Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 23, 2015 - January 12, 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
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Name: 1,2-Bis[pentabromophenyl]ethane, [Phenyl- 14C[U]]
Lot/Batch number: 3626190
Specific activity: 32.4 mCi/mmol (determined by weight assay of the precursor
DBDPEthane CAS 84852-53-9
Bibenzyl, [Phenyl-14C[U]])
Formula weight: 972
Radiochemical purity: 94.5%
Specific details on test material used for the study:
The 14C radiolabelled test substance used to prepare the test solutions for the study was received
from Perkins Elmer, Inc. on October 29, 2014, and was assigned EAG Laboratories-Easton identification
number 11975, upon receipt. The radiolabelled test substance was stored under frozen conditions. The
test substance, a solid, was identified as: 1,2-bis(pentabromophenyl)ethane, [phenyl-14C(U)]; Lot number
1930929. The test substance had a reported specific activity of 36.75 mCi/mmol, and a radiochemical
purity of 98.9% by HPLC (see Appendix 2 of Study Report).

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples were collected from each treatment and control group 4 and 1 days prior to the start of
the test after conditioning the diluter for three and seven days. 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. Additional samples of stock solutions were collected to confirm the stock concentrations
on Day 0 and 21 of the test to confirm the test stock concentrations. Additional samples were also
collected on Day 13 of the test when a leak was found in the connector of the syringe used to deliver the
test stock solution of the 294 ng/L treatment level and on Day 14 after the system resumed its normal
operation. On Day 14 of the test just prior to the scheduled sampling interval, leaks were discovered in
the connectors of the syringes used to deliver the test stock solutions of the 294 and 588 ng/L treatment
levels. Therefore, additional samples were collected when the problem was found and later on the same
day after the problem was corrected. The second set of samples served as samples for the schedule
sampling interval for that day (Day14) and the results were also used to confirm the diluter operation after
the problem was corrected. The additional samples collected on Days 13 and 14 were analyzed to
confirm the test solution concentration when the problem was found and after the problem was corrected.
All stock solution samples (1.00 mL) were collected volumetrically into glass scintillation vials and
process immediately for analysis or store refrigerated until analysis. All test solution samples (10.0 mL)
were collected volumetrically from mid-depth, placed in glass vials and processed immediately for
analysis or stored refrigerated until analysis. At each sampling interval, a second set of samples were
collected and stored as backup samples for possible analysis if needed.

Test solutions

Vehicle:
yes
Remarks:
Dimethylformamide (HPLC-grade)
Details on test solutions:
Individual stock solutions were prepared for each of the two concentrations tested, and were
prepared once during the study. Each test stock solution was prepared by mixing a calculated amount of
radiolabelled decabromodiphenyl ethane into HPLC-grade dimethylformamide (DMF) at a nominal
concentration of 2940 and 5879 ng/mL. The stock solutions were mixed by inversion, and appeared clear
and colorless, with no visible precipitate noted. Stock solutions were stored refrigerated in glass graduate
cylinder or glass volumetric flask, and aliquots of each stock were placed in the syringe pump every 2 to
4 days during the study.
The two test substance stock solutions were injected into the diluter mixing chambers at a rate of
15.5 μL/minute where they were mixed with dilution water delivered at a rate of 155 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 treatment groups was 0.1 mL/L.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The cladoceran, Daphnia magna, was selected as the test species for this study. Daphnids are
representative of an important group of aquatic invertebrates and were selected for use in the test based
upon past history of use in the laboratory. Daphnid neonates used in the test were less than 24 hours old
and were obtained from cultures maintained by EAG Laboratories-Easton, Easton, Maryland.
Identification of the species was verified by the supplier of the original stock culture.
Adult daphnids were cultured in water from the same source and at approximately the same
temperature as used during the test. During the 2-week period immediately preceding the test, water
temperatures in the cultures ranged from 19.5 to 21.1ºC, measured with a hand-held, liquid-in-glass
thermometer. The pH of the water ranged from 8.1 to 8.4, measured with a Thermo Orion Model
Benchtop 4 Star Plus pH meter. Dissolved oxygen concentrations were 7.8 mg/L (87% of saturation),
measured with a Thermo Orion Benchtop 3 Star Plus dissolved oxygen meter.
During culture and testing, daphnids were fed a mixture of yeast, cereal grass media, and trout
chow (YCT), supplemented with a vitamin stock solution and a suspension of the freshwater green alga,
Pseudokirchneriella subcapitata. Daphnids were fed two or three times per day through Day 7 of the test
and then were fed four times per day until the last day of the test. At each feeding, each test chamber was
fed 0.80 mL of YCT, 1.5 mL of algae. In addition, each test chamber was also fed with 0.50 mL of
vitamin solution once daily. This amount of feed is equal to approximately 0.2 to 0.7 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. The concentrations of selected organic and inorganic
constituents in the YCT are measured at least annually and the results from the most recent analysis are
presented in Appendices 3, 4 and 5 of the Study Report.
The four adult daphnids used to supply neonates for the test were held for 21 days prior to
collection of the juveniles for testing, and had each produced at least one previous brood. 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. 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.

