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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 1, 2017 to January 25, 2018
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:
Organization for Economic Cooperation and Development. 2012. OECD Guidelines for Testing of Chemicals, Guideline 211: Daphnia magna Reproduction Test. Adopted 2 October 2012.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Version / remarks:
U.S. Environmental Protection Agency. 2016. Series 850 – Ecological Effects Test Guidelines, OCSPP Number 850.1300: Daphnid Chronic Toxicity Test.
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
yes
Details on sampling:
Samples were collected from each treatment and control group one day prior to the start of the test after conditioning the diluter for three 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 were also collected on Days 0 and 21 of the test and stored (refrigerated) as backup samples for possible future analyses if needed. All samples (9.0 mL) were collected volumetrically from mid-depth, placed in glass vials containing 1.0 mL of tetrahydrofuran (THF) and processed immediately for analysis.
Vehicle:
yes
Remarks:
HPLC-grade dimethylformamide (DMF)
Details on test solutions:
Individual stock solutions were prepared for each of the five concentrations tested, and were prepared four times during the study. A primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 100 mg/mL. The primary stock solution was mixed by sonication for 30 or 60 minutes, followed by inversion and appeared clear and light yellow in color with no visible precipitate. Four secondary stock solutions were prepared in DMF at nominal concentrations of 6.3, 13, 25 and 50 mg/mL by proportional dilution of the primary stock. The secondary stock solutions were mixed by inversion, and appeared clear and colorless to clear and very slightly yellow with no visible precipitate noted. Stock solutions were stored refrigerated in glass graduate cylinder or glass amber bottles, and aliquots of each stock were placed in the syringe every 1 to 2 days during the study.
The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 18.0 μL/minute where they were mixed with dilution water delivered at a rate of 180 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 tetrabromophthalic anhydride treatment groups was 0.1 mL/L.
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 19.6 to 21.2ºC, measured with a digital thermometer. The pH of the water ranged from 8.2 to 8.9, measured with a Thermo Orion 4 Star Star pH/ISE meter. Dissolved oxygen concentrations were >7.4 mg/L (>82% of saturation), measured with a Thermo Orion 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, with the exception of Days 8, 9, 15 and 16 when they were fed 3 times. Since the daphnids were fed an excess amount of feed as recommended by the guidelines, the omission of one feeding on these days had no adverse impact on the organisms. At each feeding during the test, each test chamber was fed 0.80 mL of YCT, and 1.5 mL of algae. In addition, each test chamber also received 0.5 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 4 adult daphnids used to supply neonates for the test were held for at least 14 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.
Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
Not specified
Hardness:
140 - 144 mg/L as CaCO3
Test temperature:
18.7 - 20.3 °C
pH:
8.1 - 8.4
Dissolved oxygen:
6.6 - 9.1 mg/L
Salinity:
Not applicable
Conductivity:
320 - 360 μS/cm
Nominal and measured concentrations:
nominal test concentrations of 0.63, 1.3, 2.5, 5.0 and 10 mg/L
Details on test conditions:
Environmental 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 each test chamber at the beginning of the test, approximately weekly during the test, and at the end of the test using a liquid-in-glass thermometer. Temperature also was monitored continuously in one negative control test chamber using a min/max thermometer, which were verified or calibrated prior to exposure initiation with a digital thermometer and the readings were recorded daily during the test.
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. Additional measurement was were taken on Day 7 in the 2.5 mg/L treatment replicate to monitor the dissolved oxygen concentration after the test chamber and delivery line were replaced. 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. 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. When 100% immobility occurred in a test chamber, measurements of temperature, dissolved oxygen and pH were taken in that test chamber and then discontinued.
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. Hardness and alkalinity were measured by titration based on procedures in Standard Methods for the Examination of Water and Wastewater. Specific conductance was measured using 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.

