Bis(6-methylalkyl-5-en-2-yl)dihydro-heteropolycycle

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:

2019-09-09 to 2019-12-02

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted April 13, 2004

Test guideline 2

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"

Version / remarks:

2nd Ed., February 08, 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Remarks:

GC-MS/MS

Details on sampling:

Quadruplicate samples from the only test concentration 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 only concentration during the test period, quadruplicate samples from the aged test media of only concentration and the control were collected at day 1 (after 24 hours of exposure) and at the end of the test by pouring together the contents of the additional test beakers prepared for the analytical dose verification (without Tween 80, see section 6.6) of each treatment.
The pH of all samples was adjusted to pH 10 using Sodium hydroxide solution. No further steps were performed until sample preparation.
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.

Test solutions

Vehicle:

yes

Details on test solutions:

Test Concentration:
An aqueous solution of the TEST ITEM 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 (see ´Dosage of test item´ below). This stock solution was 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 the only 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.
Because Tween 80 interferes with analytical dose verification an additional set of the only test concentration and controls was prepared at test start and at the renewal period without Tween 80 (four additional replicates in total) but still with daphnids. These solutions were used as a proxy for analytical dose verification of the aged test concentration.
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 (100µL 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 above the solubility limit of the test item in test water was tested.
Therefore, a ´stock solution´ (110 mg/L) of the test substance in test medium was prepared by mixing 110.0 and 110.5mg test item in 1100 and 1105 mL. The test medium was stirred with an excess of the test substance in a vessel for 24 hours in the dark. Afterwards the test medium was allowed to settle until phase separation for 1.5 hours. 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 used for testing.
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 3.5 to 21.5 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:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

Water Hardness: 2.5 mmol/L (= 250 mg/L) as Calcium Carbonate

Test temperature:

19.9 to 20.4 °C in the freshly prepared media; 20.4 to 20.6°C in the aged test media

pH:

7.9 to 8.0 in the freshly prepared media; 7.8 to 7.9 in the aged test media; and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

8.8 to 9.0 mg/L in the freshly prepared media; 8.5 to 9.0 mg/L in the aged test media

Nominal and measured concentrations:

Nominal: 110 mg test item/L
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 (100µL Tween 80/L) but without the test item.

Details on test conditions:

Test Environment: Controlled environment room
Measurement of pH, Dissolved Oxygen and Water Temperature: 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: 19.9 to 20.4 °C in the freshly prepared media; 20.4 to 20.6°C in the aged test media
pH-Values: 7.9 to 8.0 in the freshly prepared media; 7.8 to 7.9 in the aged test media; and thus the pH-value did not vary by more than 1.5 units
Dissolved Oxygen Concentration: 8.8 to 9.0 mg/L in the freshly prepared media;
8.5 to 9.0 mg/L in the aged test media
Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 380 to 1000 lux (measured once during the test).
Recording: Test conditions were recorded with suitable instruments and documented in the raw data.

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 concentrations

Effect concentrations 1

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

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

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 in the only test concentration of nominal 110 mg test item/L.

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 0.707mg 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:

No statistical analysis was performed. The EC50 could not be quantified due to the absence of toxicity of the test item and were determined to be higher than the highest test concentration. The NOEC and the LOEC were determined directly from the raw data.

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 8.5 mg O2/L in the control and test vessels at the end of the test.

Conclusions:

The toxic effect of the test item to Daphnia magna was assessed in a semi-static limit test. The 48-hour NOEC was determined to be ≥110 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 concentration and the maintenance of the exposure concentration during the test were examined in the analytical part. All reported results refer to one concentration (loading rate) since a supersaturated solution via a water accommodated fraction was prepared.

Executive summary:

Title:

Acute Toxicity to Daphnia magna in a Semi-Static 48-hour Immobilisation Limit Test

Purpose:

The purpose of this study was to determine the influence of the test item on the mobility of Daphnia magna. For this purpose, young daphnids (< 24 hours old) were exposed in a semi-static test for 48 hours to the only concentration of nominal 110 mg/L under defined conditions for 48 hours. The recorded effects were the mobility of the daphnids after 24 and 48 hours. This limit-test is performed in compliance with the test guidelines in order to demonstrate that the test item has no toxic effects on the daphnids up to this concentration. The test method of application and the test system are recommended by the test guidelines and Daphnia magna is the recommended test species. The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium.

Guidelines:

OECD Guideline for Testing of Chemicals No. 202: "Daphnia sp., Acute Immobilisation Test" adopted April 13, 2004

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

SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

Materials and Methods

Test Item:  Bis(6-methylalkyl-5-en-2-yl)dihydro-heteropolycycle; batch no.: SS-Sa-11074-85;

Test Species: Daphnia magna, clone 5; 3.5 to 21.5 hours old.

Source: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture.

Test Design:  This study encompassed three treatment groups (one test item concentration at nominal 110 mg/L, one solvent control (Tween) and one control) each containing 20 individuals. The mobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours.

Endpoints: Number of immobile organisms after 24 and 48 hours

Test Concentrations:

An aqueous solution of the test item 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 (see ´Dosage of test item´ below). This stock solution was 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 the only 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. Because Tween 80 interferes with analytical dose verification an additional set of the only test concentration and controls was prepared at test start and at the renewal period without Tween 80 (four additional replicates in total) but still with daphnids. These solutions were used as a proxy for analytical dose verification of the aged test concentration.

Test Conditions:

Water temperature: 19.9 to 20.6 °C; pH value: 7.8 to 8.0; dissolved oxygen concentration: 8.5 to 9.0 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 380 to 1000 lux; and thus were within the ranges requested by guideline OECD 202

Results

Biological Test Results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and in the only test concentration of nominal 110 mg test item/L.

Analytical test results:

The quantification of the test item in the test samples was performed using liquid/ liquid extraction followed by analysis via gas chromatography with MS/MS detection. The concentrations of the test item were determined in two of the quadruplicate test media samples from only concentration and in two of the quadruplicate control samples from all sampling times. The mean measured value in the freshly prepared supersatured solution (WAF) of nominal 110 mg test item/L from test start and the first renewal period after 24 hours is 57.3 µg test item/L. The mean measured value in the 24 hours aged solution taken after 24 hours and 48 hours is below LOQ (=20 µg test item/L).

Conclusion:

The toxic effect of the test item to Daphnia magna was assessed in a semi-static limit test. The 48-hour NOEC was determined to be ≥110 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 concentration and the maintenance of the exposure concentration during the test were verified in the analytical part. All reported results refer to one concentration (loading rate) since a supersatured 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.