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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:
2017-10-11 to 2018-
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The freshwater alga, Raphidocelis subcapitata, was selected as the test species for this study. The species is representative of an important group of freshwater algae, and was selected for use in the test based upon a past history of use, and ease of culturing in the laboratory. Original algal cultures were obtained from the University of Texas at Austin, and have been maintained in culture medium at EAG Laboratories, Easton, Maryland since June 2017. Algal cells used in this test were obtained from EAG Laboratories – Easton cultures that had been actively growing in culture medium under similar environmental conditions as used in this test for at least two weeks prior to test initiation. Algal cells for this study were taken from a culture that had been transferred to fresh medium four days prior to test initiation.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Measurements of temperature, pH, and light intensity are presented in Tables 2, 3, and 4, respectively (see results). Temperatures remained within the 24 ± 2°C range established for the test.
pH:
Measurements of temperature, pH, and light intensity are presented in Tables 2, 3, and 4, respectively (see results). The pH of all test solutions at test initiation was 7.4. At test termination, pH measurements in samples of test solution obtained by pooling the replicates of each experimental group ranged from 7.5 to 9.9. The pH increased by more than 1.5 units over the course of the study, however, the photosynthetic activity of the test organism increases pH over time. The increase observed in pH was not considered to be detrimental to the study since growth in the control replicates achieved all validity criteria.
Nominal and measured concentrations:
WAF LOADING RATES:
Nominal
Negative Control
1.0 µg a.i./L
2.6 µg a.i./L
6.4 µg a.i./L
16 µg a.i./L
40 µg a.i./L
100 µg a.i./L
Details on test conditions:
Environmental Conditions
Test chambers were held in an environmental chamber at a temperature of 24 ± 2ºC. The temperature of a container of water adjacent to the test chambers in the environmental chamber was measured continuously using an Amega Scientific Corporation centralized monitoring system. The algae were held under continuous cool-white fluorescent lighting throughout the test. The target light intensity was 6,000 lux ± 10%. Light intensity was measured at test solution level at five locations surrounding the test flasks at test initiation using a SPER Scientific 840006C light meter. The pH of the medium in each treatment and control group was measured at test initiation and exposure termination using a Thermo Orion A214 pH meter. At test initiation, pH was measured in the individual batches of test solution prepared for each treatment and control group. At exposure termination, pH was measured in pooled samples of test solution collected from each of the remaining replicates of each treatment and control group.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
6.4 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.1 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.8 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.3 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.3 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Yield

RESULTS AND DISCUSSION

Observations and Measurements

Measurements of temperature, pH, and light intensity are presented in Tables 2, 3, and 4, respectively. Temperatures remained withinthe 24 ± 2°C range established for the test. The pH of all test solutions at test initiation was 7.4. At test termination, pH measurements in samples of test solution obtained by pooling the replicates of each experimental group ranged from 7.5 to 9.9. The pH increased by more than 1.5 units over the course of the study, however, the photosynthetic activity of the test organism increases pH over time. The increase observed in pH was not considered to be detrimental to the study since growth in the control replicates achieved all validity criteria. The light intensity measured on Day 0 ranged from 5,900 to 6,540 lux, which was within the desired range of 6,000 lux ± 10%.

Table 2 Temperature Measurements

 

Time

(Days)

Temperature (°C)1

Mean

Low

High

0

24.15

23.87

24.25

1

24.30

24.18

24.37

2

24.35

24.25

24.37

3

24.33

24.18

24.37

0-3

24.28

23.87

24.37

1 Temperature was continuously monitored in a container of water located adjacent to the study with an Amegaview Scientific centralized monitoring system.

Table 3 pH Measurements

Nominal WAF

Loading Rates

(µg a.i./L)

pH Measurements

Day 01

Day 32

Negative Control

7.4

9.7

1.0

7.4

9.9

2.6

7.4

9.9

6.4

7.4

9.6

16

7.4

7.8

40

7.4

7.5

100

7.4

7.5

1    Day 0 samples were collected from the individual batches of test solution prepared for each treatment and control group at test initiation.

2    Day 3 samples were from pooled samples of test solution collected from the three replicates per treatment group and the six replicates in the control group at test termination.

 

Table 4 Light Intensity Measurements on Day 0

Shaker Table ID#

Light Intensity (lux)1

Measurement

No. 1

Measurement

No. 2

Measurement

No. 3

Measurement

No. 4

Measurement

No. 5

 

Mean ± S.D.

