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Toxicity to aquatic algae and cyanobacteria

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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):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 Inhibition1,2

Mean Cell Density (cell s/mL

Percent Inhibition1,2

Mean Cell Density (cells/mL)

Percent Inhibition1,2

Mean Cell Density (cells/mL)

Percent Inhibition1,2

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

Area1

Percent

Inhibition1,2

Mean

Area1

Percent

inhibition1,2

Mean

Area1

Percent

Inhibition1,2

Mean

Area1

Percent

Inhibition1,2

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 Rate1

Percent

Inhibition1,2

Mean

Growth Rate1

Percent

Inhibition1,2

Mean

Growth Rate1

Percent

Inhibition1,2

Mean

Growth Rate1

Percent

Inhibition1,2

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, EbC50 and ErC50 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.