Biological Observations and Measurements
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 to maintain position in the water column, uncoordinated swimming or cessation of feeding). Immobility was defined as unable to swim within 15 seconds after gentle agitation of the test vessel, 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 approximately three times per week during the test (e.g., every Monday, Wednesday and Friday). The body length and the dry weight of each surviving first-generation daphnid were measured at the end of the test.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival, reproduction & growth
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival, reproduction & growth
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival, reproduction & growth
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival, reproduction & growth
Details on results:
Measurement of Test Concentrations
Nominal concentrations selected for use in the study were 0.63, 1.3, 2.5, 5.0 and 10 mg/L.
During the course of the test, the appearance of the test solutions at these nominal concentrations was observed in both the diluter mixing chambers, where test substance stocks and dilution water were combined prior to delivery to the test chambers, and in the test chambers. The test solutions in the mixing chambers and test chambers appeared clear and colorless during the test, with no evidence of precipitation observed in any control or treatment solution.
The measured concentrations of samples collected to verify the diluter system prior to the test ranged from 112 to 118% of nominal concentrations (Table 2). Samples of the test solutions collected during the test had measured concentrations that ranged from 96.4 to 120% of nominal concentrations. When the measured concentrations of test solution samples collected on Days 0, 7, 13 and 21 of the test were averaged for each treatment group, the mean measured test concentrations were 0.67, 1.4, 2.7, 5.4 and 11 mg/L, which represented 106, 108, 108, 108 and 110% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.

Water Chemistry Measurements
Water temperatures were within the 20 ± 1°C range established for the test. Dissolved oxygen concentrations remained ≥73% of saturation (6.6 mg/L). Measurements of pH ranged from 8.0 to 8.4 during the test. Measurements of specific conductance, hardness and alkalinity were comparable between the control and treatment group and did not appear to be influenced by tetrabromophthalic anhydride concentration. TOC in the dilution water at test initiation and termination was <2 mg C/L. Light intensity at test initiation was 1040 lux at the surface of the water of one representative test chamber.

Survival and Clinical Observations
After 21 days of exposure, survival in both the negative control and solvent control groups were 95%. There was 100% immobility in replicate A of the 2.7 mg/L treatment group on Day 17. However, this immobility was limited to only replicate A and was not consistent with survival in the remaining replicates or with survival at higher test concentrations. Therefore the immobility in replicate A of the 2.7 mg/L treatment group was not considered to be treatment related, and the data from this replicate was excluded from the data analysis. Survival in the pooled control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups at test termination was 95, 90, 100, 93, 90 and 90%, respectively. Fisher’s Exact test indicated no statistically significant decrease in survival in any of the treatment groups in comparison to the pooled control (p > 0.05). Consequently, the NOEC for survival was 11 mg/L and the LOEC was >11 mg/L. Because there was less than 50% immobility in any of the treatment groups during the test, which precluded the statistical calculation of an EC50 value. The 21-day EC50 value was estimated to be >11 mg/L, the highest concentration tested.
Daphnids in the negative control, solvent control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups that survived to test termination generally appeared normal. The observations of lethargy, discoloration and/or small in stature were also noted in the solvent control 0.67, 2.7, 5.4 and 11 mg/L treatment groups. One daphnid in the 11 mg/L treatment group was noted with hemorrhage on the side of the body. However, these incidental observations were limited to one or two daphnids per test group and they are not considered to be treatment related effects.

