of 48 hours. All samples (except the DCM application solutions) were diluted by a factor of two with acetonitrile. Additional samples of the control and of the dilution solvent were taken at each sampling without any sample treatment, except on day 14 where only the application solutions were sampled.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.
Vehicle:
yes
Remarks:
dimethylformamide (DMF)
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 203 mg test item/L dimethylformamide was prepared once a week by dissolving
10.1 mg of the test item in 49.75 mL DMF (Day 0)
10.5 mg of the test item in 51.72 mL DMF (Day 7) and
11.2 mg of the test item in 55.17 mL DMF (Day 14).
These stock solutions were diluted in a series of dilutions with the solvent using the same spacing factor as used to define the nominal test concentrations to allow the addition of the same volume of solvent (100 µL/L) to each test medium. The test media were prepared just before introduction of the daphnids (= start of the test) and each test medium renewal. A defined volume of the stock solution and its dilutions was mixed into test water to obtain the test media of the desired test concentrations. The test item was solved into the test medium as homogeneously as possible.
- Controls: A solvent control ran in addition to the control.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide (DMF); The solvent additive was chosen based upon its solubility properties and its relative lack of toxicity to Daphnia.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): The solvent additive concentration was the same in all test media and the solvent control, and did not exceed 100 µL/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The appearance of the test item in the test media was determined in all freshly prepared and aged test media of all test concentrations. There were no remarkable observations. All test media were clear and colourless.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Strain/clone: clone 5
- Age at study initiation (mean and range, SD): 3.5 to 5.5 hours old
- Stage and instar at study initiation: neonates
- Method of breeding: The Daphnia were bred in the laboratories of ibacon under similar temperature and light conditions as 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 all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of the test facility.
- Source: in-house laboratory culture
- Feeding during test
- Food type: Green algae of the species Desmodesmus subspicatus, freshly grown in the laboratories of ibacon.
- Amount: The amount of food was based on the concentration of total organic carbon (TOC) in the algal food suspension. The approximate daily amounts of algal TOC/Daphnia/day were as follows: 0.1 mg on days 0-3, 0.15 mg on days 4-7 and 0.2 mg on days 8-20.
- Frequency: The test Daphnia were fed daily.

ACCLIMATION
- Acclimation period: 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:
21 d
Hardness:
214 to 231 mg CaCO3/L
Test temperature:
18.9 to 19.4 °C in the freshly prepared media;
18.9 to 19.5 °C in the aged media
pH:
7.8 to 8.0 in the freshly prepared media;
7.9 to 8.2 in the aged media
Dissolved oxygen:
8.9 to 9.1 mg/L in the freshly prepared media;
9.2 to 9.7 mg/L in the aged media
Nominal and measured concentrations:
nominal: 20.3, 6.44, 2.05, 0.65 and 0.206 µg test item/L (each in 100 µL DMF/L)
time-weighted average: 19.9, 5.43, 1.68, 0.526, 0.170 µg test item/L
Details on test conditions:
TEST SYSTEM
- Test vessel: beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass beakers of 100 mL volume with approximately 60 mL test medium.
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): The test media of all test concentrations and of the control were renewed on Days 2, 4, 7, 9, 11, 14, 16 and 18 of the exposure period (every Monday, Wednesday and Friday). At these dates the test Daphnia were carefully transferred from test vessels with aged test media into new test vessels with freshly prepared test media of the corresponding concentrations. The handling and the allocation of the treatments was randomised on every test media renewal.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (Elendt "M4")
- Alkalinity: 0.9 mmol/L
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 190 to 280 lux
- Test Environment: Controlled environment room