AQL#16

6,130

5,910

5,900

6,540

6,040

6,104 ± 261.8

1  Measurements taken at five locations surrounding the test flasks at test solution level.


The toxicity of 1,12-Dodecanediol Bismethacrylate (CAS #72829-09-5) to R. subcapitata was determined by evaluating changes in cell density over a 72-hour exposure period. Cell densities were used to calculate growth rates for each 24-hour interval of exposure and yields at 72 hours of exposure. Mean cell densities, growth rates, and yields and their corresponding percent inhibition values after 72 hours of exposure are presented in Table 5. NOEC values and 72-hour ECx, ErCx, and EyCxvalues and their corresponding 95% confidence limits for cell density, growth rate, and yield, respectively, are presented in Table 6. ECx and ErCx values estimated at 24 and 48 hours of exposure are presented in Appendix 5. Individual replicate data for each growth parameter are presented in Appendices 6 and 7.

Table 6 NOEC, ECx, ErCx, and EyCxValues After 72 Hours of Exposure

 

Endpoint

 

Based on Nominal WAF Loading Rates

 

Growth Rate:

 

 

 

72- hour ErC501

 

17µg a.i./L

 

95% Confidence Interval

 

14 to 21µg a.i./L

 

 

 

 

 

72- hour ErC101

 

6.4 µg a.i./L

 

95% Confidence Interval

 

4.2 to 9.7 µg a.i./L

 

 

 

 

 

72- hour NOEC2

 

6.4 µg a.i./L

 

Cell Density:

 

 

 

72- hour EC501

 

8.1 µg a.i./L

 

95% Confidence Interval

 

4.7 to 14 µg a.i./L

 

 

 

 

 

72- hour EC101

 

3.8 µg a.i./L

 

95% Confidence Interval

 

1.4 to 10 µg a.i./L

 

 

 

 

 

72- hour NOEC2

 

2.6 µg a.i./L

 

Yield:

 

 

 

72- hour EyC501

 

8.3 µg a.i./L

 

95% Confidence Interval

 

5.2 to 13 µg a.i./L

 

 

 

 

 

72- hour EyC101

 

4.3 µg a.i./L

 

95% Confidence Interval

 

1.8 to 10 µg a.i./L

 

 

 

 

 

72- hour NOEC2

 

2.6 µg a.i./L

 

1  ErCx,ECx,and EyCxvalues were estimated, when possible, using non-linear regression with treatment response and nominal WAF loading rates.

 

2  NOEC values were determined based on the results of statistical comparisons between treatment groups and the negative control.

Appendix 5

EC50 and ErC50Values at 24 and 48 Hours of Exposure Based on Nominal WAF Loading Rates

 

Endpoint

 

24 Hours

48 Hours

 

Growth Rate:

 

 

 

 

ErC501

 

10 µg a.i./L

14 µg a.i./L

 

95% Confidence Interval

 

7.0 to 15 µg a.i./L

12 to 15 µg a.i./L

 

 

 

 

 

 

Cell Density:

 

 

 

 

EC501

 

8.0 µg a.i./L

6.1 µg a.i./L

 

95% Confidence Interval

 

4.8 to 13 µg a.i./L

3.9 to 9.6 µg a.i./L

 

 

 

 

 

 

1  ErCxandECxvalues were estimated, when possible, using non-linear regression with treatment response and nominal WAF loading rates.

 

 

Appendix 6

 

Cell Density and Yield by Replicate Over the 72-Hour Exposure Period

 

Nominal WAF Loading Rate

(µg a.i./L)

 

 

Replicate

Cell Density (cells/mL)1,2

Yield (cells/mL)3

24 Hours

48 Hours

72 Hours

72 Hours

Negative Control

A

85,387

611,187

2,796,242

2,786,242

 

(0.0)

B

95,189

894,117

3,409,155

3,399,155

 

 

C

114,528

654,299

2,834,469

2,824,469

 

 

D

101,225

793,933

3,688,907

3,678,907

 

 

E

90,372

600,150

2,994,231

2,984,231

 

 

F

127,030

899,115

3,815,614

3,805,614

 

1.0

A

96,217

841,727

3,071,559

3,061,559

 

 

B

110,161

1,024,345

3,427,900

3,417,900

 

 

C

106,325

901,655

3,823,609

3,813,609

 

2.6

A

99,136

815,015

3,619,708

3,609,708

 

 

B

100,060

919,579

3,679,031

3,669,031

 

 

C

80,829

564,166

3,104,815

3,094,815

 

6.4

A

88,645

595,124

2,731,545

2,721,545

 

 

B

51,832

273,693

2,086,182

2,076,182

 

 

C

67,143

357,927

2,324,145

2,314,145

 

16

A

15,204

22,369

83,617

73,617

 

 

B

19,651

66,209

398,485

388,485

 

 

C

23,689

81,682

448,073

438,073

 

40

A

10,313

9,890

14,804

4,804

 

 

B

12,214

10,387

15,624

5,624

 

 

C

11,989

10,192

23,689

13,689

 

100

A

8,785

7,706

7,199

0

 

 

B

10,988

8,637

14,025

4,025

 

 

C

12,644

8,260

7,350

0

 

1   The initial cell density of the stock culture was determined and an inoculum volume was administered to each test chamber to yield a cell density of approximately 10,000 cells/mL at test initiation (0 hours).