Reproduction
Reproduction in replicate A of the 2.7 mg/L treatment group was excluded from the analysis since the immobility of all first generation daphnid in this replicate was suspect. The first day of brood production in the negative and solvent control replicates and in all tetrabromophthalic anhydride treatment replicates was Day 8 or 9 of the test, indicating that there was no apparent delay in the onset of production at any tetrabromophthalic anhydride concentration tested. The reproduction rate of the first brood in the negative control, solvent control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups was 0.125, 0.125, 0.129, 0.122, 0.128, 0.125 and 0.121, respectively.
During the course of the test, there were total of 3, 0, 3, 6, 10, 3 (Replicate A excluded), 0 and 2 immobile, and 5, 0, 5, 0, 36, 27 (Replicate A excluded), 3 and 15 aborted/shed eggs neonates noted in the negative control, solvent control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups, respectively. No males or ephippia were produced during the test. Adult daphnids in the negative and solvent control groups produced an average of 15.2 and 14.4 live young per reproductive day, respectively. There was no significant difference in reproduction between the negative and solvent control groups. Therefore, the control data were pooled for comparison with the treatment groups. Adult daphnids in the pooled control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups produced an average of 14.8, 14.0, 14.1, 13.9, 14.8 and 14.6 live young per reproductive day, respectively. Dunnett’s test indicated there were no statistically significant decreases in mean neonate production per reproductive day in any of the treatment groups in comparison to the pooled control (p ≤ 0.05).
Adult daphnids in the negative and solvent control groups produced an average of 201 and 192 live young per adult at the beginning of the test, respectively. The control data were pooled for comparison with the treatment groups because there was no significant difference in reproduction between the negative and solvent control groups. Adult daphnids in the pooled control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups produced an average of 196, 192, 197, 188, 186 and 191 live young per adult at the beginning of the test, respectively.
Consequently, based on the results of reproduction measured as the mean number of live neonates produced per reproductive day and per adult at the beginning of the test, the NOEC was 11 mg/L and the LOEC was >11 mg/L. Based on the mean number of live neonates produced per reproductive day observed in the treatment groups, the 21-day EC10 and EC50 values were both >11 mg/L, the highest concentration tested. The 21-day EC10 and EC50 values based on mean number of live neonates
produced per adult at the beginning of the test was >11 mg/L, the highest concentration tested. Since the 95% confidence interval was outside of the data range used for the calculation, it was not reported.

Growth
Daphnids in the negative control group averaged 4.8 mm in length and 1.40 mg in dry weight, while daphnids in the solvent control group averaged 4.8 mm in length, and 1.43 mg in dry weight. There were no significant differences in the growth parameters between the negative and solvent control groups. Therefore, the control data were pooled for comparison with the treatment groups. Daphnids in the pooled control, 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups had mean lengths of 4.8, 4.8, 4.8, 4.8, 4.7 and 4.7 mm, respectively, and mean dry weights of 1.42, 1.48, 1.43, 1.39, 1.44 and 1.44 mg, respectively. Consequently, the NOEC for growth (length and dry weight) was 11 mg/L, and the LOEC was >11 mg/L.
Results with reference substance (positive control):
Negative and solvent control used for this study.
Reported statistics and error estimates:
Jonckhere-Terpstra step down trend test indicated there were no statistically significant decreases in mean neonate produced per adult at the beginning of the test.
No statistically significant decreases (p > 0.05) in mean length or mean dry weight were detected in any of the treatment groups from the pooled control (Jonckhere-Terpstra step down trend test for length and Dunnett’s one-tailed test for dry weight).

Means and Ranges of Water Quality Measurements Taken During the 21-Day Exposure to Tetrabromophthalic Anhydride

Mean Measured Concentration (mg/L)

Mean ± Std. Dev. and Range of Measured Parameters1

Temperature2(°C)

Dissolved Oxygen3(mg/L)

pH

Hardness4

(mg/L as CaCo3)

Alkalinity4

(mg/L as CaCO3)

Conductivity4(μS/cm)

Negative Control

19.2 ± 0.20

(18.9 – 19.5)

8.6 ± 0.31

(7.9 – 9.0)

8.2 ± 0.17

(8.0 – 8.4)

140 ± 6

(132 – 144)

181 ± 4

(178 – 186)

341 ± 16

(328 – 362)

Solvent Control

19.2 ± 0.19

(18.8 – 19.5)

8.5 ± 0.38

(7.9 – 9.0)

8.2 ± 0.16

(8.0 – 8.4)

--

--

--

--

--

--

0.67

19.4 ± 0.20

(19.2 – 19.8)

8.3 ± 0.69

(6.9 – 9.0)

8.2 ± 0.13

8.1 -8.4)

--

--

--

--

--

--

1.4

19.4 ± 0.40

(18.7 – 20.3)

8.2 ± 0.64

(7.1 – 9.1)

8.3 ± 0.10

(8.2 – 8.4)

--

--

--

--

--

--

2.7

19.3 ± 0.13

(19.1 – 19.5)

8.0 ± 0.90

6.6 – 9.1)

8.3 ± 0.10

(8.2 – 8.4)

--

--

--

--

--

--

5.4

19.2 ± 0.14

(19.0 – 19.4)

8.2 ± 0.77

(7.0 – 9.1)

8.3 ± 0.10

(8.2 – 8.4)

--

--

--

--

--

--

11

19.4 ± 0.24

(19.1 – 20.0)

8.4 ± 0.59

(7.3 – 9.1)

8.2 ± 0.05

(8.2 – 8.3)

141 ± 2

(140 – 144)

175 ± 4

(168 – 178)

340 ± 16

(320 – 360)

1Total organic carbon measured in the dilution water at test initiation and temperature was <2 mg C/L.

