Reaction mass of 2-[[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]methyl]oxirane and 1,3-bis[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]propan-2-ol and 1-[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]-3-[4-[[4-[3-[4-[[3,5-dimethyl-4-(oxiran-2-ylmethoxy)phenyl]methyl]-2,6-dimethyl-phenoxy]-2-hydroxy-propoxy]-3,5-dimethyl-phenyl]methyl]-2,6-dimethyl-phenoxy]propan-2-ol

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

: deviations in terms of control mortality (> 20% mortality observed). Not considered to affect study integrity and suitable justification provided in endpoint study record. Neonates produced were also only counted every 2-3 days rather than daily.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Deviations:

yes

Remarks:

: deviations in terms of control mortality (> 20% mortality observed). Not considered to affect study integrity and suitable justification provided in endpoint study record.

GLP compliance:

yes

Analytical monitoring:

yes

Remarks:

TOC Analysis

Details on sampling:

- Concentrations: 0.025, 0.05, 0.1, 0.2 and 0.4 mg/L (Nominal Loading rates)
- Sampling method: Samples collected from each treatment and control group at test initiation, at the start and end of weekly renewal cycles, and at the end of study. Samples were analysed for TOC, as is more relevant to show stability issues in complex, multi-component substances.
- Sample storage conditions before analysis: The samples were collected from mid-depth into glass vials, acidified with 2N HCl, and stored under refrigeration prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: As the test substance was poorly water soluble, individual test substance solutions were prepared in the form of water accomadated fractions (WAFs) at each loading rate. To perform this, calculated amounts of the test substance were were added to 13L of dilution water in large aspirator bottles, which were subsequently sealed. These solutions were stirred for approximately 48 hours using magnetic stirrers. Following this period, the vessels were left to stand for a period of 1 hour, before the aqueous (WAF) layer was drawn off by use of a spigot and tubing.
- Controls: The negative controls were prepared using the same methodology.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test solution appeared clear and colorless with no visible precipitate after settling, prior to drawing into the test chambers.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: Taken from cultures maintained at the testing laboratory.
- Age of parental stock (mean and range, SD): At least 13 days.
- Feeding during test: Fed daily
- Food type: A mixture of yeast, cereal grass media and trout chow (YCT), supplemented with a vitamin stock solution and a suspension of fresh green algae (Raphidocelis subcapitata).
- Amount: At each feeding during the test, each test chamber was fed 0.50 mL of YCT, 1.0 mL of algae and 0.40 mL of vitamin solution. This amount of feed is equal to approximately 0.60 mg C/daphnid/day. While this amount of feed exceeds the OECD guideline recommended amount of 0.1 to 0.2 mg C/daphnid/day, an excess amount was fed in order to maintain sufficient feed in the system to support acceptable reproduction rates.


ACCLIMATION
- Acclimation period: At least 13 days
- Acclimation conditions (same as test or not): Same conditions as on study
- Type and amount of food: Same as on study.
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): The adults showed no signs of disease or stress and no ephippia were produced during the holding period.


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: To initiate the test, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers until each chamber contained 10 daphnids. Each group of neonates then was impartially assigned to a control or treatment group and individual neonates were transferred to each test chamber to initiate the test. All transfers were made below the water surface using wide-bore pipettes.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

139 +/- 6 mg/L as CaCO3 (controls) and 139 +/- 11 mg/L as CaCO3 (0.4 mg/L).

Test temperature:

Within the range 20 +/- 1°C

pH:

8 to 8.9 during the test

Dissolved oxygen:

>/= 60% of air saturation (5.4 mg/L)

Salinity:

Not determined

Conductivity:

356 +/- 27 µS/cm (controls) and 364 +/- 18 µS/cm (0.4 mg/L)

Nominal and measured concentrations:

Nominal: 0, 0.025, 0.05, 0.1, 0.2 and 0.4 mg/L
Measured: Not determined as substance is a multi-component substance and tested using WAF preparations. Stability confirmed using TOC analysis.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Type (delete if not applicable): open (loosely covered with glass petri dishes)
- Material, size, headspace, fill volume: Approximately 200 mL per vessel
- Aeration: No
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Obtained from an on-site well. Water was passed through a sand filter, and then stored and aerated. Prior to use, the water was further filtered and sterilised using UV-sterilisation.
- Total organic carbon: < 1 mgC/L
- Particulate matter: None
- Metals: Various detected or below detection limits (all at low concentrations deemed not to have influenced the study outcomes).
- Pesticides: Various detected or below detection limits (all at low concentrations deemed not to have influenced the study outcomes).
- Chlorine: 4.3 mg/L
- Alkalinity: 177 mg/L as CaCO3
- Conductivity: 347 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: Measured during the 4 weeks prior to test initiation, with various quality measurements measured throughout the testing period.

