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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 15, 2017 tio February 1, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
Organization for Economic Cooperation and Development. 2013. OECD Guidelines for Testing of Chemicals, Guideline 210: Fish, Early-life Stage Toxicity Test. Adopted 26 July 2013.
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Version / remarks:
U.S. Environmental Protection Agency. 2016. Series 850 - Ecological Effects Test Guidelines, OCSPP Number 850.1400: Fish Early Life Stage 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:
Water samples were collected from one test chamber of each treatment and control group two days prior to test initiation to confirm the operation of the diluter. Water samples were collected from alternating replicate test chambers of each treatment and control group on Days 0, 6, 14, 21, 28 and 33 (test termination) to determine concentrations of the test substance in the test chambers. Due to precipitate noted in the 4.0 and 10 mg/L treatment groups, two sets of samples were collected on Day 0. One set was analyzed prior to centrifugation and the second set was analyzed after centrifugation to remove the precipitate. Additional samples were collected on Day 13 of the test when precipitate was noted in the 4.0 and 10 mg/L treatment groups and were stored refrigerated for possible future analysis if needed. On Day 26, additional samples were collected from all replicates of the 4.0 mg/L treatment group when a dilution water delivery to the mixing chamber of this treatment group was interrupted and again after the rotameter was replaced and the delivery of dilution water to the mixing chamber resumed. These samples were analyzed to confirm the test concentration. All samples (9.00 mL) were collected at mid-depth in the test chambers, placed in glass vials containing 1.00 mL of tetrahydrofuran (THF) and processed immediately for analysis or stored refrigerated until processed for analysis.
Vehicle:
yes
Details on test solutions:
Stock solutions were prepared five times during the test. At each preparation, a primary stock solution was prepared in HPLC-grade DMF at a nominal concentration of 100 mg/mL. The solution was sonicated for approximately 60 minutes, followed by inversion to mix and appeared clear and yellow.
Additional stock solutions at nominal concentrations of 2.6, 6.4, 16 and 40 mg/mL were prepared by proportional diluted aliquots of primary stock solution in DMF. The secondary stock solutions were mixed by inversion and appeared clear and colorless, clear and slightly yellow, clear and light yellow to clear and yellow in color. Stock solutions were stored under refrigerated conditions and fresh aliquots were placed in the syringe pumps every two days during the test. The stock solutions were delivered to the diluter mixing chambers (at a rate of 20 μL/minute) where they were mixed with dilution water (at a rate of 200 mL/minute) to achieve the desired test concentrations of 0.26, 0.64, 1.6, 4.0 and 10 mg/L. The solvent control was prepared by injecting HPLC-grade DMF into 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):
Pimephales promelas
Details on test organisms:
The fathead minnow, Pimephales promelas, was selected as the test species for this study. Fathead minnows are one of the preferred fish species to test the toxicity of test substances during the early life-stages of fish (1, 2, 3). This species was selected for use in the test based upon past use and ease of handling in the laboratory. Fathead minnow embryos used in the test 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. The embryos were removed from the spawning substrates and examined under a dissecting microscope to select healthy, viable specimens at approximately the same stage of development (between gastrula and neurula). Embryos collected for use in the test were from 15 individual spawns and were <24 hours old when the test was initiated. To initiate the test, groups of 1 to 3 embryos were impartially distributed among incubation cups until each cup contained 20 embryos. One cup was placed in each treatment and control test chamber.
Newly-hatched larvae were fed live brine shrimp nauplii (Artemia sp.) three times per day during the first seven days of post-hatch. Thereafter, they were fed live brine shrimp nauplii three times per day on weekdays and at least two times per day on weekends. Brine shrimp nauplii were obtained by hatching cysts purchased from Brine Shrimp Direct, Ogden, Utah. The concentrations of selected pesticides and organic and inorganic constituents in the Artemia cysts are measured annually and the results from the most recent analysis are presented in Appendix 3. Fish were not fed for approximately 48 hours prior to the termination of the test to allow for clearance of the digestive tracts before weight measurements were made. To ensure that the feeding rate per fish remained constant, rations were adjusted at least weekly to account for losses due to mortality.
Biomass loading at the end of the test, based on the mean wet weight of the negative control group, was 0.029 g of fish per liter of test solution that passed through the test chamber during a 24-hour period. Instantaneous loading (the total wet weight of fish per liter of water in the tank) at the end of the test was 0.30 g fish/L.
Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
33 d
Remarks on exposure duration:
5-day hatching period plus a 28-day post-hatch growth period
Post exposure observation period:
Not specified
Hardness:
136 - 148 mg/L as CaCO3
Test temperature:
24.0 - 24.7 °C
pH:
8.1 - 8.3
Dissolved oxygen:
6.9 - 8.2 mg/L
Salinity:
Not applicable
Conductivity:
326 - 374 μS/cm
Nominal and measured concentrations:
mean measured concentrations of 0.27, 0.67, 1.7, 4.3 and 10 mg/L
Details on test conditions:
EXPERIMENTAL DESIGN
Fathead minnow embryos were exposed to a geometric series of five test concentrations, a negative (dilution water) control and a solvent control (0.1 mL/L HPLC-grade dimethylformamide) under flow-through conditions. The exposure period included a 5-day embryo hatching period, and a 28-day post-hatch juvenile growth period. Nominal test concentrations were 0.26, 0.64, 1.6, 4.0 and 10 mg/L. The concentrations were selected in consultation with the Sponsor, and were based on exploratory range-finding toxicity data. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at the beginning of the test, at weekly intervals during the test and at test termination.
Delivery of the test solutions to the test chambers was initiated four days prior to test initiation in order to achieve equilibrium of the test substance. Four replicate test chambers were maintained in each treatment and control group, with one incubation cup in each test chamber. Each incubation cup contained 20 embryos, resulting in a total of 80 embryos per treatment. At test initiation, embryos <24 hours old were impartially distributed to incubation cups and exposed to test solution in the test chambers. After a 5-day embryo hatching period, the larvae were released into the test chambers, where exposure continued during a 28-day post-hatch juvenile growth period. Observations of the effects of tetrabromophthalic anhydride on time to hatch, hatching success, growth, and survival were used to calculate the no-observed-effect-concentration (NOEC) and the lowest-observed-effectconcentration (LOEC). The effective concentrations for an x percent effect (EC10 and EC20) for each endpoint were also estimated and reported when possible.