2Temperature monitored continuously during the test ranged from 19.1 to 19.8 °C, measured using a min/max thermometer.

3A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20 °C in freshwater.

4-- = no measurements scheduled.

 

Summary of Survival, Reproduction and Growth of Daphnia magna Exposed to Tetrabromophthalic Anhydride for 21 Days

Mean Measured Concentration (mg/L)

Percent Adult Survival1

Production Rate of First Brood (day-1)

Mean No. Neonates Per Reproductive Day ± Std. Dev.1

Mean No. Neonates Per Adult at Beginning of Test ± Std. Dev.1

Mean Length ± Std. Dev. (mm)1

Mean Dry Weight ± Std. Dev. (mg)1

Negative Control

Solvent Control2

Pooled Control

0.67

1.4

2.7

5.4

11

95

95

95

90

100

932

90

90

0.125

0.125

0.125

0.129

0.122

0.128

0.125

0.121

15.2 ± 1.6

14.4 ± 0.67

14.8 ± 1.2

14.0 ± 2.4

14.1 ± 0.79

13.9 ± 2.2

14.8 ± 1.1

14.6 ± 0.73

201 ± 15.2

192 ± 24.2

196 ± 19.3

192 ± 30.4

197 ± 11.1

188 ± 42.2

186 ± 31.8

191 ± 14.7

4.8 ± 0.050

4.8 ± 0.050

4.8 ± 0.046

4.8 ± 0.050

4.8 ± 0.058

4.8 ± 0.058

4.7 ± 0.096

4.7 ± 0.096

1.40 ± 0.095

1.43 ± 0.045

1.42 ± 0.071

1.48 ± 0.10

1.43 ± 0.067

1.39 ± 0.050

1.44 ± 0.12

1.44 ± 0.060

1There were no significant difference from the pooled control in survival (Fisher;s Exact test, p>0.05); in mean number of neonates produced per reproductive day (Dunnett’s one-tailed test, p>0.05); in mean number of neonates produced per adult at the beginning of the test (Jonckheere-Terpstra step down trend test, p>0.05); in mean length (Jonckheere-Terpstra step down trend test, p>0.05) and in dry weight (Dunnett;s one-tailed test, p>0.05).

2The survival data of the 2.7 mg/L treatment replicate A was excluded from the analysis of survival at test end due to unexpected immobility of all first generation daphnids in this replicate on Day 17 of the test.

 

Validity criteria fulfilled:
yes
Conclusions:
The cladoceran, Daphnia magna, was exposed to tetrabromophthalic anhydride at mean measured concentrations of 0.67, 1.4, 2.7, 5.4 and 11 mg/L under flow-through conditions for 21 days.
There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤11 mg/L. Consequently, the NOEC for the test was 11 mg/L. The LOEC was >11 mg/L and the MATC was estimated to be >11 mg/L.
The EC50 for immobility and EC10 and EC50 values for reproduction were greater than 11 mg/L, the highest concentration since there were less than 50% reduction in immobility and reproduction.
Executive summary:

The objective of the study was to determine the effects of tetrabromophthalic anhydride on the survival, growth and reproduction of the cladoceran, Daphnia magna, during a 21-day exposure period under flow-through test conditions.

 

The protocol was based on procedures outlined in the OECD Guidelines for Testing of Chemicals, Guideline 211: Daphnia magna Reproduction Test and the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines, OCSPP Number 850.1300: Daphnid Chronic Toxicity Test.

