Benzene, ethenyl-, ar-bromo derivs.

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from November 16, 2004 to November 23, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to OECD Guideline 201 and mostly in accordance with GLP standards.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Remarks:

with the following exceptions: The characterization and the stability of the test substance under conditions of storage at the test site were not determined in compliance with GLP Standards.

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples of the test solutions were collected at approximately 0 and 96 hours to measure the concentrations of the test substance. Samples at test initiation were collected from the individual batches of test solution prepared for each treatment and control group prior to addition of the algae. At exposure termination, samples were collected from the pooled replicated from each treatment and control group.
- Sample storage conditions before analysis: All samples were collected in glass vials and were processed on the day of collection and analysed as soon as possible.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:A primary stock solution was prepared by dissolving DBS in freshwater algal medium at a nominal concentration of 10 mg/L. The stock was inverted at least 20 times, sonicated and stirred on a magnetic stir plate with a Teflon® coated stir bar for approximately 24 hours to mix and appeared clear and colorless. The primary stock was proportionally diluted with freshwater algal medium to prepare the four additional test solutions at nominal concentrations of 0.63, 1.3,2.5 and 5.0 mg/L. Test concentrations were not corrected for percent active ingredient in the test substance.
- Controls: A negative control was performed
- Evidence of undissolved material: All test solutions appeared clear and colorless

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater green alga, Selenastrum capricornutum
- Strain:Printz (UTCC 37)
- Source (laboratory, culture collection): University of Toronto Culture Collection
- Age of inoculum : cultures had been actively growing in culture medium for at least two weeks prior to test initiation
- Method of cultivation: The algal cells were cultured and tested in freshwater algal medium. The culture was last transferred to fresh medium four days prior to test initiation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

not measured

Test temperature:

Temperatures ranged from 23.8 to 25.2°C and were within the 24 ± 2°C range established for the test. The minimum and maximum temperatures measured continuously throughout the study were 23 and 24°C, respectively.

pH:

The pH of the test solutions at test initiation was 8.0 and at exposure termination ranged from 8.4 to 9.4. The pH tended to increase relative to increases in algal densities, which is typical for tests conducted with Selenastrum capricornutum.

Dissolved oxygen:

not measured

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/L. Samples collected on Day 0 had recoveries of 62, 59, 63, 63 and 67% of nominal concentrations, respectively. Recoveries in all samples collected at 96 hours, were

