3,3'-[methylenebis(oxymethylene)]bisheptane

Long-term toxicity to aquatic invertebrates

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:

From April 09, 2019 to August 28, 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)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Lambiotte & Cie - 1812101500R
- Expiration date of the lot/batch: 10 December 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Initially stored at approximately 4 ˚C in the dark under nitrogen from receipt on 17 December 2018 until 14 January 2019 when it was stored at room temperature in the dark under nitrogen

Analytical monitoring:

yes

Details on sampling:

- Concentration: 0.0104 mg/L
- Sampling method: Water samples were taken from the control, solvent control and the test group for immediate quantitative analysis. Samples of the fresh test preparations were taken from the bulk test preparations on Days 0, 5, 12 and 20 and of the expired test preparations (replicates pooled) on Days 1, 6, 13 and 21. Duplicate sets of samples were taken on each occasion and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: All samples were analyzed on the day of sampling. Duplicate sets of samples were also taken on each occasion and stored refrigerated for future analysis (if required).

Vehicle:

yes

Remarks:

dimethylformamide

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (definitive test)
An initial solvent stock solution was prepared by dissolving 10.4 mg of test item in a final volume of 10 mL of DMF to give a 1.04 mg/mL solvent stock solution. A further solvent stock solution was prepared by dissolving 2 mL of the 1.04 mg/mL stock concentration in a final volume of 20 mL of DMF to give a 0.104 mg/mL solvent stock solution. An aliquot (200 μL) of the 0.104 mg/mL solvent stock solution was dispersed in 2 liters of test water with the aid of magnetic stirring for approximately 5 minutes to produce the test concentration of 0.0104 mg/L. The solvent stocks were prepared weekly and stored refrigerated when not in use.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Stage and instar at study initiation: 1st instar
- Method of breeding: parthenogenesis
- Source: in-house
- Feeding during test: yes
- Food type: mixture of algal suspension (Raphidocelis subcapitata formally known as Pseudokirchneriella subcapitata) and GM300 fish food suspension
- Amount: Each daphnid received between 5 and 20 μL of an algal suspension throughout the test with 20 μL of GM300 fish food suspension provided on Days 0, 1, 2 and 3 of the test.
- Frequency: daily

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:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

250 to 270 mg/L

Test temperature:

18 to 22°C

pH:

7.6 - 8.4

Dissolved oxygen:

≥8.1 mg O2/L

Nominal and measured concentrations:

For the definitive test: nominal concentration of 0.0104 mg/L
The geometric mean measured test concentration was determined to be 0.0056 mg/L (54% of nominal concentration)

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass vessels
- Type: closed (covered with a plastic lid)
- Material, size, headspace, fill volume: single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels
- Aeration: no
- Renewal rate of test solution: The test preparations were renewed daily.
- No. of organisms per vessel: single daphnid per vessel
- 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
Reconstituted Water – Elendt M7 Medium

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods
- Light intensity: 756 to 1001 lux

EFFECT PARAMETERS MEASURED:
Immobilization
Reproduction
Body Length

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.000104, 0.00104 and 0.0104 mg/L
- Results used to determine the conditions for the definitive study: No immobilization or sub-lethal effects were observed in the test concentrations of 0.000104 and 0.00104 mg/L; however, 20% immobilization was observed at 0.0104 mg/L. Sub-lethal effects of exposure were observed in two daphnids in the solvent control. The response (in the adults) was pale.
Based on this information a "Limit Test" at a concentration of 0.0104 mg/L was selected for the definitive test.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Body length

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Body length

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Body length

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Details on results:

- Lethal Effects on the Parental Generation (P1)
Low levels of mortality (10%) were observed in the control and solvent control and no mortalities were apparent at the test concentration of 0.0056 mg/L.
As this test was conducted as a “Limit Test” and no effects were apparent, the LOEC could not be determined.

- Sub-lethal Effects on the Parental Generation (P1)
No sub-lethal effects of exposure were observed in the control, solvent control and at the 0.0056 mg/L test concentration.
After 21 days the length of each surviving adult was determined. The results showed that there were no statistically significant differences (P≥0.05) between the pooled control and the 0.0056 mg/L test group in terms of length of the daphnids after 21 days exposure to the test item.
The NOEC values (body length) based on the geometric mean measured test
concentration was calculated using the Student t-test procedure incorporating Shapiro-Wilk's test on normal distribution and Levene’s test on variance homogeneity at 21 days. As this test was conducted as a “Limit Test” and no effects were apparent, the LOEC value could not be determined.

- Effects on Reproduction
After 21 days there were no statistically significant differences (P≥0.05) between the pooled control and the 0.0056 mg/L test group in terms of the numbers of live young produced per surviving adult. The NOEC values (reproduction) based on the geometric mean measured test
concentration was calculated using the Student t-test procedure incorporating Shapiro-Wilk's test on normal distribution and Levene’s test on variance homogeneity at 21 days. As this test was conducted as a “Limit Test” and no effects were apparent, the LOEC value could not be determined.

- Effects on the Filial Generation (F1)
Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, all "filial" daphnids produced by the control, solvent control groups and 0.0056 mg/L test group were observed to be of normal appearance.
Young were first produced in the control and solvent control groups on Day 8 of the test. There were no unhatched eggs or dead young observed in the control, solvent control or
0.0056 mg/L test group throughout the test.