 

Daphnids were exposed to a geometric series of five test concentrations and a negative control (dilution water) and solvent control (0.1 mL HPLC-grade dimethylformamide/L). Four replicate test chambers were tested for each treatment and control group. Each replicate contained one compartment with five daphnids, resulting in a total of 20 daphnids in each treatment and control group. Nominal test concentrations were selected in consultation with the Sponsor based on a non-GLP solubility trial and exploratory range finding toxicity data. The nominal test concentrations were 0.63, 1.3, 2.5, 5.0 and 10 mg/L. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at test initiation, at approximately weekly intervals during the test and at test termination.

 

Delivery of the test substance to the test chambers was initiated approximately 4 days prior to the introduction of the daphnids to the test water in order to achieve equilibrium of the test substance in the test chambers. At test initiation, neonate daphnids were impartially assigned to exposure chambers.

First-generation daphnids were observed daily during the test for immobility, the onset of reproduction, and clinical signs of toxicity. Following the onset of reproduction, the numbers of second-generation daphnids were counted three times per week (e.g., on Monday, Wednesday and Friday), and at test termination (Day 21). Body lengths and dry weights of the surviving first-generation daphnids were measured at the end of the exposure period. Observations of the effects of tetrabromophthalic anhydride on survival, reproduction and growth were used to determine the no-observed-effect concentration (NOEC), the lowest-observed-effect concentration (LOEC) and the maximum acceptable toxicant concentration (MATC). An EC10 value was calculated for reproduction and EC50 values were calculated based on reproduction and on first-generation immobility at test termination, when possible.

 

RESULTS

Survival and Clinical Observations

After 21 days of exposure, survival in both the negative control and solvent control groups were 95%. There was 100% immobility in replicate A of the 2.7 mg/L treatment group on Day 17. However, this immobility was limited to only replicate A and was not consistent with survival in the remaining replicates or with survival at higher test concentrations.

Therefore the immobility in replicate A of the 2.7 mg/L treatment group was not considered to be treatment related, and the data from this replicate was excluded from the data analysis. Survival in the pooled control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups at test termination was 95, 90, 100, 93, 90 and 90%, respectively. Consequently, the NOEC for survival was 11 mg/L and the LOEC was >11 mg/L. Because there was less than 50% immobility in any of the treatment groups during the test, which precluded the statistical calculation of an EC50 value. The 21-day EC50 value was estimated to be >11 mg/L, the highest concentration tested.

Daphnids in the negative control, solvent control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups that survived to test termination generally appeared normal. The observations of lethargy, discoloration and/or small in stature were also noted in the solvent control 0.67, 2.7, 5.4 and 11 mg/L treatment groups. One daphnid in the 11 mg/L treatment group was noted with hemorrhage on the side of the body. However, these incidental observations were limited to one or two daphnids per test group and they are not considered to be treatment related effects.

 

Reproduction

Reproduction in replicate A of the 2.7 mg/L treatment group was excluded from the analysis since the immobility of all first generation daphnid in this replicate was suspect. The first day of brood production in the negative and solvent control replicates and in all tetrabromophthalic anhydride treatment replicates was Day 8 or 9 of the test, indicating that there was no apparent delay in the onset of production at any tetrabromophthalic anhydride concentration tested. The reproduction rate of the first brood in the negative control, solvent control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups was 0.125, 0.125, 0.129, 0.122, 0.128, 0.125 and 0.121, respectively.

During the course of the test, there were total of 3, 0, 3, 6, 10, 3 (Replicate A excluded), 0 and 2 immobile, and 5, 0, 5, 0, 36, 27 (Replicate A excluded), 3 and 15 aborted/shed eggs neonates noted in the negative control, solvent control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups, respectively. No males or ephippia were produced during the test.

Adult daphnids in the negative and solvent control groups produced an average of 15.2 and 14.4 live young per reproductive day, respectively. There was no significant difference in reproduction between the negative and solvent control groups. Therefore, the control data were pooled for comparison with the treatment groups. Adult daphnids in the pooled control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups produced an average of 14.8, 14.0, 14.1, 13.9, 14.8 and 14.6 live young per reproductive day, respectively.