Test Apparatus
A continuous-flow diluter was used to deliver each concentration of the test substance, a solvent (HPLC-grade dimethylformamide) control, and a negative (dilution water) control. Syringe pumps (Harvard Apparatus, Holliston, Massachusetts) were used to deliver the five test substance stock solutions and HPLC-grade dimethylformamide (DMF) for the solvent control into mixing chambers assigned to each treatment and the solvent control. The syringe pumps were calibrated prior to the test. The stock solutions were diluted with well water in the mixing chambers in order to obtain the desired test concentrations. The flow of dilution water to the mixing chambers was controlled by rotameters, which were calibrated prior to test initiation and verified at approximately weekly intervals during the test. The flow of test water from each mixing chamber was split and allowed to flow into four replicate test chambers. The proportion of the test water that was split into each replicate was checked prior to the test and at approximately weekly intervals during the test to ensure that flow rates varied by no more than ±10% of the mean for the four replicates. The diluter flow rate was adjusted to provide approximately 10 volume additions of test water in each test chamber per day. The general operation of the diluter was checked visually at least two times per day during the test and at least once at the end of the test. Periodically during the test, all organisms were transferred to clean test chambers to prevent the buildup of bacterial/fungal growth.
The test was conducted in a temperature-controlled environmental chamber designed to maintain the target test temperature throughout the test period. The test chambers were 9-L glass aquaria filled with approximately 7 L of test solution. The depth of the test water in a representative test chamber was approximately 15.5 cm. Test chambers were labeled with the project number, test concentration and replicate. Embryos were held in incubation cups constructed from glass cylinders approximately 50 mm in diameter with 425 μm nylon screen mesh attached to the bottom with silicone sealant. The cups were suspended in the water column of each test chamber and attached to a rocker arm. The reciprocating motion of the rocker arm (4 rpm) facilitated circulation of test water around the embryos during incubation.