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations on mortality of adults and offspring occurrence were made daily. The number of offspring was counted at each water renewal and determined for each day. Dead offspring was counted if possible at each water renewal. Dead adults and offspring were removed at each water renewal day. The date of first eggs, of first brood and the number of offspring per adult in each treatment replicate was documented. Additionally, any signs of intoxication (e.g. pale adults, aborted eggs) were recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.15
- Range finding study
- Test concentrations: please see "Any other information on materials and methods incl. tables"
- Results used to determine the conditions for the definitive study: please see "Any other information on materials and methods incl. tables"
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
5.43 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: reproduction per introduced adult
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
5.43 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: reproduction per surviving adult
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
5.43 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.53 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: reproduction per introduced adult
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
9.62 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: reproduction per surviving adult
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 5.43 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
17 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: reproduction per introduced adult
Remarks on result:
other: 95 % Confidence intervals: 10.3 - 28.2 µg/L
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
26.4 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: reproduction per surviving adult
Remarks on result:
other: 95 % Confidence intervals: 12.8 - 54.4 µg/L
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 5.43 - < 19.9 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % Confidence intervals: not determinable
Details on results:
- Other biological observations: Apart from the reported mortality and the reduced reproduction, dead offspring were observed during the test. The dead offspring are given in Tables 4 to 10. Dead offspring was observed more frequently in the concentration of 5.43 mg test item/L. According to the Guideline, these dead offspring are not considered in the evaluation of the results of reproduction.
- Mortality of control: no
- Other adverse effects control: no
- Immobilisation of control: no
Reported statistics and error estimates:
The mortality of adult daphnia was tested with the Step-down Cochran-Armitage test on a concentration-response pattern.
Since a Cochran-Armitage trend test revealed a significant trend in mortality, the evaluation was based on reproduction (total number of live offspring) per introduced adult. The data for reproduction per surviving adult are also reported.
The difference between the control and the solvent control was tested with a two sample Mann-Whitney U-test procedure for reproduction per introduced adult and by student t-test for surviving adult.
The difference between the control and the solvent control was tested with a Fisher´s exact binominal test for immobility.
For all endpoints there were no differences between the control and the solvent control and both controls were pooled for all further analyses.
The data for reproduction per introduced adult were not normally distributed, variance homogeneity was given, but no linear trend was revealed and therefore the NOEC and the LOEC for reproduction per introduced adult was evaluated by the Multiple Sequential U-test after Bonferroni-Holm.
The data for reproduction per surviving adult were normally distributed, variance homogeneity was given, but no linear trend was revealed and therefore the NOEC and the LOEC for reproduction per introduced adult was evaluated by Dunnett´s t-test after.
The NOEC and the LOEC for mortality were determined directly from the raw data.
The EC50, EC20 and EC10 for reproduction per introduced adult were determined by logit analysis. The EC50, EC20 and EC10 for reproduction per surviving adult were determined by non-linear regression (3 parametric normal CDF). The LC50, LC20, LC10 for mortality could not be calculated by statistical analyses and were determined from the raw data.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.
Validity criteria fulfilled:
yes
Conclusions:
The study was conducted under GLP according to OECD Guideline 211 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 long-term toxicity of the test substance towards daphnia.
In a semi-static test neonates (< 24 h) of the freshwater invertebrate Daphnia magna was exposed to the test substance at the concentrations of nominal 20.3, 6.44, 2.05, 0.65 and 0.206 µg/L (each in 100 µL DMF/L), a solvent control (100 µL DMF/L) and a control for a period of 21 days under defined conditions. Using LC-MS/MS detection time weighted average concentrations of 19.9, 5.43, 1.68, 0.526, 0.170 µg test item/L were determined, correspondingly. Based on the obtained results, the 21 d NOEC was determined to be 5.43 µg test item/L for the observed effect parameters mortality of adults and number of offspring per introduced adult and per survived adult. Hence, the chronic toxicity NOECs determined for Daphnia magna were clearly above the water solubility of < 0.53 µg/L (Holzaht-Grimme, 2019).
Executive summary:

The study was performed under GLP according to OECD TG 211. Due to the poor water solubility (Holzaht-Grimme, 2019) and the hydrolysis (Holzaht-Grimme, 2020) of the test material the OECD Series on Testing and Assessment, No. 23 "Guidance Documenton Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals" has also been taken into account.

The purpose of this study was to evaluate the influence of the test item, Bis(2,6-diisopropylphenyl)carbodiimide, on mortality and reproduction of Daphnia magna during an exposure period of 21 days. Therefore, female Daphnia (3.5 to 5.5 hours old) taken from an in-house laboratory culture of the test facility, were exposed in a semi-static test to aqueous test media containing the test item at concentrations of 0.206, 0.65, 2.05, 6.44 and 20.3 µg test item/L (each in 100 µL DMF/L) ), a solvent control (100 µL DMF/L) and a control, corresponding to time weighted average concentrations of 0.170, 0.526, 1.68, 5.43 and 19.9 µg test item/L, for a period of 21 days under defined conditions. Following several independent pre-experiments to determine the solubility and stability of the test material in the test medium and the need to develop a reproducible analytical method, dimethylformamide (DMF) was used as solvent additive to achieve stable and reproducible concentrations in the test medium. The mortality of adults, the number of offspring per introduced adult and the number of offspring per surviving adult were compared with corresponding parameters in the controls.