Adult daphnids in the negative and solvent control groups produced an average of 201 and 192 live young per adult at the beginning of the test, respectively. The control data were pooled for comparison with the treatment groups because there was no significant difference in reproduction between the negative and solvent control groups. Adult daphnids in the pooled control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups produced an average of 196, 192, 197, 188, 186 and 191 live young per adult at the beginning of the test, respectively. Consequently, based on the results of reproduction measured as the mean number of live neonates produced per reproductive day and per adult at the beginning of the test, the NOEC was 11 mg/L and the LOEC was >11 mg/L. Based on the mean number of live neonates produced per reproductive day observed in the treatment groups, the 21-day EC10 and EC50 values were both >11 mg/L, the highest concentration tested. The 21-day EC10 and EC50 values based on mean number of live neonates produced per adult at the beginning of the test was >11 mg/L, the highest concentration tested.

 

Growth

Daphnids in the negative control group averaged 4.8 mm in length and 1.40 mg in dry weight, while daphnids in the solvent control group averaged 4.8 mm in length, and 1.43 mg in dry weight. There were no significant differences in the growth parameters between the negative and solvent control groups. Therefore, the control data were pooled for comparison with the treatment groups. Daphnids in the pooled control, 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups had mean lengths of 4.8, 4.8, 4.8, 4.8, 4.7 and 4.7 mm, respectively, and mean dry weights of 1.42, 1.48, 1.43, 1.39, 1.44 and 1.44 mg, respectively. Consequently, the NOEC for growth (length and dry weight) was 11 mg/L, and the LOEC was >11 mg/L.

 

CONCLUSIONS

The cladoceran, Daphnia magna, was exposed to tetrabromophthalic anhydride at mean measured concentrations of 0.67, 1.4, 2.7, 5.4 and 11 mg/L under flow-through conditions for 21 days.

There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤11 mg/L. Consequently, the NOEC for the test was 11 mg/L. The LOEC was >11 mg/L and the MATC was estimated to be >11 mg/L.

The EC50 for immobility and EC10 and EC50 values for reproduction were greater than 11 mg/L, the highest concentration since there were less than 50% reduction in immobility and reproduction.

Description of key information

21 day NOEC (survival, reproduction & growth): 11 mg/L

21 day EC50 >11 mg/L based on survival, reproduction & growth.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
11 mg/L

Additional information

The objective of the study was to determine the effects of tetrabromophthalic anhydride on the survival, growth and reproduction of the cladoceran, Daphnia magna, during a 21-day exposure period under flow-through test conditions.

Daphnids were exposed to a geometric series of five test concentrations and a negative control (dilution water) and solvent control (0.1 mL HPLC-grade dimethylformamide/L). Four replicate test chambers were tested for each treatment and control group. Each replicate contained one compartment with five daphnids, resulting in a total of 20 daphnids in each treatment and control group. The nominal test concentrations were 0.63, 1.3, 2.5, 5.0 and 10 mg/L. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at test initiation, at approximately weekly intervals during the test and at test termination.

 

Delivery of the test substance to the test chambers was initiated approximately 4 days prior to the introduction of the daphnids to the test water in order to achieve equilibrium of the test substance in the test chambers. At test initiation, neonate daphnids were impartially assigned to exposure chambers.

First-generation daphnids were observed daily during the test for immobility, the onset of reproduction, and clinical signs of toxicity. Following the onset of reproduction, the numbers of second-generation daphnids were counted three times per week (e.g., on Monday, Wednesday and Friday), and at test termination (Day 21). Body lengths and dry weights of the surviving first-generation daphnids were measured at the end of the exposure period.

 

RESULTS

Survival and Clinical Observations

After 21 days of exposure, survival in both the negative control and solvent control groups were 95%. There was 100% immobility in replicate A of the 2.7 mg/L treatment group on Day 17. However, this immobility was limited to only replicate A and was not consistent with survival in the remaining replicates or with survival at higher test concentrations. Therefore the immobility in replicate A of the 2.7 mg/L treatment group was not considered to be treatment related, and the data from this replicate was excluded from the data analysis. Survival in the pooled control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups at test termination was 95, 90, 100, 93, 90 and 90%, respectively. Consequently, the NOEC for survival was 11 mg/L and the LOEC was >11 mg/L. Because there was less than 50% immobility in any of the treatment groups during the test, which precluded the statistical calculation of an EC50 value. The 21-day EC50 value was estimated to be >11 mg/L, the highest concentration tested.

