Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 January 2019 to 16 December 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Chemical name (IUPAC, synonym or trade name: 2-dimethylamino-2-(4-methyl-benzyl)-1-(4-morpholin-4-yl-phenyl)-butan-1-one
CAS number: 119344-86-4
EC number 438-340-0
Identification: Omnirad 379
Appearance: Slightly yellow powder
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and both control groups.

Sampling:
- Frequency: One day before the start of exposure to check the functioning of the system (samples were taken from the solvent control and the test concentrations only). At the start, after 7, 14 and 21 days of exposure.
- Volume: 1.8 mL
- Storage: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.

Reserve samples of 1.8 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test Samples: The samples were analyzed on the day of sampling. To this end, the samples were diluted in a 9:1 (v:v) ratio with 1% formic acid in acetonitrile and subsequently analyzed. If necessary, the samples were further diluted with 0.1% formic acid in 10/90 (v/v) acetonitrile/test-medium to obtain concentrations within the calibration range. Only the results that were within the calibrated range were reported. All solutions containing the test item were protected from light.

Stock and Spiking Solutions: Analytical stock solutions of the test item were prepared in N,N-Dimethylformamide at concentrations of 1000 and 4000 mg/L. Spiking solutions in N,N-Dimethylformamide were prepared from a stock solution or dilutions of a stock solution.

Calibration Solutions:
Range-finding test: Three working solutions in the concentration range of 4.0 - 40 mg/L were prepared in N,N-Dimethylformamide from two stock solutions. Working solutions were diluted with 0.1% formic acid in 10/90 (v/v) acetonitrile/test-medium to obtain calibration solutions in the concentration range of 0.004 – 1.00 mg/L.

Final test 1: Five working solutions in the concentration range of 0.4 - 100 mg/L were prepared in N,N-Dimethylformamide from two stock solutions. Working solutions were 100-fold diluted with 0.1% formic acid in 10/90 (v/v) acetonitrile/test-medium to obtain calibration solutions in the concentration range of 0.004 – 1.00 mg/L.

Final test 2: Six working solutions in the concentration range of 0.2 - 100 mg/L were prepared in N,N-Dimethylformamide from two stock solutions. Working solutions were 100-fold diluted with 0.1% formic acid in 10/90 (v/v) acetonitrile/test-medium to obtain calibration solutions in the concentration range of 0.002 – 1.00 mg/L.

Quality Control (QC) Samples: For each QC sample, 1.8 mL blank medium was spiked with test item at a target concentration of 3, 5, 10, 1000 or 10000 µg/L. The QC samples were treated similarly as the test samples. Blank QC samples consisting of blank medium were treated similar to the QC samples spiked with test item and test samples.

Calibration Curves
Calibration curves were constructed using five or six concentration levels. For each level, duplicate responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration weighting factor. If necessary, two data points were excluded from the curve constructed during the final test 1 and 2 since the back calculated accuracy was > 15% from the nominal concentration. The coefficient of correlation (r) was
> 0.99 for each curve.

Samples
QC Samples
The chromatograms of the blank QC samples showed no peak at the retention time of the test item, except for analysis on 17 Jan 2019. Also, for the same day of preparation mean accuracies for the QC samples at concentration level 0.005 mg/L and 0.01 mg/L were 125% and 127% respectively. The higher recoveries obtained were considered to have no influence on the analyzed concentrations in samples because comparable values were obtained for next day of analysis. For other days of analysis recoveries for QC samples were in the specified range 70-110%.

During the final test 1 on the 21 Mar 2019 QC at higher level was just below the criteria 70-110%, at 69%. Results of the analyzed samples were in good agreement with previous sampling days, therefore accuracy for high QC was considered not to have influence on the validity of the results. The main recovery obtained for the other days of the analysis, on all concentration levels, were within the specified criterion range.

For the final test 2, accuracy of the QC samples at concentration level of 0.003 mg/L was assessed. Accuracy obtained on 07 May 2019, was too low due to the preparation error; therefore, QC samples were prepared again on the same day. The main recovery obtained for the new QC samples at concentration level of 0.003 mg/L was within the specified rage, demonstrating that the analytical method is adequate for the determination of the test item at low concentration levels. The main recovery obtained for the other days of the analysis, on all concentration levels, were within the specified criterion range.
Vehicle:
yes
Details on test solutions:
Preparation of Test Solutions
The test item batch was a slightly yellow powder with a purity of 96.416%. Weighing of test item and preparation of stock solutions was performed under dimmed light.
Stock solutions were kept in the dark. No correction was made for the purity/composition of the test item.