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile, 250-mL Erlenmeyer flasks plugged with foam stoppers containing 100 mL of test or control mediu
- Type (delete if not applicable): closed
- Initial cells density: 10000 cells/mL
- Control end cells density: 3,195,748 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified Wildlife International, Ltd. well water. The test medium then was prepared by adding appropriate volumes of the stock nutrient solutions to purified well water, NANOpure® water, (NH4C1 15 mg/L, MgC12.6H2O 12 mg/L, CaC12.2H2O 18 mg/L, MgSO4.7H2O 15 mg/L, KH2PO4 1.6 mg/L, FeC13.6H2O 0.08 mg/L, Na2EDTA•2H2O 0.1 mg/L, H3BO3 0.185 mg/L, MnC12.4H20 0.415 mg/L ZnC12 3 µg/L, CoC12.6H2O 1.5 µg/L, CuC12.2H2O 0.01 µg/L, Na2MoO4.2H2O 7 µg/L and
NaHCO3 50 mg/L.)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Wildlife International, Ltd. well water
- Pesticides, organics and metals:
In µg/L: Aldrin < 0.0020, Heptachlor Epoxide < 0.0020, Alpha BHC < 0.0020, Malathion < 0.39, Beta BHC < 0.012, Merphos < 0.39, Bolstar < 0.39, Methoxychlor < 0.020, Chlordane < 0.068, Methyl Parathion < 0.39, Coumaphos < 0.62, Mevinphos < 0.39, Delta BHC < 0.029, Mirex < 0.0098, Demeton-O < 0.39, Naled < 0.58, Demeton-S < 0.39, o,p-DDD < 0.0039, Diazinon < 0.39, o,p-DDE < 0.0039, Dichlorvos < 0.39, o,p-DDT < 0.0039, Dieldrin < 0.0049, p,p-DDD < 0.0039, Disulfoton < 0.39, p,p-DDE < 0.0039, Dursban (Chlorpyrifos) < 0.39, p,p-DDT < 0.0039, Endosulfan I < 0.0039, PCB-1016 < 0.20, Endosulfan II < 0.0049, PCB-1221 < 0.39, Endosulfan Sulfate ,< 0.0088, PCB-1232 < 0.098, Endrin <0.0039, PCB-1242 < 0.20, EPN < 0.78, PCB-1248 < 0.29, Ethion < 0.39, PCB-1254 < 0.20, Ethoprop < 0.39, PCB-1260
< 0.29, Ethyl Parathion < 0.39, Phorate < 0.39, Famphur < 0.48, Ronnel < 0.39, Fensulfothion < 0.87, Stirophos < 0.39, Fenthion < 0.39, Telodrin < 0.0020, Gamma BHC – Lindane < 0.0020, Tokuthion < 0.39, Guthion (Azinphos-methyl) < 0.78, Toxaphene < 0.29, HCB < 0.0020, Trichloronate < 0.39, Heptachlor < 0.0020 and Trithion < 0.39
In mg/L: Aluminum <0.0413, Antimony < 0.0085, Arsenic <0.0049, Barium <0.005, Beryllium <0.00034, Bromide <2.0, Cadmium <0.00087, Calcium 36.2, Chloride 3.4, Chromium < 0.0022, Cobalt <0.0016, Copper <0.0021, Fluoride 0.58, Iron <0.0453, Lead <0.0093, Magnesium 13.7, Manganese <0.005, Mercury <0.00016, Nickel <0.0038, Nitrate Nitrogen <0.40, Potassium 6.20, Selenium <0.0047, Silver < 0.0018, Sodium 19.3, Sulfate 4.7, Thallium <0.0089, Vanadium <0.0017, and Zinc <0.0041
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: yes, the medium was sterilized by filtration (0.22 µm) prior to use.
- Adjustment of pH: ). The pH of the medium was adjusted to 7.9 using 0.1N NaOH
- Photoperiod: 24 hours/day
- Light intensity and quality: Cool-white fluorescent lighting at an intensity of 4300 ± 10% lux. Light intensity was measured at five locations surrounding the test flasks on the shaker table at test initiation. Light intensity was measured using a SPER Scientific 840006C light meter. The light intensity ranged from 4100 to 4620 lux, which was within the desired range of 4300 ± 10% lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The concentration of algal cells was verified using a haemacytometer and microscope, and 1.0 mL of the inoculum was added to each test chamber to achieve a nominal concentration of approximately 10,000 cells/mL at test initiation.

TEST CONCENTRATIONS
- Range finding study: A range finding toxicity study was performed, the results were not presented in the reporting of the study.
- Test concentrations: Nominal test concentrations selected were 0.63, 1.3, 2.5, 5.0 and 10 milligrams DBS per liter (mg/L).

Reference substance (positive control):

not required

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: cell density

Remarks on result:

other: 5.4-7.0 mg/L (95% CL)

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

7.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: cell density

Remarks on result:

other: 6.5-8.2 mg/L (95% CL)

Duration:

72 h

Dose descriptor:

other: NOAEC

Effect conc.:

0.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: cell density

Duration:

96 h

Dose descriptor:

other: NOAEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: cell density

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 5.4-6.9 mg/L (95% CL)

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

6.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 6.2-7.7 mg/L (95% CL)

Duration:

72 h

Dose descriptor:

other: NOAEC

Effect conc.:

0.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

other: NOAEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 8.5-9.7 mg/L (95% CL)

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CL not calculable

Duration:

72 h

Dose descriptor:

other: NOAEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

other: NOAEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): YES
- Observation of abnormalities (for algal test): some enlarged cells were noted for the 10 mg/L treatment group
- Any stimulation of growth found in any treatment: NO.