Non-GLP Range-Finding Test
Exploratory range-finding toxicity tests were conducted prior to the definitive test to evaluate the effect of tetrabromophthalic anhydride on test organisms. In the range-finding test, embryos <24 hours old were impartially distributed to incubation cups and exposed to a series of test concentrations in the test chambers at concentrations of 0.081, 0.27, 0.90, 3.0 and 10 mg/L, a negative control, and a solvent control, with two replicates each containing 25 embryos under flow-through conditions for 19 days (5 days hatch and 14 days post-hatch).
A primary stock solution at 100 mg/mL was prepared by dissolving a calculated amount of test substance (20.000 g) in 200 mL of HPLC-grade dimethylformamide (DMF). The primary stocks were sonicated for approximately 60 minutes followed by inversion to mix and appeared clear and light green in color. Secondary stock solutions (110 mL) at nominal concentrations of 0.81, 2.7, 9.0 and 30 mg/mL were prepared by proportionally diluting aliquots of the primary stock solution in DMF and inverted at least 20 times to mix and appeared clear and colorless, clear and faintly green to clear and light green. Aliquots of each stock were placed in the syringe pump every 3 to 4 days during the range finding test. The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 10.0 μL/minute where they were mixed with dilution water delivered at a rate of 100 mL/minute to achieve the desired test concentrations of 0.081, 0.27, 0.90, 3.0 and 10 mg/L. 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.
After a 5-day embryo hatching period, the larvae were released into the test chambers, where exposure continued during a 14-day post-hatch juvenile growth period. At the end of the 14-day growth period, measurements of wet weight for each treatment and control group were made. Mean percent hatching success in the negative control, solvent control group and in the of 0.081, 0.27, 0.90, 3.0 and 10 mg/L treatment groups was 98, 100, 100, 100, 96, 100 and 100%, respectively. Mean percent post-hatch survival in the negative control, solvent control group and in the of 0.081, 0.27, 0.90, 3.0 and 10 mg/L mg/L treatment groups was 88, 94, 98, 94, 94, 96 and 90%, respectively. The mean wet weight in the negative control, solvent control group and in the of 0.081, 0.27, 0.90, 3.0 and 10 mg/L treatment groups was 27.5, 27.0, 27.8, 25.1, 25.9, 28.2 and 27.5 mg, respectively.
Based on the results of the preliminary range-finding tests, the nominal test concentrations of 0.26, 0.64, 1.6, 4.0 and 10 mg/L were selected in consultation with the Sponsor for the definitive test.

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 test initiation using a SPER Scientific Model 840006 light meter.
The target test temperature during the test was 25 ± 1°C. Temperature was measured in each test chamber at the beginning of the test, 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.
Dissolved oxygen and pH were measured in alternating replicates of each treatment and control group at the beginning of the test, weekly during the test, and at the end of the test. Measurements of dissolved oxygen were made using a Thermo Orion Star A213 dissolved oxygen meter and pH was measured 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 concentration treatment group at the beginning of the test, 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.

Biological Observations and Measurements
During the first day of exposure, embryos were observed twice for mortality and eggs with fungus. Thereafter, until hatching was complete, observations of embryo mortality and the removal of dead embryos were performed once daily. When hatching reached >90% in the control groups on Day 5 of the test, the larvae were released to their respective test chambers and the post-hatch period began. Any unhatched embryos were kept in the egg cups until they hatched and were released into the test chamber, or until death of the embryo occurred. During the 28-day post-hatch exposure period, the larvae were observed daily to evaluate the numbers of mortalities and the numbers of individuals exhibiting clinical signs of toxicity or abnormal behavior. From these observations, time to hatch, hatching success, and post-hatch growth and survival were evaluated. Hatching success was calculated as the percentage of embryos that hatched successfully. Post-hatch survival was calculated as the number of larvae surviving to test termination divided by the total number of embryos that hatched successfully.
Post-hatch growth of the fathead minnows was evaluated at the conclusion of the 28-day post-hatch exposure period. Total length for each surviving fish was measured to the nearest 1 mm using a metric ruler, and wet and dry weights were measured to the nearest 0.1 mg using an analytical balance. Fish were placed in an oven at approximately 60 °C for approximately 46 hours to obtain dry weight data.
Reference substance (positive control):
no
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: hatching success, survival & growth
Key result
Duration:
33 d
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: hatching success, survival & growth
Details on results:
Measurement of Test Concentrations
Nominal concentrations selected for use in the study were 0.26, 0.64, 1.6, 4.0 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. Due to precipitate noted in the mixing chamber of the 4.0 mg a.i./L treatment group and in the mixing chamber and test chambers of the 10 mg/L treatment group on Day 0 of the test, impellors were placed in the mixing chambers of all treatment groups to aid the mixing of the stock solutions and dilution water prior to deliver to the test chambers. On Day 0, two sets of samples were collected and processed for analyses before and after centrifugation to remove the precipitate. Measured concentrations in the centrifuged samples on Day 0 ranged from 96.4 to 99.6% of nominal and in the uncentrifuged samples of the same samples ranged from 95.9 to 131% of nominal. Since these results indicated centrifugation had little impact on measured concentrations, samples were analyzed without centrifugation during the remaining of the study. Additional samples were collected on Day 13 of the test when precipitate was noted in the 4.0 and 10 mg/L treatment groups were not analyzed since the analytical prior to and after centrifugation of samples on Day 0 suggested that the centrifuged and uncentrifuged samples had comparable recoveries. Additional samples collected from all replicates of the 4.0 mg/L treatment group on Day 26 when the rotameter used to control the dilution water delivered to the mixing chamber of this treatment group malfunctioned had percent recoveries that ranged from 330 to 422% of nominal. Samples recoveries ranged from 98.7 to 101% of nominal after the rotameter was replaced and the delivery of dilution water to the mixing chamber resumed. Since the interruption was brief (at most 15 hours) in comparison to the exposure period of 33 days and the results confirmed that the diluter resumed its normal operation, the results were excluded from the calculation of mean measured test concentrations. When the results of the measured concentrations on Days 0, 6, 14, 21, 28 and 33 of the test were averaged the mean measured test concentrations of the 0.26, 0.64, 1.6, 4.0 and 10 mg/L was 0.27, 0.67, 1.7, 4.3 and 10 mg/L, respectively, representing 104, 105, 106, 108 and 100% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.