Range-Finding Test
Stock solutions of 5,000 µg/mL were prepared in dimethylformamide daily. Lower stock solutions, i.e. 50 and 500 µg/mL were prepared by diluting the highest stock with DMF. All stock solutions were clear. Any residual volumes were discarded.

Final Test 1
Stock solutions of 5,000 µg/mL were prepared in DMF three times a week. Lower stock solutions, i.e. 500, 900, 1,600 and 2,800 µg/mL were prepared by diluting the highest stock
with DMF. All stock solutions were clear. Any residual volumes were discarded.

Final Test 2
Stock solutions of 4,000 µg/mL were prepared in DMF three times a week. Lower stock solutions, i.e. 38, 120, 390 and 1,250 µg/mL were prepared by diluting the highest stock with
DMF. All stock solutions were clear. Any residual volumes were discarded.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Source: In-house laboratory culture with a known history.
- Reason for selection: This system has been selected as an internationally accepted invertebrate species.
- 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: To initiate the test, young daphnids < 24 hours old were selected, from parental daphnids greater than two weeks old.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
Between 179 and 196 mg/L expressed as CaCO3
Test temperature:
Between 19 and 22°C
pH:
Between 7.1 and 8.3
Dissolved oxygen:
Between 1.3 and 8.8 mg/L
Nominal and measured concentrations:
- Test Item:
Final test 1: 50, 90, 160, 280 and 500 µg/L;
Final test 2: 3.8, 12, 39, 125 and 400 µg/L

Controls:
Blank-control: Test medium without test item or other additives;
Solvent-control: Test medium without test item but with 0.1 mL DMF/L
Details on test conditions:
Test Procedure and Conditions:-
- Test duration: 21 days
- Test type: Flow-through, with continuous renewal of test media
- Test vessels: Stainless steel vessel with a medium volume of 1.5 L containing four mesh containers. Covered with a Perspex plate.
- Medium: M7
- Experimental design: At the start of the experiment (nominal day 0) twenty neonate daphnids, less than one day old, per group were divided over four stainless steel mesh containers (five daphnids per mesh).
- Light: 16 h photoperiod daily
- Feeding: Daily, a quantity of a Chlorella sp. suspension is added as feed for the daphnids providing a ration of ca. 0.4 mg C/daphnid/day.

Measurements and Recordings:-
Parental daphnids:
-Condition: Every day, the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed.
-Presence of eggs in the brood pouch: Every day.
-Body length: At the end of the test.

Offspring:
- Appearance first brood When observed.
- Newborn daphnids Every day, the number of newborn young was counted and the condition of the young recorded. Thereafter the young were removed.
- Presence of unhatched eggs When observed.
- Incidence of immobility When observed.

Test medium:
- Temperature, oxygen and pH: Once a week in all test solutions. Additionally, the temperature was continuously monitored in a temperature-control vessel.
- Hardness: Once a week in both control groups and the highest test concentration.
- Light: At the start and the end of the test.

Breeding:-
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing them individually in 50 ml M7-medium (range-finding test and final test 1) or by placing about 250 of them into 5 litres of medium in an all-glass culture vessel (final test 2).
- Maximum age of the cultures: 4 weeks
- Monitoring of the individual cultures: Three times a week the young are counted and the parental daphnids are transferred to new media.
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Feeding: Daily, a suspension of fresh water algae.
- Validity of the cultures: Historical data on the reproductive capacity are based on the numbers of living young counted three times a week in the individual cultures and tested to meet the validity criteria for survival and reproduction.
- Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).
- Composition of medium M7: Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis:
Macro salts:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