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
N/A

Test conditions

Hardness:
138 +/- 2, range 136 - 140 (mg/L as CaCO3); Negative control
Test temperature:
20.3 degrees C +/- 0.13, range 20.1 - 20.4; Negative control
pH:
8.2 +/- 0.10, range 8.1 - 8.3; Negative control
Dissolved oxygen:
8.8 +/- 0.26, range 8.5 - 9.1; Negative control
Salinity:
N/A
Conductivity:
404 +/- 55, range 354 - 483; Negative control
Nominal and measured concentrations:
295 ng/L (nominal); 255.5 +/- 61.7 ng/L (measured)
588 ng/L (nominal); 355.5 +/- 73.2 ng/L (measured)
Details on test conditions:
Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that
emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a
photoperiod of 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
was measured at the water surface of one representative test chamber at the beginning of the test using a
SPER Scientific Model 840006 light meter.
The target test temperature during the test was 20 ± 1°C. Temperature was measured in one
replicate test chamber at the beginning of the test and in each test chamber at approximately weekly
during the test, and at the end of the test using a digital thermometer. Temperature also was monitored
continuously in one negative control test chamber using a validated environmental monitoring system
(Amegaview Central Monitoring System), which was calibrated prior to exposure initiation and verified
or calibrated approximately weekly during the test with a digital thermometer. The recorder was verified
prior to test initiation and approximately weekly during the test using a digital thermometer.
Dissolved oxygen was measured in one replicate test chamber of each treatment and control
group at the beginning of the test, approximately three times per week during the test, and at the end of
the test. Measurements typically rotated between the two replicates in each treatment or control group at
each measurement interval. Measurements of pH were made in one replicate test chamber of each
treatment and control group at the beginning of the test, approximately weekly during the test, and at the
end of the test. Measurements typically rotated between the two replicates in each treatment or control
group at each measurement interval, with the exception of the measurements of pH on Day 21 were
inadvertently made in the same replicate as the previous measurements (i.e., measurements of pH on Day
21 were made in replicate A instead of in replicate B). Dissolved oxygen was measured using a Thermo
Orion Star A213 dissolved oxygen meter, and measurements of pH were made using a Thermo Orion
Dual Star pH/ISE meter.
Hardness, alkalinity and specific conductance were measured in alternating replicates of the
negative control (dilution water) and the highest test concentration at the beginning of the test,
approximately weekly during the test and at the end of the test. However, the measurements of hardness,
alkalinity and specific conductance on Day 14 were inadvertently made in the same replicate as in the
previous measurement (i.e., they were made in replicate B instead of replicate A). Hardness and
alkalinity were measured by titration based on procedures in Standard Methods for the Examination of
Water and Wastewater (4). Specific conductance was measured using an Acorn Series Model CON6
Conductivity-Temperature meter or a Thermo Orion Star A122 portable conductivity meter. Total
organic carbon (TOC) in the dilution water at the beginning and end of the test was measured using a
Shimadzu Model TOC-VCSH total organic carbon analyzer, based on procedures in Standard Methods
for the Examination of Water and Wastewater (Reference 4 in Study Report).
Observations of each first-generation daphnid were made daily during the test. At these times,
the numbers of immobile daphnids were recorded along with any clinical signs of toxicity (e.g., inability
maintain position in the water column, discoloration (e.g., pale or dark) and cessation of feeding).
Immobility was defined as unable to swim within 15 seconds after gentle agitation of the test vessel are
considered to be immobilized (even if they can still move their antennae). The presence of eggs in the
brood pouch, aborted eggs, males or ephippia also were recorded daily. With the onset of reproduction,
neonates produced by the first-generation daphnids were counted and then discarded every Monday,
Wednesday and Friday during the test. The body length of each surviving first-generation daphnid were
measured at the end of the test.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
356 ng/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other:
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 356 ng/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other:
Key result
Duration:
21 d
Dose descriptor:
other: Max. Acceptable Toxicant Concentration
Effect conc.:
> 356 ng/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other:
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 356 ng/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 356 ng/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 356 ng/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Results with reference substance (positive control):
N/A
Reported statistics and error estimates:

Survival data was considered to be discrete-variable data, while reproduction and growth data
were considered continuous-variable data. Discrete-variable data were analyzed using Chi-square and
Fisher’s Exact test to identify treatment groups that showed a statistically significant difference (α = 0.05)
from the pooled control. All continuous-variable data were evaluated for normality using Shapiro-Wilk’s
or chi-square tests and for homogeneity of variance using Levene’s test (α = 0.01). When the data passed
the assumptions of normality and homogeneity, those treatments that were significantly different from the
pooled control means were identified using Bonferroni t-test or Dunnett’s one-tailed test (α = 0.05). All
statistical tests were performed using a personal computer with TOXSTAT (5) or SAS (6) software.
The results of the statistical analyses were used to aid in the determination of the NOEC, LOEC and
MATC. However, scientific judgment was used to determine if statistical differences were biologically
meaningful, and if the data followed a concentration-dependent response. The NOEC was defined as the
highest test concentration that produced no significant treatment-related effects on survival, reproduction or
growth. The LOEC was defined as the lowest test concentration that produced a significant
treatment-related effect on survival, reproduction or growth. The MATC was calculated as the geometric
mean of the NOEC and LOEC. EC10 and EC50 values based on reproduction and EC50 value based on
immobility observed in the first-generation daphnids at the end of the test were not determined since there
were less than 50% in percent immobility or in reproduction in any of the treatment groups from the
control groups (7, 8).

Any other information on results incl. tables

Summary Results: Based On Mean, Measured Test Concentrations

Treatment Groups: Control; Solvent Control; 256 ng/L (measured); 356 ng/L (measured)

NOEC (ng/L)

356

LOEC (ng/L)

>356

Max. Acceptable Toxicant Concentration (ng/L)

>356

21-Day EC50 – Immobility (ng/L)

>356

95% Conf. Interval (ng/L)

Not calculated

21-Day EC10 – Reproduction (ng/L)

>356

95% Conf. Interval (ng/L)

Not calculated

21-Day EC50 – Reproduction (ng/L)

>356

95% Conf. Interval (ng/L)

Not calculated

Summary of Survival, Reproduction and Growth ofDaphnia magnaExposed to 14C-DBDPE for 21 Days

Mean Measured

Concentration1

(ng/L)

Percent Adult

Survival1

Mean No. Neonates

Per Reproductive Day

+/- Std. Dev.

Mean No. Neonates

Per Adult at

The Beginning of the Test

+/- Std. Dev.

Mean Length

+/- Std. Dev.

(mm)

Negative Control

90

11.8 +/- 1.3

161.3 +/- 18

4.7 +/- 0.050

Solvent Control

90

11.0 +/- 0.72

144.8 +/- 4.5

4.6 +/- 0.15

Pooled Control

90

11.4 +/- 1.1

153.0 +/- 15

4.7 +/- 0.12

256

95

12.5 +/- 0.41

172.0 +/- 11

4.7 +/- 0.050

356

100

12.8 +/- 0.29

178.8 +/- 4.1

4.8 +/- 0.082

1There were no statistically-significant differences in percent survival (Fisher’s Exact test, p>0.05), reproduction (Dunnett one-tailed test, p>0.05) or length (Bonferroni t-test, p>0.05) when compared to the pooled control.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The cladoceran, Daphnia magna, was exposed to 14C-decabromodiphenyl ethane at mean
measured concentrations of 256 and 356 ng/L under flow-through conditions for 21 days. There were no
statistically significant treatment-related effects on survival, reproduction or growth at concentrations
≤356 ng/L. Consequently, the NOEC, based on the results of the study, was 356 ng/L. The LOEC was
>356 ng/L and the MATC was calculated to be >356 ng/L. The 21-day EC50 value for adult immobility
was >356 ng/L, the highest concentration tested. The 21-day EC10 and EC50 values for reproduction was
>356 ng/L, the highest concentration tested. The test concentration was limited by the solubility in the test medium and was the maximum concentration that could be obtained.
Therfore the substance does not show any effects in the study at the maximum obtainable concentration in the test medium that was 356 ng/L.
Executive summary:

The cladoceran, Daphnia magna, was exposed to 14C-decabromodiphenyl ethane at mean

measured concentrations of 256 and 356 ng/L under flow-through conditions for 21 days. There were no

statistically significant treatment-related effects on survival, reproduction or growth at concentrations

≤356 ng/L. Consequently, the NOEC, based on the results of the study, was 356 ng/L. The LOEC was

>356 ng/L and the MATC was calculated to be >356 ng/L. The 21-day EC50 value for adult immobility

was >356 ng/L, the highest concentration tested. The 21-day EC10 and EC50 values for reproduction was

>356 ng/L, the highest concentration tested.

The test concentration was limited by the solubility in the test medium and was the maximum concentration that could be obtained.

Therfore the substance does not show any effects in the study at the maximum obtainable concentration in the test medium that was 356 ng/L.