Physical and Chemical Measurements of Water
Water temperatures were within the 25 ± 1 °C range established for the test. Dissolved oxygen concentrations remained ≥84% of saturation
(6.9 mg/L). Measurements of specific conductance, hardness and alkalinity were comparable between the negative control and treatment group and did not appear to be influenced by tetrabromophthalic anhydride concentration. Light intensity at test initiation was 523 lux at the surface of the water of one representative test chamber.

Time to Hatch and Hatching Success
Daily observations of the embryos indicated that there were no apparent differences in time to hatch between the control groups and any of the tetrabromophthalic anhydride between the control groups and the 0.27, 0.67, 1.7, 4.3 and 10 mg/L treatment groups. The majority of fathead minnow embryos in the control and treatment replicates hatched on Days 4 and 5 of the test. Hatching reached >90% in the control and all treatment groups on Day 5 of the test, at which time the larvae were released to their respective test chambers.
Hatching success in the negative and solvent control groups was 98 and 100%, respectively. There were no statistically significant differences in hatching success between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Hatching success in the pooled control, 0.27, 0.67, 1.7, 4.3 and 10 mg/L treatment groups was 99, 96, 95, 98, 95 and 96%, respectively. Consequently, the NOEC for hatching success was 10 mg/L and the LOEC was >10 mg/L. Because there were no reductions in hatching success ≥10% from the pooled control, the EC10 and EC20 values based on hatching success were both >10 mg/L, the highest concentration tested.

Larval Survival and Clinical Observations
Larval survival in the negative and solvent control groups was 81 and 88%, respectively. There were no statistically significant differences in larval survival between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Larval survival in the pooled control, 0.27, 0.67, 1.7, 4.3 and 10 mg/L treatment groups was 84, 87, 82, 82, 91 and 90%, respectively. Consequently, the NOEC for larval survival was 10 mg/L and the LOEC was >10 mg/L. Because there were no reductions in survival ≥10% from the pooled control, the EC10 and EC20 values based on survival were both >10 mg/L, the highest concentration tested.
In general, the majority of the fish in the control groups and in the 0.27, 0.67, 1.7, 4.3 and 10 mg/L treatment groups appeared normal throughout the test. There were observations of organisms with small stature, erratic swimming, loss of equilibrium, lying on the bottom of the test chambers, weak appearance, discoloration, pin-eyed, and with a curled or crooked spine. However, these observations were infrequent and were also noted in the controls, and were not considered to be treatment related.

Growth
The NOEC for growth was 10 mg/L and the LOEC was >10 mg/L. The EC10 and EC20 values for growth were >10 mg/L, the highest concentration tested.
Results with reference substance (positive control):
Negative and Solvent Controls used.
Reported statistics and error estimates:
Jonckheere-Terpstra step down trend test indicated there were no statistically significant reductions in total length or wet weight among fish in any of treatment groups from the pooled control (p > 0.05) or in dry weight among fish in any of the treatment groups from the negative control or solvent control (p > 0.05).
Fisher’s Exact test indicated there were no statistically significant decreases in survival in any of the treatment groups in comparison to the pooled controls (p > 0.05).
Fisher’s Exact test indicated that there were no statistically significant decreases in hatching success in any of the tetrabromophthalic anhydride treatment groups when compared to the pooled controls (p > 0.05).