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16 hours light/ 8 hours darkness
- Light intensity:Ambient laboratory light was used to illuminate the test systems.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Survival and clinical observations, reproduction and growth. Neonates produced by adult Daphnids were counted every two to three days. The OECD guideline prefers that these are counted daily, but does not specifiy that this is a definitive requirement. The counting regime was considered adequate and the results valid, and in line with the EPA OPPTS 850.1300 guideline.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes
- Test concentrations: 0.11, 0.36, 1.2, 2.0, 3.0, 4.1, 14 and 45 mg/L nominal loading rates.
- Results used to determine the conditions for the definitive study: All first generation daphnids at >/= 1.2 mg/L were immobile by Day 9 of the range finding phase.

Reference substance (positive control):

not required

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

0.29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

0.34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: A clear reduction in dry weight was observed at the 0.4 mg/L loading rate. However, growth rate data from this loading rate was excluded form statistical analysis due to a marked reduction in survival. No other statistical significance with respect to body weight and length was observed at the remaining test loading rates.

- Mortality of control: The percentage of parent daphnids in the controls that appear to be immobilized at the end of the test in the negative control should be ≤20%, it was 30%. However, the percentage of parent daphnids in the 0.025, 0.050 and 0.20 mg/L loading rate of the TMBPF DGE epoxy resin treatment group that appear to be immobilized at the end of the test were ≤20%, indicating that the daphnid neonates used to initiate the study were healthy. Additionally, high reproduction rates in the negative control and treatment groups further demonstrated the health of the organisms. Finally, statistical comparisons assuming control survival of <20% did not change the NOEL and LOEL for survival. Therefore, the study was judged to be valid.

- Other adverse effects control: None

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

- Effect concentrations exceeding solubility of substance in test medium: Not noted

Results with reference substance (positive control):

No reference test required as not a standard requirement of the relevant OECD guideline

Reported statistics and error estimates:

Test endpoints analyzed statistically for first-generation daphnids were survival, reproduction, and growth (length and dry weight).

The reproduction endpoint was analyzed as the number of live young produced per 21-day surviving adult; and the number of live young produced per parent animal alive at the beginning of the test excluding parental accidental mortality (i.e., non-substance related mortality caused by an accidental incidence or other known cause) and/or inadvertent mortality (i.e., non-substance related mortality with no known cause)

Because the mortality of the first generation daphnids in the control were not treatment related and had no known cause, these first generation daphnids were excluded from the analysis of reproduction.

The immobile first generation daphnids in the treatment concentrations were also excluded as the mortality did not follow a concentration response pattern and were not considered to be treatment related. Consequently, the reproduction endpoint was analyzed based on the mean number of live young produced per 21-day surviving adult. Neonates produced by those first-generation daphnids that did not survive the full 21 days were excluded from analysis of reproduction.

TOC analysis results:

Measurements of TOC in the fresh solutions collected from the newly prepared WAFs of each test solution at test initiation and at the beginning of one renewal day each week on Days 0, 3, 10 and 17 of the test ranged from < LOQ to 2.037 mg C/L. Measurements of TOC in the old solutions collected from one alternating replicate of each treatment and control group at the end of one renewal day each week and at test termination on Days 3, 6, 13 and 21 of the test ranged from < LOQ to 2.869 mg C/L. Due to the low nominal loading rates, TOC content of the newly prepared test solutions were generally below or slightly above the method limit of quantitation (LOQ) of 1.00 mg C/L. The TOC contents of the old solutions appeared to increase slightly due to the addition of feed during the test. The results of the study were based on the nominal loading rates.

Validity criteria fulfilled:

yes

Remarks:

See 'details on results field' for more information.

Conclusions:

A study was conducted to assess the toxicity of the test substance to Daphnia magna over a period of 21 days, using nominal loading rates of 0.025, 0.05, 0.1, 0.2 and 0.4 mg/L under semi-static conditions. A statistically significant decrease in survival was noted at a the 0.4 mg/L loading rate, with no statistical differences noted in growth or reproduction at

Description of key information

A study was conducted to assess the toxicity of the test substance to Daphnia magna over a period of 21 days, using nominal loading rates of 0.025, 0.05, 0.1, 0.2 and 0.4 mg/L under semi-static conditions. A statistically significant decrease in survival was noted at the 0.4 mg/L loading rate, with no statistical differences noted in growth or reproduction at </= 0.2 mg/L. Consequently, the NOEL was set as 0.2 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.2 mg/L