The quantification of the test item Bis(2,6-diisopropylphenyl)carbodiimide in the test samples was performed using liquid chromatography with MS/MS detection.In the freshly prepared test media 100 % of the nominal test concentrations were found (average of all test concentrations). In the aged test media 70 % of nominal were found (average of all test concentrations).Correct dosing of the test item could be demonstrated.

Based on the obtained results, the following endpoints were determined refering to the time-weighted mean measured concentrations of the test material:

EC50(21d) mortality > 5.43 < 19.9 µg/L

EC10(21d) mortality > 5.43 µg/L

NOEC (21d) mortality = 5.43 µg/L

EC50(21d) reproduction per introduced adult = 17.0 µg/L

EC10(21d) reproduction per introduced adult = 3.53 µg/L

NOEC (21d) reproduction per introduced adult = 5.43 µg/L

EC50(21d) reproduction per surviving adult = 26.4 µg/L

EC10(21d) reproduction per surviving adult = 9.62 µg/L

NOEC (21d) reproduction per surviving adult = 5.43 µg/L

Description of key information

Long-term toxicity to aquatic invertebrates: NOEC (21d) = 5.43 mg/L (time-weighted mean measured) for Daphnia magna based on mortality, reproduction per introduced adult and reproduction per surviving adult (semi-static, OECD 211, GLP)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
5.43 µg/L

Additional information

The study was performed under GLP according to OECD TG 211. Due to the poor water solubility (Holzaht-Grimme, 2019) and the hydrolysis (Holzaht-Grimme, 2020) of the test material the OECD Series on Testing and Assessment, No. 23 "Guidance Documenton Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals" has also been taken into account.

The purpose of this study was to evaluate the influence of the test item, Bis(2,6-diisopropylphenyl)carbodiimide, on mortality and reproduction of Daphnia magna during an exposure period of 21 days. Therefore, female Daphnia (3.5 to 5.5 hours old) taken from an in-house laboratory culture of the test facility, were exposed in a semi-static test to aqueous test media containing the test item at concentrations of 0.206, 0.65, 2.05, 6.44 and 20.3 µg test item/L (each in 100 µL DMF/L) ), a solvent control (100 µL DMF/L) and a control, corresponding to time weighted average concentrations of 0.170, 0.526, 1.68, 5.43 and 19.9 µg test item/L, for a period of 21 days under defined conditions. Following several independent pre-experiments to determine the solubility and stability of the test material in the test medium and the need to develop a reproducible analytical method, dimethylformamide (DMF) was used as solvent additive to achieve stable and reproducible concentrations in the test medium. The mortality of adults, the number of offspring per introduced adult and the number of offspring per surviving adult were compared with corresponding parameters in the controls.

The quantification of the test item Bis(2,6-diisopropylphenyl)carbodiimide in the test samples was performed using liquid chromatography with MS/MS detection. In the freshly prepared test media 100 % of the nominal test concentrations were found (average of all test concentrations). In the aged test media 70 % of nominal were found (average of all test concentrations).Correct dosing of the test item could be demonstrated.

Based on the obtained results, the following endpoints were determined refering to the time-weighted mean measured concentrations of the test material:

EC50(21d) mortality > 5.43 < 19.9 µg/L

EC10(21d) mortality > 5.43 µg/L

NOEC (21d) mortality = 5.43 µg/L

EC50(21d) reproduction per introduced adult = 17.0 µg/L

EC10(21d) reproduction per introduced adult = 3.53 µg/L

NOEC (21d) reproduction per introduced adult = 5.43 µg/L

EC50(21d) reproduction per surviving adult = 26.4 µg/L

EC10(21d) reproduction per surviving adult = 9.62 µg/L

NOEC (21d) reproduction per surviving adult = 5.43 µg/L

Hence, the chronic toxicity NOECs determined for Daphnia magna were clearly above the water solubility of < 0.53 µg/L (Holzaht-Grimme, 2019).