Means and Ranges of Water Quality Measurements Taken During the 33-Day Exposure to Tetrabromophthalic Anhydride

Mean Measured Concentration (mg/L)

Mean ± SD and Range of Measured Parameters

Temperature1(°C)

DO2

(mg/L)

pH

Hardness

(mg/L as CaCO3)

Alkalinity

(mg/L as CaCO3)

Conductivity (μS/cm)

Negative Control

24.5 ± 0.27

(24.1 – 25.0)

8.1 ± 0.13

(7.9 – 8.2)

8.2 ± 0.06

(8.1 – 8.3)

139 ± 9

(128 – 148)

178 ± 4

(172 – 182)

348 ± 11

(330 – 361)

Solvent Control

24.4 ± 0.22

(24.1 – 24.7)

7.7 ± 0.38

(7.3 – 8.2)

8.2 ± 0.04

(8.2 – 8.3)

--

--

--

--

--

--

0.27

24.4 ± 0.22

(24.0 – 24.7)

7.6 ± 0.55

(6.9 – 8.2)

8.2 ± 0.04

(8.1 – 8.2)

--

--

--

--

--

--

0.67

24.4 ± 0.20

(24.0 – 24.7)

7.9 ± 0.43

(7.2 – 8.2)

8.3 ± 0.05

(8.2 – 8.3)

--

--

--

--

--

--

1.7

24.4 ± 0.23

(24.0 – 24.6)

7.7 ± 0.48

(7.1 – 8.2)

8.3 ± 0.05

(8.2 – 8.3)

--

--

--

--

--

--

4.3

24.5 ± 0.21

(24.2 – 24.7)

7.7 ± 0.34

(7.2 – 8.2)

8.2 ± 0.06

(8.1 – 8.3)

--

--

--

--

--

--

10

24.4 ± 0.21

(24.0 – 24.6)

7.7 ± 0.49

(6.9 – 8.2)

8.2 ± 0.04

(8.2 – 8.3)

142 ± 4

(136 – 148)

180 ± 1

(178 – 180)

351 ± 16

(326 – 374)

1Temperature measured continuously during the test ranged from 24.26 to 25.03 °C.

2A dissolved oxygen concentration of 4.9 mg/L represents 60% saturation at 25 °C in freshwater.

-- = no measurements scheduled.

 

Summary of Hatching Success, Larval Survival and Growth of Fathead Minnows Exposed to Tetrabromophthalic Anhydride

Mean Measured Concentration (mg/L)

Hatching Success (%)1

Post-Hatch Survival (%)1

Mean Total length ± Std. Dev. (mm)2

Mean Wet Weight ± Std. Dev. (mg)2

Mean Dry Weight ± Std. Dev. (mg)4

Negative Control

Solvent Control

Pooled Control

0.27

0.67

1.7

4.3

10

98

100

99

96

95

98

95

96

81

88

84

87

82

82

91

90

24.5 ± 0.76

24.5 ± 0.60

24.2 ± 0.63

24.2 ± 0.30

23.8 ± 0.50

24.7 ± 0.47

23.9 ± 0.66

23.8 ± 0.36

103.3 ± 6.62

100.5 ± 2.27

101.9 ± 4.82

99.1 ± 1.85

93.5 ± 3.64

110.4 ± 4.32

97.3 ± 7.70

98.8 ± 1.90

22.6 ± 1.5

20.3 ± 0.50

--3

20.5 ± 0.72

18.9 ± 0.75

22.4 ± 0.87

18.8 ± 0.97

20.2 ± 0.61

NOEC (mg/L)

LOEC (mg/L)

10

>10

10

>10

10

>10

10

>10

10

>10

1There were no significant differences in hatching success of survival from the pooled control (Fisher’s Exact test, p>0.05).

2No statistically significant difference were found in total length or wet weight from the pooled control (Jonckheere-Terpstra step down trend test, p>0.05).

3A statistically significant difference was found between negative control and solvent control dry weight. The treatment dry weight data were compared to the negative control and solvent control separately.

4No statistically significant difference found in dry weight from the negative control or solvent control (Jonckheere-Terpstra step down trend test, p>0.05).