The following salts and vitamins were added to freshly prepared ISO medium to reach the following
concentrations:
Salts: H3BO3 0.73 mg/L
FeSO4.7H2O 0.25 mg/L
MnCl2.4H2O 0.092 mg/L
LiCl 0.078 mg/L
RbCl 0.018 mg/L
SrCl2.6H2O 0.039 mg/L
Na2MoO4.2H2O 0.016 mg/L
NaBr 0.0041 mg/L
CuCl2.2H2O 0.0043 mg/L
ZnCl2 0.013 mg/L
CoCl2.6H2O 0.010 mg/L
KI 0.0033 mg/L
Na2SeO3 0.0022 mg/L
NH4VO3 0.00059 mg/L
Na2EDTA.2H2O 0.64 mg/L
Na2SiO3.5H2O 7.6 mg/L
NaNO3 0.28 mg/L
KH2PO4 0.15 mg/L
K2HPO4 0.19 mg/L
Vitamins: Thiamine hydrochloride 75.0 µg/L, B12 1.0 µg/L and Biotin 0.75 µg/L.
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
64 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: for mortality, reproduction and growth
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
115 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
65 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
103 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
172 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Details on results:
Final Test 1:
Measured Concentrations
Analyses of samples taken from the target concentrations on a weekly basis showed that measured concentrations were slightly below target but relatively stable. Effect parameters were based on the average exposure concentrations.

Condition of Parental Daphnids
The two control groups were compared for mortality and combined for statistical analysis since no significant difference was found between the control groups. One out of the forty parental daphnids exposed to the pooled control died during the test period. Hence, parental mortality did not exceed 20% in the control. Statistical analysis showed that mortality was different from the control group at the two highest test concentrations.
The Cochran-Armitage test procedure (α = 0.05; one sided greater) detected a significant trend in mortality. Therefore, the effect parameters for reproductive output (NOEC and EC) were reported per introduced parent. In the control, the presence of eggs in the brood pouch was recorded for the first time on Day 4 and the first brood appeared on day 7. The first recording of young was similar to the control in test concentrations up to and including 125 µg/L. At 222 and 349 µg/L the first young were recorded on Day 11 and Day 15, respectively. On Day 11 and Day 12 most eggs in the daphnids exposed to 349 µg/L were observed to be outside the brood pouch. These eggs did not hatch.

Reproduction
The two control groups were compared for reproduction and combined for statistical analysis since no significant difference was found between the control groups. On average, 248 young were produced per introduced daphnid in the pooled control. Statistical analysis showed that the reproduction of the daphnids was significantly different from the control group at all test concentrations.

Body Length
The two control groups were compared for body length. Statistical analysis was performed using the solvent control group only since a significant difference was found between the control groups.

Final Test 2:
Final test 2 was performed because no NOEC for reproduction and length was found with the range of concentrations used in final test 1.

Measured Concentrations
Analyses of samples taken from the target concentrations on a weekly basis showed that measured concentrations were below target but relatively stable. Effect parameters were based on the average exposure concentrations.

Condition of Parental Daphnids
The two control groups were compared for mortality and combined for statistical analysis since no significant difference was found between the control groups. Four out of the forty parental daphnids exposed to the pooled control died during the test period, see Table 11. Hence, parental mortality did not exceed 20% in the control. Statistical analysis showed that mortality was different from the control group at the highest test concentrations.

The Cochran-Armitage test procedure (α = 0.05; one sided greater) detected a significant trend in mortality. Therefore, the effect parameters for reproductive output (NOEC and EC) were reported per introduced parent. In the control, the presence of eggs in the brood pouch was recorded for the first time on Day 5 and the first brood appeared on day 7-8. The first recording of young was similar to the control in test concentrations up to and including 64 µg/L. At 187 µg/L the first young were recorded on Day 12. On Day 11 a number of aborted eggs were observed in this concentration.

Reproduction
The two control groups were compared for reproduction and combined for statistical analysis since no significant difference was found between the control groups. On average, 183 young were produced per introduced daphnid in the pooled control. Statistical analysis showed that the reproduction of the daphnids was significantly different from the control group at 187 µg/L.

Body Length
The two control groups were compared for body length and combined for statistical analysis since no significant difference was found between the control groups. Statistically significant reduction of growth was found at 187 µg/L.

In conclusion, under the conditions of the present study Omnirad 379 did not affect parental mortality, reproduction and growth of Daphnia magna at an average exposure concentration of 64 µg/L, after 21 days of exposure (NOEC).

Exposure to an average concentration of 187 µg/L induced significant increase of parental mortality and significant inhibition of reproduction and growth of Daphnia magna, after 21 days of exposure (LOEC).

