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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-03-13 to 2019-09-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline; EU Method; GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
yes
Remarks:
The quantification of the test item methyl-bis(2-arylpropyl)dihydro-heteropolycycle in the test samples was performed using liquid-liquid extraction with the organic solvent Cyclohexane followed by analysis via gas chromatography with MS/MS detection.
Details on sampling:
Quadruplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test and at day 1.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, quadruplicate samples from the aged test media of all test concentrations and the control were collected at day 1 (after 24 hours of exposure) and at the end of the test from the additional test beakers prepared for the analytical dose verification (without Tween 80) of each treatment.
All samples were adjusted to pH10 with 1 M sodium hydroxide solution before storage.
Samples of the solvent control were not taken since Tween 80 is interfering with the analytical method and would therefore not have been reasonably analysable.
All samples were stored in a freezer (≤ - 20 °C), protected from light, until analysis was performed

Test solutions

Vehicle:
yes
Remarks:
Tween 80, 100µL/L
Details on test solutions:
An aqueous solution of methyl-bis(2-arylpropyl)dihydro-heteropolycycle in test media with a concentration (nominal: 110 mg/L) as close to saturation as possible (referred to as a ´stock solution´) was obtained first. This stock solution and its dilutions of 1:2, 1:4, 1:8 and 1:16 were prepared for test start and at the renewal period after 24 hours. Samples of the freshly prepared solutions for analytical dose verification were taken before Tween 80 was added to each test concentration 100 µL/L, after which the test organisms were introduced for testing. Additionally, a solvent control (100 µL Tween 80/L) and a control were tested. These concentrations refer to nominal concentrations of 110, 55, 27.5, 13.75 and 6.875 mg test item/L. Because Tween 80 interferes with analytical dose verification an additional set of all test concentrations and controls was prepared at test start and at the renewal period without Tween 80 but still with daphnids. These solutions were used as a proxy for analytical dose verification of the aged test concentrations. Control: In the control, test water was used without addition of the solvent and the test item.
Solvent Control: In the solvent control, test water was used with the solvent (100 µL Tween 80/L) but without the test item.
Dosage of Test Item:
The test item is not well soluble in test medium. To avoid physical effects of undissolved test item on the daphnids, no concentrations far above the solubility limit of the test item in test water were tested. Therefore, a ´stock solution´ (nominal 110 mg/L) of methyl-bis(2-arylpropyl)dihydro-heteropolycycle in test water was prepared by mixing 110 and 115 mg test item into 1000 and 1045.45 mL test water at test start and 112.2 and 112.8 mg test item into 1020.0 and 1025.45 mL test water on Day 1. The test medium was stirred with an excess of methyl-bis(2-arylpropyl)dihydro-heteropolycycle in a vessel for 24 hours in the dark. Afterwards the test medium was allowed to settle until phase separation for 1 hour. This ´stock solution´ was withdrawn from a tap at the center of the vessel, after first flushing the tap and tubing with a small aliquot, and then mixed with test water to produce the desired dilutions of 1:2, 1:4, 1:8 and 1:16. The test media were prepared just before introduction of the daphnids (= start of the test) and test medium renewal on Day 1.
Appearance of the Test Item in Test Medium:There were no remarkable observations.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Species:Daphnia magna (Straus), clone 5
Age at Test Start: From 8.50 to 23.75 hours old
Sex: Female
Origin: The Daphnia introduced in the test were taken from ibacon's in house laboratory culture.
Breeding Conditions: The Daphnia were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental Daphnia was performed in Elendt M4 medium. The test organisms were not first brood progeny. The Daphnia in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
Water Hardness: 2.5 mmol/L (= 250 mg/L) as Calcium Carbonate
Test temperature:
19 - 20 °C at test start;
19 - 20 °C at test end
pH:
7.9 to 8.0 at test start;
7.9 to 8.0 at test end; and thus the pH-value did not vary by more than 1.5 units
Dissolved oxygen:
8.7 to 9.2 mg/L at test start;
8.8 to 9.0 mg/L at test end
Nominal and measured concentrations:
An aqueous solution of methyl-bis(2-arylpropyl)dihydro-heteropolycycle in test media with a concentration (nominal: 110 mg/L) as close to saturation as possible (referred to as a ´stock solution´) was obtained first. This stock solution and its dilutions of 1:2, 1:4, 1:8 and 1:16 were prepared for test start and at the renewal period after 24 hours. Samples of the freshly prepared solutions for analytical dose verification were taken before Tween 80 was added to each test concentration at 100 µL/L, after which the test organisms were introduced for testing. Additionally, a solvent control (100 µL Tween 80/L) and a control were tested.
These concentrations refer to nominal concentrations of 110, 55, 27.5, 13.75 and 6.875 mg test item/L.
Because Tween 80 interferes with analytical dose verification an additional set of all test concentrations and controls was prepared at test start and at the renewal period without Tween 80 but still with daphnids. These solutions were used as a proxy for analytical dose verification of the aged test concentrations.