 

Validity criteria fulfilled:
yes
Conclusions:
Fathead minnows (Pimephales promelas) were exposed to tetrabromophthalic anhydride at mean measured concentrations of 0.27, 0.67, 1.7, 4.3 and 10 mg/L under flow-through conditions for 33 days (a 5-day hatching period plus a 28-day post-hatch growth period). There were no statistically significant treatment-related effects on hatching success, survival or growth at concentrations ≤10 mg/L. Consequently, the NOEC, based on all endpoints, was 10 mg/L. The LOEC was >10 mg/L.
Because there were no reductions in hatching success and survival ≥10% from the pooled control, the EC10 and EC20 values based on hatching success, survival and growth (measured as total length, wet and dry weight) were >10 mg/L, the highest concentration tested.
Executive summary:

The objective of the study was to determine the effects of tetrabromophthalic anhydride on the time to hatch, hatching success, survival, and growth of fathead minnows, Pimephales promelas, during early life-stage development under flow-through conditions.

 

The study protocol was based on procedures outlined in the OECD Guidelines for Testing of Chemicals, Guideline 210: Fish, Early-life Stage Toxicity Test and U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines, OCSPP Number 850.1400: Fish Early-Life Stage Toxicity Test.

 

Fathead minnow embryos were exposed to a geometric series of five test concentrations, a negative (dilution water) control and a solvent control (0.1 mL/L HPLC-grade dimethylformamide) under flow through conditions. The exposure period included a 5-day embryo hatching period, and a 28-day post-hatch juvenile growth period. Nominal test concentrations were 0.26, 0.64, 1.6, 4.0 and 10 mg/L. The concentrations were selected in consultation with the Sponsor, and were based on exploratory range-finding toxicity data.

Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at the beginning of the test, at weekly intervals during the test and at test termination.

 

Delivery of the test solutions to the test chambers was initiated four days prior to test initiation in order to achieve equilibrium of the test substance. Four replicate test chambers were maintained in each treatment and control group, with one incubation cup in each test chamber. Each incubation cup contained 20 embryos, resulting in a total of 80 embryos per treatment. At test initiation, embryos <24 hours old were impartially distributed to incubation cups and exposed to test solution in the test chambers. After a 5-day embryo hatching period, the larvae were released into the test chambers, where exposure continued during a 28-day post-hatch juvenile growth period. Observations of the effects of tetrabromophthalic anhydride on time to hatch, hatching success, growth, and survival were used to calculate the no-observed-effect-concentration (NOEC) and the lowest-observed-effect concentration (LOEC). The effective concentrations for an x percent effect (EC10 and EC20) for each endpoint were also estimated and reported when possible.

 

Fathead minnows (Pimephales promelas) were exposed to tetrabromophthalic anhydride at mean measured concentrations of 0.27, 0.67, 1.7, 4.3 and 10 mg/L under flow-through conditions for 33 days (a 5-day hatching period plus a 28-day post-hatch growth period). There were no statistically significant treatment-related effects on hatching success, survival or growth at concentrations ≤10 mg/L. Consequently, the NOEC, based on all endpoints, was 10 mg/L. The LOEC was >10 mg/L.

 

Because there were no reductions in hatching success and survival ≥10% from the pooled control, the EC10 and EC20 values based on hatching success, survival and growth (measured as total length, wet and dry weight) were >10 mg/L, the highest concentration tested.

Description of key information

33 day NOEC, based on all endpoints, was 10 mg/L. The LOEC was >10 mg/L.

EC10 and EC20 values based on hatching success, survival and growth (measured as total length, wet and dry weight) were >10 mg/L, the highest concentration tested.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
10 mg/L

Additional information

The objective of the study was to determine the effects of tetrabromophthalic anhydride on the time to hatch, hatching success, survival, and growth of fathead minnows, Pimephales promelas, during early life-stage development under flow-through conditions.

 

Fathead minnows (Pimephales promelas) were exposed to tetrabromophthalic anhydride at mean measured concentrations of 0.27, 0.67, 1.7, 4.3 and 10 mg/L under flow-through conditions for 33 days (a 5-day hatching period plus a 28-day post-hatch growth period). There were no statistically significant treatment-related effects on hatching success, survival or growth at concentrations ≤10 mg/L. Consequently, the NOEC, based on all endpoints, was 10 mg/L. The LOEC was >10 mg/L.

Because there were no reductions in hatching success and survival ≥10% from the pooled control, the EC10 and EC20 values based on hatching success, survival and growth (measured as total length, wet and dry weight) were >10 mg/L, the highest concentration tested.