Benzene, ethenyl-, ar-bromo derivs.

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 10, 2005 to March 21, 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study followed relevant guidelines and Good Laboratory Practice Standards, but it did not fully meet the validity criteria as noted below.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Version / remarks:

draft guidlines

GLP compliance:

yes

Remarks:

with the following exceptions: the characterization of the test substance, and the stability of the test substance under the conditions of storage at the test site, were not determined in accordance with GLP standards.

Analytical monitoring:

yes

Details on sampling:

Samples were collected from one test chamber of each treatment and control group three days prior to the start of the test to confirm the conditioning of the diluter. Samples also were collected from alternating replicate test chambers in each treatment and control group at test initiation, at the approximate mid-point of the test (48 hours) and at test termination to measure concentrations of the test substance. All samples were collected at mid-depth, placed in glass vials, and analyzed as soon as possible without storage.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: One stock solution was prepared for each of the five concentrations tested. A primary stock solutions was prepared in DMF at a nominal concentration of 100 mg DBS/mL. Aliquots of the primary stock solution were proportionally diluted with DMF to prepare four secondary stock solutions at concentration of 6.3, 13, 25 and 50 mg/mL. All stocks were mixed by inversion and ranged in appearance from clear and colorless to clear and light yellow. The stock solutions were injected into the diluter mixing chambers (at a rate of 12.5 µL/minute) where they were mixed with well water ( at a rate of 125 mL/minute) to achieve the desired test concentrations. The test solutions appeared clear and colorless in the test chambers and the diluter mixing chambers at test initiation and termination. The solvent control was prepared by injecting DMF into the mixing chamber for the solvent control.
- Controls: A solvent control and negative control were included in the study.
- Chemical name of vehicle : Dimethyl formamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): The concentration of DMF in the solvent control and all test substance treatment groups was 0.1 mL/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): An oily surface layer was evident in the diluter mixing chambers for the 10 mg/L solution at test initiation and the 2.5, 5.0 and 10 mg/L solutions by test termination.

Loading was defined as the total wet weight of fish per liter of test water that passed through the test chamber in 24 hours and was 0.027 g fish/L/day. Instantaneous loading was 0.16 g fish/L of test water present in the test chambers at any given time.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead Minnow
- Strain: Pimephales promelas
- Source: Fathead minnows used in the test were obtained from cultures maintained at Wildlife International, Ltd., Easton, Maryland.
- Age at study initiation: Fish were reported as from the same year class
- Length at study initiation (length definition, mean, range and SD): The length of the longest fish was no more than twice the length of the shortest fish. The average total length of 10 negative control fish measured at the end of the test was 3.1 cm, with a range of 2.7 to 3.5 cm.
- Weight at study initiation (mean and range, SD): The average wet weight (blotted dry) of 10 negative control fish measured at the end of the test was 0.24 g, with a range of 0.11 to 0.35 g.
- Feeding during test: No feeding at least two days prior to the test and during the test.

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): the fathean minnows were held prior to the test in water from the same sourve and at approximately the same temperature as used in the test. During the test, water temperatures ranged from 21.7 to 22.5C, measured with a hand-held thermometer. The pH of the water ranged from 8.1 to 8.4, measured with a Fisher Scientific Accumet Model 915 pH meter. Dissolved oxygen ranged from 7.2 to 8.3 mg/L (≥83% of saturation), measured with a Yellow Springs Instruments Model 51B dissolved oxygen meter.
- Type and amount of food: During the holding period, the fish were fed daily a commercially-prepared diet supllieb by Zeigler Brothers Inc. (Gardners Pennsylvania) supplemented with brine shrimo nauplii (Artemia sp.) supplied by Brine Shrimp Direct, (Ogden, Utah)
- Feeding frequency: Daily
- Health/mortality: During the 14-day period prior to the test the fish showed no signs of disease or stress.

At test initiation, the fathead minnows were collected from the culture tank and impartially distributed two at a time to the test chambers until each contained 10 fish.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Hardness was measured at test initiation at 136 mg/L as CaCO3.

Test temperature:

Water temperature was measured in each test chamber at the beginning and end of the test, and was within the 22 ± 1 °C range established for the test.

pH:

The pH was measured in alternating replicate test chambers of each treatment and control group at the beginning and end of the test and at approximately 24-hour intervals during the test. Measurements of pH ranged from 8.3 to 8.5.