The mean measured values of the freshly prepared media are:
4135 μg test item/L in the WAF stock solution
2040 μg test item/L in the 1:2 dilution of WAF stock solution
685 μg test item/L in the 1:4 dilution of WAF stock solution
288 μg test item/L in the 1:8 dilution of WAF stock solution
118 μg test item/L in the 1:16 dilution of WAF stock solution

The mean measured values of the 24 hours aged media are:
3638 μg test item/L in the WAF stock solution
1748 μg test item/L in the 1:2 dilution of WAF stock solution
510 μg test item/L in the 1:4 dilution of WAF stock solution
201 μg test item/L in the 1:8 dilution of WAF stock solution
Details on test conditions:
Test Units
Glass beakers of 100 mL volume containing approximately 60 mL of test medium. The test vessels were covered with a lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.

Identification: Each test unit was uniquely identified with serial number, treatment and replicate number.

Test Conditions
Test Environment: Controlled environment room

The water temperature, pH-values and the dissolved oxygen concentrations were determined in all freshly prepared and aged test media of each treatment group.
Water Temperature: 19.4 to 20.3 °C in the freshly prepared media; 19.4 to 19.9°C in the aged test media
pH-Values: 7.9 to 8.0 in the freshly prepared media; 7.9 to 8.0 in the aged test media; and thus the pH-value did not vary by more than 1.5 units
Dissolved Oxygen: 8.7 to 9.2 mg/L in the freshly prepared media; 8.8 to 9.0 mg/L in the aged test media
Light Regime: 16 h light: 8 h dark
Light Intensity: The light intensity was 450 to 630 lux (measured once during the test).
Reference substance (positive control):
yes
Remarks:
For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 110 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
55 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
110 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item concentration of 55 mg test item/L. At the concentration of 110 mg test item/L, eight animals were immobile at test end.
Results with reference substance (positive control):
In the most recent test with the reference item potassium dichromate the EC50 after 24 hours was determined to be 0.932 mg test item/L, indicating that the sensitivity of the Daphnia was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).
Reported statistics and error estimates:
The 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated by probit analysis.
The NOEC and LOEC after 24 and 48 hours were calculated by Fisher’s Exact Binomial Test (24h) and Step-down Cochran Armitage Test (48h).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Control Immobilisation Rate was 0 % and furthermore no daphnid showed signs of disease or stress. Dissolved Oxygen Concentration was above or equal 8.8 mg O2/L in the control and test vessels at the end of the test.
Conclusions:
The toxic effect of the test item methyl-bis(2-arylpropyl)dihydro-heteropolycycle to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was determined to be 55.0 mg test item/L. The 48-hour LOEC was determined to be 110 mg test item/L and the 48-hour EC50 value was determined to be > 110 mg test item/L. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal concentrations (loading rate) since a supersaturated stock solution via a water accommodated fraction was prepared.
Executive summary:

The toxic effect of the test item methyl-bis(2-arylpropyl)dihydro-heteropolycycle to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was determined to be 55.0 mg test item/L. The 48-hour LOEC was determined to be 110 mg test item/L and the 48-hour EC50 value was determined to be > 110 mg test item/L. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal concentrations (loading rate) since a supersaturated stock solution via a water accommodated fraction was prepared.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

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