Nominal concentrations selected for use in this study were 0.63,1.3, 2.5, 5.0 and 10 mg/L. Samples collected on Day 0 had recoveries of62, 59,63,63 and 67% of nominal concentrations, respectively. Recoveries in all samples collected at 96 hours, were
- Effect concentrations exceeding solubility of substance in test medium: YES - This is not discussed in the present report; however, the dominant (85%) dibromostyrene constituent has a water solubility of 6.91 mg/L according to the key study on solubility (endpoint summary 4.8). Therefore, some of the reported EC50s for the present study exceed this level. This may be because the ECs are reported in terms of nominal concentrations, while actual measured concentrations in the present study were much less.

Reported statistics and error estimates:

Please refer to subheading Statistical analyses under section Any other information on materials and methods incl. tables.

Table 1
Mean Cell Density and Percent Inhibition

Nominal	24 Hours		48 Hours		72 Hours			96 Hours
Test Concentration (mg/L)	MeanCell Density(cells/mL)	Percent Inhibition^1,2	Mean Cell Density (cell s/mL	Percent Inhibition^1,2	Mean Cell Density (cells/mL)	Percent Inhibition^1,2	Mean Cell Density (cells/mL)		Percent Inhibition^1,2
Negative Control	17,157		117,044		757,937		3,195,748
0.63	15,737	8.3	103,376	12	700,128	7.6	3,146,431		1.5
1.3	16,675	2.8	100,729	14	631,686	17	2,940,067		8.0
2.5	14,996	13	101,600	13	579,896*	23	2,962,938		7.3
5.0	11,443	33	84,895	27	487,148*	36	2,692,040		16
10	14,046	18	12,862	89	58,219*	92	529,676*		83

¹Calculations were performed using SAS Version 8.02. Manual calculations may differ slightly.²Percent inhibition was calculated relative to the negative control replicates.

* Statistically significant difference (p<0.05) at 72 and 96 hours from the negative control replicates using Dunnett's test.

 

Table 2
Mean Area Under the Growth Curve (Biomass) and Percent Inhibition

Nominal	0 - 24 Hours		0 - 48 Hours		0 - 72 Hours		0 - 96 Hours
Test Concentration (mg/L)	Mean Area^1	Percent Inhibition^1,2	Mean Area^1	Percent inhibition^1,2	Mean Area^1	Percent Inhibition^1,2	Mean Area^1	Percent Inhibition^1,2
Negative Control	85,880		1,456,288		11,716,064		58,920,292
0.63	68,848	20	1,258,212	14	10,660,264	9.0	56,578,968	4.0
1.3	80,104	6.7	1,248,956	14	9,797,940	16	52,418,980	11
2.5	59,956	30	1,219,108	16	9,157,056*	22	51,431,068	13
5.0	22,296	`74	938,348	36	7,562,868*	35	45,473,124*	23
10	48,552	43	131,448	91	744,424*	94	7,559,168*	87

¹Calculations were performed using SAS Version 8.02. Manual calculations may differ slightly.

²Percent inhibition was calculated relative to the negative control replicates.

* Statistically significant difference (p<0.05) at 72 and 96 hours from the negative replicates using Dunnett's test.

 

 

Table3
Mean Growth Rate and Percent Inhibition

Nominal	0 - 24 Hours                                      0 - 48 Hours				0 - 72 Hours		0 - 96 Hours
Test Concentration (mg/L)	Mean Growth Rate^1	Percent Inhibition^1,2	Mean Growth Rate^1	Percent Inhibition^1,2	Mean Growth Rate^1	Percent Inhibition^1,2	Mean Growth Rate^1	Percent Inhibition^1,2
Negative Control	0.0223		0.0512		0.0600		0.0600
0.63	0.0187	16	0.0485	5.3	0.0590	1.7	0.0599	0.11
1.3	0.0213	4.5	0.0480	6.2	0.0575	4.1	0.0592	1.3
2.5	0.0168	25	0.0483	5.8	0.0564	6.0	0.0592	1.3
5.0	0.0068	69	0.0445	13	0.0538*	10	0.0582	2.9
10	0.0140	37	0.0052	90	0.0237*	61	0.0412*	31

¹Calculations were performed using SAS Version 8.02. Manual calculations may differ slightly.