Dissolved oxygen:

Dissolved oxygen was measured in alternating replicate test chambers of each treatment and control group at the beginning and end of the test and at approximately 24-hour intervals during the test. Dissolved oxygen concentrations remained ≥8.4 mg/L (≥97% of saturation) throughout the test.

Salinity:

NA

Nominal and measured concentrations:

Nominal concentrations selected for use in this study were 0.63, 1.3, 2.5, 5.0 and 10 mg DBS/L. When the measured concentrations of the test samples collected at 0, 48 and 96 hours were averaged, the mean measured concentrations for the study were 0.25, 0.66, 0.86, 1.9 and 3.0 mg/L, representing 40, 51, 34, 38 and 30% of nominal concentrations, respectively.

Details on test conditions:

TEST SYSTEM:

A continuous-flow diluter was used to deliver each concentration of the test substance, a solvent (dimethyl formamide) control, and a negative (well water) control. Syringe pumps (Harvard Apparatus, South Natick, Massachusetts) were used to deliver the five test substance stock solutions and dimethyl formamide (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 mixed with dilution 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 the test. The flow of test water from each mixing chamber was split and allowed to flow into replicate test chambers.

The diluter was adjusted so that each test chamber received at least six volume additions of test water every 24 hours. The general operation of the diluter was checked visually at least two times per day during the test and once on the final day of the test.

Test chambers were 25-L stainless steel aquaria filled with approximately 15 L of test water. The depth of the test water in a representative chamber was 18.5 cm. Test chambers were indiscriminately positioned in a temperature-controlled water bath set to maintain the desired test temperature. The water bath was enclosed in a plexiglass ventilation hood in order to minimize any potential for cross-contamination.

TEST CONDITIONS:

Hardness, temperature, pH, DO, and test material concentration were measured as noted above. Specific conductance and alkalinity were measured at test initiation at 320 umhos/cm and 178 mg/L as CaCO3, respectively. The freshwater used for culturing and testing was sourced from an on-site well, which was passed through a sand filter, aerated, and filtered with 0.45 µm.

Fluorescent light bulbs that emit wavelengths similar to natural sunlight (Colortone 50) were used for illumination of the culture and test chambers. A photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting.

Observations were made periodically to determine the number of mortalities. The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 3, 24, 48, 72 and 96 hours after test initiation.

Reference substance (positive control):

not required

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

6.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 5.0-10 mg/L (95% CL)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

7.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 5.0-10 mg/L (95% CL)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

7.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 5.0-10 mg/L (95% CL)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL could not be calculated

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Duration:

96 h

Dose descriptor:

other: no mortality concentration

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Suitability of analytical methods: Quantitative recoveries of fortified control samples (92 to 102%) indicate that the analytical method utilized was acceptable in recovering DBS from aqueous solutions. Measured concentrations of DBS in the test solution samples were low, but were consistent throughout the test. Possible causes of the low recoveries were low water solubility, adsorption to glass surfaces and particles in the test solutions, and polymerization of the styrene double bond. Utilizing a flow-through system with at least six volume turnovers each day and a solvent to aid in solubilization of the test substance maximized the amount of DBS that could be maintained in the water.

- Mortality and signs of toxicity: Daily observations of mortality and signs of toxicity observed during the test are presented in Table 5. All fathead minnows in the negative and solvent control groups appeared normal throughout the test. All fathead minnows in the 0.63 mg/L treatment group also appeared normal throughout the test, with the exception of one fish exhibiting erratic swimming behavior at test termination. This fish was noted with a curved spine, apparently caused by injury. Therefore, the erratic swimming was not considered to be treatment-related. At test termination, percent mortality in the 1.3, 2.5, 5.0 and 10 mg/L treatment groups was 0, 0, 20 and 100%, respectively. While no mortalities occurred among fish at 1.3 and 2.5 mg/L, all fish in those treatment groups exhibited signs of toxicity by test termination, including lethargy, lying on the chamber bottom and surfacing. Consequently, the no-mortality concentration was 2.5 mg/L and the NOEC was considered to be 0.63 mg/L. LC50 values at 24, 48, 72 and 96 hours were determined from the mortality data.

Reported statistics and error estimates:

The results of the study were based on the nominal test concentrations. The mortality data were analyzed using the computer program of C. E. Stephan. The program was designed to calculate the LC50 value and the 95% confidence interval by probit analysis, the moving average method, and binomial probability with nonlinear interpolation. In this study, the binomial method was used to calculate the 48, 72 and 96-hour LC50 values. There was <50% mortality in any treatment group at 24 hours. Therefore, the 24-hour LC50 value, the no mortality concentration and the NOEC were determined by visual interpretation of the mortality and observation data.

