reaction mass of 2-[[1-(chloromethyl)-2-[[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methoxy]ethoxy]methyl]oxirane and cis-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane and trans-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

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:

6 Feb - 8 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-[[1-(chloromethyl)-2-[[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methoxy]ethoxy]methyl]oxirane

Molecular formula:

C17H29ClO5

IUPAC Name:

2-[[1-(chloromethyl)-2-[[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methoxy]ethoxy]methyl]oxirane

Constituent 2

Reference substance name:

cis-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Cas Number:

1197197-64-0

Molecular formula:

C14H26O4

IUPAC Name:

cis-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Constituent 3

Reference substance name:

trans-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Cas Number:

158307-92-7

Molecular formula:

C14H26O4

IUPAC Name:

trans-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

impurity 1

Reference substance name:

[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methanol

Molecular formula:

C11H20O3

IUPAC Name:

[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methanol

impurity 2

Reference substance name:

[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methanol

Molecular formula:

C14H25ClO4

IUPAC Name:

[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methanol

impurity 3

Reference substance name:

2-[[3-chloro-2-[[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methoxy]propoxy]methyl]oxirane

Molecular formula:

C20H34Cl2O6

IUPAC Name:

2-[[3-chloro-2-[[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methoxy]propoxy]methyl]oxirane

impurity 4

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot No.of test material: TX7E5501
- Physical state: pale yellow liquid
- Purity: 87.6%
- Expiration date of the lot/batch: 26 May 2022
- Storage condition of test material: ambient

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were collected from the batches of test solution prepared for each treatment and control group at the beginning of the test, and from two of the four replicate test chambers in each treatment and control group at 48 hours.
- Sampling method: Samples (5.0 mL) were collected from mid-depth, placed in glass vials containing 5 mL of 0.2% formic acid in methanol, and processed immediately for analysis.
- Sample storage conditions before analysis: not stored

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Method: Test solutions were prepared by weighing the required mass of Heloxy 107 on a Teflon disc and quantitatively adding the disc to 2L volumetric flasks containing 1L of dilution water. The lowest test concentration (6.3 mg/L) was filled to 2L. The flasks were covered using glass stoppers and the test solutions were stirred with Teflon coated stir bars overnight. All test solutions appeared clear and colorless during the test
- Controls: dilution water without test substance
- Evidence of undissolved material: No

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Cladoceran; Water flea
- Source: Cultures maintained by the test laboratory with known history
- Age at study initiation: < 24 hours, from parental daphnids of more than two weeks old
- Culturing: Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. Adult daphnids in the cultures were held for at least 14 days prior to collection of the juveniles for testing. The adults showed no signs of disease or stress during the holding period, no ephippia were produced, and mortality was <10% in the two-day period prior to test initiation. During the 14-day period preceding the test, water temperatures ranged from 19.5 to 20.4 °C. The pH of the water ranged from 8.1 to 8.4, and dissolved oxygen concentrations were ≥ 7.6 mg/L. Daphnids in the cultures were fed a mixture of yeast, cereal grass media and trout chow, supplemented with a vitamin stock solution and a suspension of the freshwater green algae, Raphidocelis subcapitata. The adults were fed prior to test initiation, but neonates were not fed during the test.

FEEDING DURING TEST
- Food type: Not fed during test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

132 mg CaCO3/L

Test temperature:

19.1 - 20.0 °C

pH:

8.3 - 8.5

Dissolved oxygen:

8.6 - 8.9

Conductivity:

357 μS/cm

Nominal and measured concentrations:

Nominal: Negative control (0), 6.3, 13, 25, 50, and 100 mg formulation/L
Measured: see Table 1. Nominal concentrations were used to determine effect concentrations. Test substance was a mixture, and the two predominant components showed reasonable stability over the course of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beaker, loosely covered wtih a plastic petri dish
- Fill volume: approximately 220 mL
- Depth: 6.9 cm
- Aeration: dilution water aerated prior to filling
- Renewal: None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 40-m deep well located at testing lab. Water was passed through a sand filter and pumped into a storage tank and aerated with spray nozzles. Water was filtered to 0.45 μm and UV-sterilized prior to testing. Periodic water analysis, Attachments 1 and 2.
- Total organic carbon: < 1 mg C/L
- Pesticides: all < LOQ
- Alkalinity: 170 mg CaCO3/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: most recent analysis on samples collected 7 Dec 2016
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h:8h with 30-min transitions
- Light intensity: 554 lux at surface of water of one representative chamber at test initiation
EFFECT PARAMETERS MEASURED : Mortality and immobilization at 24h and 48 h
RANGE-FINDING STUDY
- Test concentrations: 0.1, 1.2, 3.7, 11, 33 and 100 mg formulation/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

71 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: Lethargy (see Table 2)
- Mortality of control: None

Reported statistics and error estimates:

The immobility data were analyzed using the computer program of C. E. Stephan. Based on the immobility pattern in this study, nonlinear interpolation was used to calculate the 24 and 48-hour EC50 values and binominal probability was used to calculate the 95% confidence intervals (CI, 50-100 mg/L)

Any other information on results incl. tables

Table 2. Cumulative percent mortality/immobility.

Nominal concentration	Number Immobile/ Observations¹	Number Immobile/ Observations	Number Immobile/ Observations	Cumulative percent immobilized
Time	~3.5 hr	24 hrs	48 hrs
Negative control	0 / 20 AN	0 / 20 AN	0 / 20 AN	0
6.3	0 / 20 AN	0 / 20 AN	0 / 20	0
13	0 / 20 AN	1 / 1 Q:AN, 18 AN	1 / 1 Q:C, 18 AN	5
25	0 / 20 AN	0 / 20 AN	0 / 20 AN	0
50	0 / 20 AN	0 / 20 AN	0 / 11 C,   9 AN	0
100	0 / 20 AN	12 / 8 C	20 / --	100

1, Observations:

AN = appear normal;

Q,AN = trapped at water surface but appear normal after gentle submersion;

Q,C = trapped at water surface and appear lethargic after gentle submersion;

C = lethargy

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Control immobilization was < 10% at the end of the test (0%). The dissolved oxygen concentration was ≥ 3 mg/L in the control and test vessels throughout the test (≥8.6 mg/L)

Conclusions:

The 48-hr EC50 of Heloxy 107 to Daphnia magna was 71 mg/L (OECD 202).

Executive summary:

The 48-hour EC50 of Heloxy 107 to Daphnia magna was examined in a static test conducted according to OECD 202. Test solutions were prepared at nominal concentrations of 6.3, 13, 25, 50 and 100 mg formulation/L. Concentrations were verified analytically for the two major components of the test material. Concentrations of the two predominant components ranged from 94.4 to 111% of nominal test concentrations. The 48-hour EC50 is 71 mg/L based on the  nominal concentration.  The test was conducted according to internationally accepted test guidelines and was GLP compliant. It is reliable without restriction and suitable for Risk Assessment, Classification and Labeling, and PBT Analysis.