2,2',6,6'-Tetrabromo-4,4'-isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane and 2,4,6-tribromophenol

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:

19/07/2016 - 11/01/2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The substance was stored under ambient conditions.
The test substance, a solid, was identified as: F-3014;
Batch number 290130349;
CAS number 158725-44-1.
The test substance had an expiration date of July, 2017

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Duplicate samples were collected from each treatment and control group eight and nine days prior to the start of the test after conditioning the diluter for three and two days, respectively. Additional samples were collected from the negative and solvent control groups prior to test initiation to confirm there were not measured concentrations of test substance in the control solutions. Duplicate water samples also were collected from alternating replicate test chambers in each treatment and control group at the beginning of the test, at approximately weekly intervals during the test and at the end of the test to measure concentrations of the test substance. Samples of the stock solutions being delivered to the diluter were collected for analysis on Day 11 of the study. Additional duplicate samples of test solutions were collected on Day 14 due to a test solution delivery malfunction which impacted one replicate test chamber
of the 25, 50 and 100 μg/L treatment groups. All samples were collected from mid-depth. Samples to be analyzed without centrifugation were collected into glass French square bottles using glass volumetric pipettes, while samples to be analyzed after centrifugation were collected into plastic centrifuge tubesusing glass graduated cylinders.

- Sample storage conditions before analysis:At each sampling interval one set of samples were processed immediately for analysis and one set of samples were stored for possible future analysis.

Vehicle:

yes

Remarks:

DMF

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Individual stock solutions were prepared for each of the five concentrations tested, and were prepared six times during the study. Test solution concentrations were not adjusted for the active ingredient of the test substance during preparation, and are based on the test substance as received. The primary stock was sonicatedfor approximately 10 or 15 minutes and appeared clear and colorless, with no visible precipitates. Four secondary stock solutions were prepared in DMF at nominal concentrations of 63, 130, 250 and 500 μg/mL by proportional dilution of the primary stock. Stock solutions were stored refrigerated in glass amber bottles with Teflon®-lined lids, and aliquots of each stock were placed in the syringe every one to four days during the study.
The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 10.00 μL/minute where they were mixed with dilution water delivered at a rate of 100 mL/minute to achieve the desired test concentrations.
- Controls: The negative control received dilution water only. The solvent control was prepared by delivering HPLC-grade DMF to the mixing chamber for the solvent control.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):A primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 1000 μg/mL. The concentration of DMF in the solvent control and all F-3014 treatment groups was 0.1 mL/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):The stock solutions were mixed by inversion, and appeared clear and colorless.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: cladoceran
- Justification for species other than prescribed by test guideline: no
- Source: cultures maintained by EAG Laboratories, Easton, Maryland
- Age of parental stock (mean and range, SD): Daphnid neonates used in the test were less than 24 hours old
- Feeding during test: yes
- Food type: mixture of yeast, cereal grass media, and trout chow (YCT), supplemented with a vitamin stock solution and a suspension of the freshwater green alga, Raphidocelis subcapitata.
- Amount:At each feeding, each test chamber was fed 0.75 mL of YCT, 1.5 mL of algae and 0.50 mL of vitamin solution. This amount of feed is equal to approximately 0.71 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 flow-through system to support acceptable reproduction rates.
- Frequency: Daphnids were fed two or three times per day through Day 6 of the test and then were fed four times per day until the last day of the test.

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not):Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test.
- Type and amount of food: as in the test
- Feeding frequency: as in the test
- Health during acclimation (any mortality observed):Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7-day period prior to the test. 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:
The five adult daphnids used to supply neonates for the test were held for 24 days prior to collection of the juveniles for testing, and had each produced at least one previous brood. . 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 5 daphnids. Each group of neonates then was impartially assigned to a control or treatment group and the neonates were transferred to the test compartments to initiate the test. All transfers were made below the water surface using wide-bore pipettes.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

21-day exposure period under flow-through test conditions. The delivery system and the test chambers were placed in a temperature-controlled environmental chamber to maintain the target water temperature throughout the test period.