Sublethal observations / clinical signs:

Table 5 (Reproduced from study report)

Cumulative Mortality and Observations

Nominal Test Concentration (mg/L)	Replicate	No. Exposed	3 Hours		24 Hours		48 Hours		72 Hours		96 Hours		Cumulative Percent Mortality
			No. Dead^1	Effects^2	No. Dead^1	Effects^2	No. Dead^1	Effects^2	No. Dead^1	Effects^2	No. Dead^1	Effects^2
Negative Control	A	10	0	10 AN	0	10 AN	0	10 AN	0	10 AN	0	10 AN	0
	B	10	0	10 AN	0	10 AN	0	10 AN	0	10 AN	0	10 AN
Solvent Control	A	10	0	10 AN	0	10 AN	0	10 AN	0	10 AN	0	10 AN	0
	B	10	0	10 AN	0	10 AN	0	10 AN	0	10 AN	0	10 AN
0.63	A	10	0	10 AN	0	10 AN	0	10 AN	0	10 AN	0	9 AN; 1 E^3	0
	B	10	0	10 AN	0	10 AN	0	10 AN	0	10 AN	0	10 AN
1.3	A	10	0	10 AN	0	10 AN	0	7 AN; 3 C	0	4 R; 6 C	0	4 R; 5 C; 1 A	0
	B	10	0	10 AN	0	10 AN	0	5 AN; 4 C; 1 A	0	5 R; 5 C	0	1 R; 9 C
2.5	A	10	0	10 AN	0	10 AN	0	6 C; 3 R; 1 AN	0	8 R; 2 C	0	10 C	0
	B	10	0	10 AN	0	10 AN	0	7 C; 3 R	0	7 R; 3 C	0	5 R; 5 C
5.0	A	10	0	10 C	0	8 R; 2 C	0	8 R; 1 A; 1 E	0	9 R; 1 A	2	7 R; 1 C	20
	B	10	0	10 C	0	6 R; 4 C	0	10 R	0	9 R; 1 C	2	8 R
10	A	10	0	10 C	3	7 R	9	1 A	9	1 R	10	--	100
	B	10	0	10 C	3	7 R	10	--	10	--	10	--

1 Cumulative number of dead fish.

2 Observed Effects: AN = appear normal; C = lethargy; R = lying on bottom of chamber; A = surfacing; E = erratic swimming.

3 This fish apparently was injured, causing a curved spine and erratic swimming.

Validity criteria fulfilled:

no

Remarks:

OECD 203 requires that the results be based on measured concentrations, if they differ from nominal by more than 20%. The present study used nominal concentrations, even though the measured concentrations differed by more than 20% from nominal.

Conclusions:

Fathead minnows (Pimephales promelas) were exposed for 96 hours under flow-through conditions to five nominal concentrations of dibromostyrene (DBS) ranging from 0.63 to 10 mg/L. Based on the nominal test concentrations, the 96-hour LC50 value was 6.2 mg/L, with a 95% confidence interval of 5.0 to 10 mg/L. The no mortality concentration was 2.5 mg/L and the NOEC was 0.63 mg/L. Study reportedly followed OECD 203 guidelines, but as noted above, results were reported in terms of nominal concentrations even though the measured concentrations differed from nominal by more than 20%. In such a case, the validity criteria required that results be reported in terms of measured concentrations. Therefore the LC50 values may be somewhat overestimated.

Description of key information

Fathead minnows (Pimephales promelas) were exposed for 96 hours under flow-through conditions to five nominal concentrations of dibromostyrene (DBS) ranging from 0.63 to 10 mg/L. Based on the nominal test concentrations, the 96-hour LC50 value was 6.2 mg/L, with a 95% confidence interval of 5.0 to 10 mg/L. The no mortality concentration was 2.5 mg/L and the NOEC was 0.63 mg/L. Study reportedly followed OECD 203 guidelines, but as noted above, results were reported in terms of nominal concentrations even though the measured concentrations different from nominal by more than 20%. In such a case, validity criteria require that results be reported in terms of measured concentrations. Therefore the LC50 values may be somewhat overestimated.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

6.2 mg/L

Additional information

Palmer SJ et al (2005) was considered reliable and adequate for assessment and use as the key study for this data requirement. The study was conducted in accordance with the guideline OECD 203 and in compliance with GLP. The study was assigned a reliability score of 1.