Results with reference substance (positive control):

N/A

Reported statistics and error estimates:

The "No Observed Effect Concentration" (NOEC) for the number of parental generation Daphnia mortalities for the control, solvent control and each test group was estimated by inspection of the mortality data. For reproduction, the Numbers of live young produced per surviving adult over the duration of the test for the pooled control and the test group were compared using the Student t-test procedure incorporating Shapiro-Wilk's test on normal distribution and Levene’s test on variance homogeneity. Results from the pooled control and the test group Daphnia length data,
determined for the surviving daphnids on termination of the test, were compared using the Student t-test procedure incorporating Shapiro-Wilk's test on normal distribution and Levene’s test on variance homogeneity.

 

Endpoint		Concentration mg/L (95% confidence limits)
Immobilization	EC10	>0.0056 (not determined)*
	EC20	>0.0056 (not determined)*
	EC50	>0.0056 (not determined)*
	No Observed Effect Concentration	0.0056
	Lowest Observed Effect Concentration	Not determined
Reproduction	EC10	>0.0056 (not determined)*
	EC20	>0.0056 (not determined)*
	EC50	>0.0056 (not determined)*
	No Observed Effect Concentration	0.0056
	Lowest Observed Effect Concentration	Not determined
Body Length	EC10	>0.0056 (not determined)*
	EC20	>0.0056 (not determined)*
	EC50	>0.0056 (not determined)*
	No Observed Effect Concentration	0.0056
	Lowest Observed Effect Concentration	Not determined

*Not possible to calculate 95% confidence limits for the data set as only a “Limit Test” was conducted.

Validity criteria fulfilled:

yes

Conclusions:

No effects were observed on immobilisation, reproduction and body length at the geometric mean concentration tested, 0.0056 mg/L.

Executive summary:

Introduction

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

Information provided by the Sponsor indicated the water solubility of the test item to be 0.0104 mg/L.

A solvent spike method of preparation was considered most appropriate for this test item. Based on the results of a preliminary range-finding test, Daphnia magna were exposed

(10 replicates of a single daphnid per group) to a solution of the test item at a nominal

concentration of 0.0104 mg/L for a period of 21 days. The test solutions were renewed daily throughout the test. An initial solvent stock solution was prepared by dissolving a nominal amount of test item (10.4 mg) in a final volume of 10 mL of dimethylformamide (DMF) to give a 1.04 mg/mL solvent stock solution. A further solvent stock solution was prepared by dissolving 2 mL of the 1.04 mg/mL stock concentration in a final volume of 20 mL of DMF to give a 0.104 mg/mL solvent stock solution. An aliquot (200 µL) of the 0.104 mg/mL solvent stock solution was dispersed in 2 liters of test water with the aid of magnetic stirring for approximately 5 minutes to produce the test concentration 0.0104 mg/L.

The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with an algal suspension and supplemented with GM300 fish food suspension on Days 0, 1, 2 and 3.

Results

Analysis of the fresh test preparations on Days 0, 5, 12 and 20 showed measured test concentrations to range from 0.00897 to 0.0129 mg/L (between 86 and 124% of nominal). A decline in measured test concentration of the aged test preparations on Days 1, 6, 13 and 21 was observed to be between 0.00185 and 0.00402 mg/L (between 18 and 35% of freshly prepared media concentrations). Given these results it was considered appropriate to calculate the results based on the geometric mean measured test concentrations only in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 0.0056 mg/L. Analysis of the solvent stocks confirmed that these were stable for greater than seven days when stored refrigerated.

Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentration:

Endpoint		Concentration mg/L (95% confidence limits)
Immobilization	EC10	>0.0056 (not determined)*
	EC20	>0.0056 (not determined)*
	EC50	>0.0056 (not determined)*
	No Observed Effect Concentration	0.0056
	Lowest Observed Effect Concentration	Not determined
Reproduction	EC10	>0.0056 (not determined)*
	EC20	>0.0056 (not determined)*
	EC50	>0.0056 (not determined)*
	No Observed Effect Concentration	0.0056
	Lowest Observed Effect Concentration	Not determined
Body Length	EC10	>0.0056 (not determined)*
	EC20	>0.0056 (not determined)*
	EC50	>0.0056 (not determined)*
	No Observed Effect Concentration	0.0056
	Lowest Observed Effect Concentration	Not determined

* Not possible to calculate 95% confidence limits for the data set as only a “Limit Test” was conducted.

Description of key information

Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentration:

 

Endpoint		Concentration mg/L (95% confidence limits)
Immobilization	EC10	>0.0056 (not determined)*
	EC20	>0.0056 (not determined)*
	EC50	>0.0056 (not determined)*
	No Observed Effect Concentration	0.0056
	Lowest Observed Effect Concentration	Not determined
Reproduction	EC10	>0.0056 (not determined)*
	EC20	>0.0056 (not determined)*
	EC50	>0.0056 (not determined)*
	No Observed Effect Concentration	0.0056
	Lowest Observed Effect Concentration	Not determined
Body Length	EC10	>0.0056 (not determined)*
	EC20	>0.0056 (not determined)*
	EC50	>0.0056 (not determined)*
	No Observed Effect Concentration	0.0056
	Lowest Observed Effect Concentration	Not determined

*Not possible to calculate 95% confidence limits for the data set as only a “Limit Test” was conducted.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.006 mg/L