Range-Finding Test

A total of 70% of the parental daphnids exposed to the highest nominal concentration of 500 µg/L died during the test period. No (significant) mortality was observed at any of the lower concentrations and the solvent control. Note that a total of 20% mortality is acceptable for the control and therefore considered not significant. All surviving parental daphnids in the highest test concentration were observed to be smaller when compared to the control from

Day 5 of exposure until the end of the test. In addition, these daphnids were observed to be less coloured from Day 8 of exposure until the end of the test. Further observations included one aborted egg in the solvent control on Day 8 and three aborted eggs the lowest test concentration on Day 10.

The presence of eggs in the brood pouch was recorded for the first time on Day 5 for the solvent control and the two lowest test concentrations. The first brood was observed on day 7. For the highest test concentration the presence of eggs in the brood pouch was recorded on Day 6. However, no first brood was recorded during the 10-day test period.

Samples taken from the mixing vessels at the start of the test showed that the concentrations were in agreement with target (89-109%). Samples taken from the test concentrations at Day -1 (to check the flow-through system), Day 0 and Day 3 showed that the actual test concentrations were generally below target but stable.

Test conditions during the range-finding test were maintained within the limits prescribed by the study plan, except for the temperature measured continuously in a temperature control vessel.

Validity criteria fulfilled:
yes
Remarks:
The study met the acceptability criteria prescribed by the study plan and was considered valid.
Conclusions:
The test item did not affect parental mortality, reproduction and growth of Daphnia magna at an average exposure concentration of 64 µg/L, after 21 days of exposure (NOEC).

Exposure to an average concentration of 187 µg/L induced significant increase of parental mortality and significant inhibition of reproduction and growth of Daphnia magna, after 21 days of exposure (LOEC).
Executive summary:

The objective of the test was to evaluate the effects of the test item on the mobility, growth and the reproductive capacity of Daphnia magna. For this purpose, test organisms were exposed to aqueous solutions containing the test item at various concentrations. The time of the first production of young, the number of young born, immobility and other signs of intoxication observed were compared with corresponding parameters in the controls.

The study procedures described in this report were based on the OECD guideline for Testing of Chemicals: Guideline No. 211, 2012. In addition, the procedures were designed to meet the OECD guidance document number 23, 2000.  The study was also performed under GLP conditions.

The test item batch was a slightly yellow powder with a purity of 96.416%. Weighing of test item and preparation of stock solutions was performed under dimmed light. Stock solutions were kept in the dark. The test was performed using a flow-through system with target concentrations of 3.8, 12, 39, 125 and 400 µg/L, which were based on the results of a preceding range-finding test and a first final test. A blank- and a solvent control were also included.

In the flow-through system the dilution water was dosed separately from the test item stock solutions into mixing vessels. Stock solutions in dimethylformamide (DMF) were prepared three times per week at a factor of 10,000 higher than the target concentrations. The dosed volumes of stock and the dilution water were mixed under continuous stirring in the mixing vessels before entering the test vessels. The dosing was computer controlled, and the system was checked daily. The test was performed with four replicates containing 5 daphnids per replicate for each concentration and the control groups. The study duration was 21 days. Every day the condition of the parental daphnids was recorded, during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was recorded. At the end of the test the length of the surviving parental daphnids was measured.

Samples for chemical analysis of the test item concentrations were taken one day before the start, at the start and at weekly intervals during the test. Analyses of samples taken from the target concentrations on a weekly basis showed that measured concentrations were below target but relatively stable. Effect parameters were based on the average exposure concentrations that were calculated to be 2.4, 8.3, 28, 64 and 187 µg/L. The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized below: -

NOEC for mortality 64 µg/L

EC50 for mortality 115 (87 - 153)* µg/L

NOEC for reproduction 64 µg/L

EC10 for reproduction 65 (39 - 108)* µg/L

EC50 for reproduction 103 (58 - 185)* µg/L

NOEC for growth 64 µg/L

EC10 for growth 172 (118 - 250)* µg/L.

* ( ) – 95% confidence intervals.  

In conclusion, under the conditions of the present study, the test item did not affect parental mortality, reproduction and growth of Daphnia magna at an average exposure concentration of 64 µg/L, after 21 days of exposure (NOEC).

Exposure to an average concentration of 187 µg/L induced significant increase of parental mortality and significant inhibition of reproduction and growth of Daphnia magna, after 21 days of exposure (LOEC).

Description of key information

Study conducted to recognised testing guideline with GLP certification

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
64 µg/L

Additional information