Hardness:

140-148 (mg/L as CaCO3)

Test temperature:

20-21 oC

pH:

8.0-8.4

Dissolved oxygen:

5.5-9.1

Salinity:

18.3 mg/L as sodium

Conductivity:

328-382 (μS/cm)

Nominal and measured concentrations:

TEST CONCENTRATIONS:
Nominal Mean Measured
Negative Control < LOQ
Solvent Control < LOQ
6.3 µg/L 6.6 µg/L
13 µg/L 9.9 µg/L
25 µg/L 24 µg/L
50 µg/L 60 µg/L
100 µg/L 102 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Test compartments were 300 mL glass beakers.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Test compartments were 300 mL glass beakers, approximately 6.5 cm in diameter and 12 cm in height. Nylon mesh screens covered two holes on opposite sides of each test compartment to permit test solution to flow in and out of the compartment. The depth of the test water in a representative compartment was approximately 8 cm, while the depth of water in a representative test chamber was approximately 16 cm.
- Aeration: Dissolved oxygen concentrations were 7.4 mg/L (82% of saturation),
- Type of flow-through (e.g. peristaltic or proportional diluter): proportional diluter. Test chambers were 12 L glass aquaria filled with approximately 10 L of test water. The volume in the test chambers was maintained by an overflow port on the side of the test chamber.
- Renewal rate of test solution (frequency/flow rate):The proportion of the test solution that was pumped into each replicate test chamber was checked prior to the test and approximately weekly during the test to ensure that flow rates varied by no more than ± 5% of the mean flow rate for the replicates. The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 10.00 μL/minute where they were mixed with dilution water delivered at a rate of 100 mL/minute to achieve the desired test concentrations. The negative control received dilution water only. The solvent control was prepared by delivering HPLC-grade DMF to the mixing chamber for the solvent control. The concentration of DMF in the solvent control and all F-3014 treatment groups was 0.1 mL/L.
- No. of organisms per vessel: Each replicate contained two compartments with five daphnids, resulting in a total of 20 daphnids in each treatment and control group
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:Easton Well Water
- Total organic carbon:< 1
- Particulate matter:
- Metals: Tables presented in "Any other information on materials and methods" below
- Pesticides:Tables presented in "Any other information on materials and methods" below
- Chlorine:4.3mg/L
- Alkalinity:176 – 178 mg/L as CaCO3
- Ca/mg ratio:
- Conductivity:321 – 367μS/cm
- Culture medium different from test medium:
- Intervals of water quality measurement: During the 4-Week Period Immediately Preceding the Test
Pesticides, Organics and Metals - Analyses performed by Lancaster Laboratories on samples collected on December 7, 2015


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting.
- Light intensity:1160 lux at the surface of the water

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study: yes
- Test concentrations: 8.1, 27, 90 300, 1000 mg/L
- Results used to determine the conditions for the definitive study:nominal test concentrations were 6.3, 13, 25, 50 and 100 μg F-3014/L.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

102 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 102 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

102 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

number of neonates produced per reproductive day and number of neonates produced per live adult at biginning of the test

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 102 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

number of neonates produced per reproductive day and number of neonates produced per live adult at biginning of the test

Details on results:

- Behavioural abnormalities: Daphnids in the control and treatment groups that survived to test termination generally appeared normal throughout the test. There were a few observations of daphnids in the treatment groups that appeared pale, lethargic or were observed to be floating on the surface of the test solution. However, these observations were infrequent, comparable to observations in the control groups and did not follow a dose-response pattern. Therefore, these observations considered to be non-treatment related.
- Observations on body length and weight:Daphnids in the negative control group averaged 5.1 mm in length and 1.29 mg in dry weight, while daphnids in the solvent control group averaged 5.1 mm in length and 1.38 mg in dry weight. There were no significant differences in the growth parameters between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparison with the treatment groups. Daphnids in the pooled control and the 6.6, 9.9, 24, 60 and 102 μg/L treatment groups had mean
lengths of 5.1, 5.1, 4.9, 5.0, 5.0 and 5.0 mm, respectively, and mean dry weights of 1.33, 1.19, 1.34, 1.32, 1.18 and 1.33 mg, respectively. According to Dunnett’s test, there were statistically significant decreases in mean length for the 9.9, 24 and 60 μg/L treatment groups in comparison to the pooled control (p ≤ 0.05). However, the differences were very slight (< 4%), did not follow a dose-response pattern and were likely due to the narrow standard deviations within each set of data. Therefore, the statistically significant decreases detected in the treatment groups were not considered to be biologically meaningful. There were no statistically significant decreases in mean dry weight for any F-3014 treatment group in comparison to the pooled control according to Dunnett’s test (p > 0.05). Consequently, the NOEC for growth (length and dry weight) was 102 μg/L, and the LOEC was >102 μg/L, the highest concentration tested.
- Other biological observations:Adult daphnids in the pooled control and the 6.6, 9.9, 24, 60 and 102 μg/L treatment groups produced an average of 229, 213, 210, 212, 264 and 195 live young per adult at the beginning of the test, respectively. Dunnett’s one-tailed test indicated there were no statistically significant decreases in the mean number of live young produced per adult at the beginning of the test in any F-3014 treatment group in comparison to the pooled control (p > 0.05). Consequently, based on the number of live young produced per adult at the beginning of the test, the NOEC was 102 μg/L and the LOEC was >102 μg/L, the highest concentration tested. Based on the number of live young produced per adult at the beginning of the test observed in the treatment groups, the 21-day EC10 and EC50 values were both determined to be >102 μg/L, the highest concentration tested.
- Mortality of control:After 21 days of exposure, survival in the negative control and solvent control groups was 90.0 and 95.0%, respectively. Since there were no statistically significant differences in survival between the negative and solvent control groups for any parameter tested (p > 0.05), the control data were pooled for comparisons to the treatment groups.
- Other adverse effects control:
- Abnormal responses:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium: In the study concentartion exceed the water solubility of the substance (<1 μg/L) by at least two orders of magnitude using DMF as a carrier. “When a laboratory test design has been specifically modified to accommodate the instability of test material or other factors likely to cause variability in test concentrations, and the design is judged adequate based on sufficient preliminary information, the study will not be rejected solely on the grounds that measured concentrations varied by more than 30 percent of the nominal concentration…The most important criterion is that the test levels must not experience a shift in “order”. That is, the highest test level should remain highest, the next should remain second, etc.” (9). There were no inversions of measured concentrations throughout the study. Therefore, the high variability in measured concentrations should not impact the interpretation of the study results.

Validity criteria fulfilled:

yes

Conclusions:

The cladoceran, Daphnia magna, was exposed to F-3014 at mean measured concentrations of 6.6 to 102 μg/L under flow-through conditions for 21 days. The substance concentartions exceed the water solubility value (<1 μg/L) by at least two orders of magnitude using DMF as a carrier. Nevertheless, there were no biologically meaningful, statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤102 μg/L. Consequently, the NOEC, was determined to be 102 μg/L. The LOEC and MATC were both determined to be >102 μg/L, the highest concentration tested.
The 21-day EC50 value for adult immobility was determined to be >102 μg/L, the highest concentration tested. Based on the number of live young produced per reproductive day, the 21-day EC10 and EC50 values were determined to be >102 μg/L. Based on the number of live young produced per adult at the beginning of the test, the 21-day EC10 and EC50 values were determined to be >102 μg/L.

Executive summary:

The cladoceran, Daphnia magna, was exposed to F-3014 at mean measured concentrations of 6.6 to 102 μg/L under flow-through conditions for 21 days. The substance concentartions exceed the water solubility value (<1 μg/L) by at least two orders of magnitude using DMF as a carrier. Nevertheless, there were no biologically meaningful, statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤102 μg/L.

Description of key information

The cladoceran, Daphnia magna, was exposed to F-3014 at mean measured concentrations of 6.6 to 102 μg/L under flow-through conditions for 21 days. The substance concentartions exceed the water solubility value (<1 μg/L) by at least two orders of magnitude using DMF as a carrier. Nevertheless, there were no biologically meaningful, statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤102 μg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

102 µg/L