²Percent inhibition was calculated relative to the negative control replicates.

* Statistically significant difference (p<0.05) at 72 and 96 hours from the negative control replicates using Dunnett's test.

 

Table 4
EC50, E_bC50 and E_rC50 Values Over the 96-Hour Exposure Period

		Cell Density	Area Under the Growth Curve (Biomass)			Growth Rate
Time	EC50 (mg/L)	95% Confidence Interval (mg/L)	EbC50 (mg/L)	95% Confidence Interval (mg/L)	ErC50 (mg/L)	95% Confidence Interval (mg/L)
24 Hours	>10	--^1	>10	--^1	>10	--^1
48 Hours	6.7	6.1 and 7.3	6.3	4.3 and 7.6	7.1	6.7 and 7.6
72 Hours	6.2	5.4 and 7.0	6.1	5.4 and 6.9	9.1	8.5 and 9.7
96 Hours	7.3	6.5 and 8.2	6.9	6.2 and 7.7	>10	--^1

' 95% confidence limits could not be calculated with the data obtained.

Validity criteria fulfilled:

yes

Remarks:

OECD 201: The cell concentration in the control cultures should have increased by a factor of at least 16 within three days; Disappearance of the test substance from the water into the biomass does not necessarily invalidate the test.

Conclusions:

Selenastrum capricornutum were exposed to five concentrations of DBS and evaluated for effects on cell density, area under the growth curve (biomass) and growth rate. Biomass was the most sensitive endpoint, as defined by the lowest 96-hour EC50 value. The 72-hour EbC50, based on biomass, was 6.1 mg/L, while the calculated EC50 and ErC50 values were 6.2 and 9.1 mg/L, respectively. The 96-hour EbC50, based on biomass, was 6.9 mg/L, while the calculated EC50 and ErC50 values were 7.3 and > 1 0 mg/L, respectively. The 72-hour NOAEC based on cell density and biomass was 0.63 mg/L. The 96-hour NOAEC, based on cell density and biomass was 2.5 mg/L.

Executive summary:

In a GLP compliant study conducted in accordance with OECD 201, Selenastrum capricornutum were exposed to five concentrations of DBS and evaluated for effects on cell density, area under the growth curve (biomass) and growth rate. Biomass was the most sensitive endpoint, as defined by the lowest 96-hour EC50 value. The 72-hour EbC50, based on biomass, was 6.1 mg/L, while the calculated EC50 and ErC50 values were 6.2 and 9.1 mg/L, respectively. The 96-hour EbC50, based on biomass, was 6.9 mg/L, while the calculated EC50 and ErC50 values were 7.3 and > 1 0 mg/L, respectively. The 72-hour NOAEC based on cell density and biomass was 0.63 mg/L. The 96-hour NOAEC, based on cell density and biomass was 2.5 mg/L.

Description of key information

Selenastrum capricornutum were exposed to five concentrations of DBS and evaluated for effects on cell density, area under the growth curve (biomass) and growth rate. Biomass was the most sensitive endpoint, as defined by the lowest 96-hour EC50 value. The 72-hour EbC50, based on biomass, was 6.1 mg/L, while the calculated EC50 (cell density) and ErC50 (growth rates) values were 6.2 and 9.1 mg/L, respectively. The 96-hour EbC50, based on biomass, was 6.9 mg/L, while the calculated EC50 (cell density) and ErC50 (growth rate) values were 7.3 and > 1 0 mg/L, respectively. The 72-hour NOAEC based on cell density and biomass was 0.63 mg/L. The 96-hour NOAEC, based on cell density and biomass was 2.5 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

10 mg/L

Additional information

The OECD 201, GLP compliant study by Desjardins D et al (2005) was considered adequate and reliable to fulfil the data requirement as a key study. The study was assigned a reliability score of 1.