Daphnids in the negative control, solvent control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups that survived to test termination generally appeared normal. The observations of lethargy, discoloration and/or small in stature were also noted in the solvent control 0.67, 2.7, 5.4 and 11 mg/L treatment groups. One daphnid in the 11 mg/L treatment group was noted with hemorrhage on the side of the body. However, these incidental observations were limited to one or two daphnids per test group and they are not considered to be treatment related effects.

 

Reproduction

Reproduction in replicate A of the 2.7 mg/L treatment group was excluded from the analysis since the immobility of all first generation daphnid in this replicate was suspect. The first day of brood production in the negative and solvent control replicates and in all tetrabromophthalic anhydride treatment replicates was Day 8 or 9 of the test, indicating that there was no apparent delay in the onset of production at any tetrabromophthalic anhydride concentration tested. The reproduction rate of the first brood in the negative control, solvent control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups was 0.125, 0.125, 0.129, 0.122, 0.128, 0.125 and 0.121, respectively.

During the course of the test, there were total of 3, 0, 3, 6, 10, 3 (Replicate A excluded), 0 and 2 immobile, and 5, 0, 5, 0, 36, 27 (Replicate A excluded), 3 and 15 aborted/shed eggs neonates noted in the negative control, solvent control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups, respectively. No males or ephippia were produced during the test.

Adult daphnids in the negative and solvent control groups produced an average of 15.2 and 14.4 live young per reproductive day, respectively. There was no significant difference in reproduction between the negative and solvent control groups. Therefore, the control data were pooled for comparison with the treatment groups. Adult daphnids in the pooled control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups produced an average of 14.8, 14.0, 14.1, 13.9, 14.8 and 14.6 live young per reproductive day, respectively.

Adult daphnids in the negative and solvent control groups produced an average of 201 and 192 live young per adult at the beginning of the test, respectively. The control data were pooled for comparison with the treatment groups because there was no significant difference in reproduction between the negative and solvent control groups. Adult daphnids in the pooled control and the 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups produced an average of 196, 192, 197, 188, 186 and 191 live young per adult at the beginning of the test, respectively. Consequently, based on the results of reproduction measured as the mean number of live neonates produced per reproductive day and per adult at the beginning of the test, the NOEC was 11 mg/L and the LOEC was >11 mg/L. Based on the mean number of live neonates produced per reproductive day observed in the treatment groups, the 21-day EC10 and EC50 values were both >11 mg/L, the highest concentration tested. The 21-day EC10 and EC50 values based on mean number of live neonates produced per adult at the beginning of the test was >11 mg/L, the highest concentration tested.

 

Growth

Daphnids in the negative control group averaged 4.8 mm in length and 1.40 mg in dry weight, while daphnids in the solvent control group averaged 4.8 mm in length, and 1.43 mg in dry weight. There were no significant differences in the growth parameters between the negative and solvent control groups. Therefore, the control data were pooled for comparison with the treatment groups. Daphnids in the pooled control, 0.67, 1.4, 2.7, 5.4 and 11 mg/L treatment groups had mean lengths of 4.8, 4.8, 4.8, 4.8, 4.7 and 4.7 mm, respectively, and mean dry weights of 1.42, 1.48, 1.43, 1.39, 1.44 and 1.44 mg, respectively. Consequently, the NOEC for growth (length and dry weight) was 11 mg/L, and the LOEC was >11 mg/L.

 

CONCLUSIONS

The cladoceran, Daphnia magna, was exposed to tetrabromophthalic anhydride at mean measured concentrations of 0.67, 1.4, 2.7, 5.4 and 11 mg/L under flow-through conditions for 21 days.

There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤11 mg/L. Consequently, the NOEC for the test was 11 mg/L. The LOEC was >11 mg/L and the MATC was estimated to be >11 mg/L.

The EC50 for immobility and EC10 and EC50 values for reproduction were greater than 11 mg/L, the highest concentration since there were less than 50% reduction in immobility and reproduction.