2  Mean cell density in the negative control replicates after 72 hours was 3,256,436 cells/mL, exceeding the 16X growth requirement.

3  Yield was calculated as the final cell density in the exposure period minus the initial cell density.

 

Appendix 7 Growth Rate by Replicate Over the 72-Hour Exposure Period

 

Nominal WAF Loading Rate

(µg a.i./L)

 

 

Replicate

Growth Rate (per hour)

024 Hours

0 – 48 Hours

0 – 72 Hours

Negative Control

A

0.0894

0.0857

0.0782

(0.0)

B

0.0939

0.0936

0.0810

 

C

0.1016

0.0871

0.0784

 

D

0.0965

0.0911

0.0821

 

E

0.0917

0.0853

0.0792

 

F

0.1059

0.0937

0.0826

1.0

A

0.0943

0.0924

0.0795

 

B

0.1000

0.0964

0.0811

 

C

0.0985

0.0938

0.0826

2.6

A

0.0956

0.0917

0.0818

 

B

0.0960

0.0942

0.0821

 

C

0.0871

0.0840

0.0797

6.4

A

0.0909

0.0851

0.0779

 

B

0.0686

0.0689

0.0742

 

C

0.0793

0.0745

0.0757

16

A

0.0175

0.0168

0.0295

 

B

0.0282

0.0394

0.0512

 

C

0.0359

0.0438

0.0528

40

A

0.0013

0

0.0054

 

B

0.0083

0.0008

0.0062

 

C

0.0076

0.0004

0.0120

100

A

0

0

0

 

B

0.0039

0

0.0047

 

C

0.0098

0

0

After 72 hours of exposure, inhibition of cell density in the1.0, 2.6, 6.4, 16, 40 and 100 µg a.i./Ltreatment groups (based on nominal WAF loading rates) was -6, -6, 27, 90, 99 and 100%, respectively, relative to the negative control. Inhibition of growth rate in the1.0, 2.6, 6.4, 16, 40 and 100 µg a.i./Ltreatmentgroups was -1, -1, 5, 45, 90 and 98%, respectively, relative to the negative control. Inhibition of yield in the1.0, 2.6, 6.4, 16, 40 and 100 µg a.i./Ltreatment groups was -6, -7, 27, 91, 100 and 100%, respectively, relative to the negative control(Table 5). Dunnett’s test indicated mean cell density and mean yield were significantly reduced (p < 0.05) in the6.4, 16, 40 and 100 µg a.i./Ltreatment groups when compared to the negative control. Jonckheere-Terpstra Step-Down Trend test indicated that mean growth rate was significantly reduced (p < 0.05) in the16, 40 and 100 µg a.i./Ltreatment groups when compared to the negative control. The 72-hour NOEC was determined to be 2.6 µg a.i./Lbased on statistically significant reductions in cell density and yield. The 72-hour EC50values for cell density, growth rate, and yield were determined to be 8.1, 17, and 8.3 µg a.i./L, respectively (Table 6).

After 72 hours of exposure, adherence of cells to the test chambers was not observed in any of the control or treatment groups. Flocculation or aggregation of cells was not observed in any of the experimental groups. Cells present in all treatment groups appeared normal when compared to cells in the negative control.

Conditions for the Validity of the Test

Mean cell density in the control flasks increased by a factor of 326 after three days, achieving the 16X growth criterion. The coefficient of variation of average specific growth rates in the control replicates was 2.3%, achieving the less than 7% criterion. The mean percent coefficient of variation for section-by-section specific growth rates in the control replicates was 22.4%, achieving the less than 35% criterion (Appendix 8).

Appendix 8 Specific Growth Rate of the Negative Control

 

Section-by-Section Growth Rate for Negative Control

Control Replicate

0-24 Hour
Growth Rate

24-48 Hour
Growth Rate

48-72 Hour
 Growth Rate

% Coefficient of Variation1

A

0.0894

0.0820

0.0634

17.1

B

0.0939

0.0933

0.0558

27.0

C

0.1016

0.0726

0.0611

26.6

D

0.0965

0.0858

0.0640

20.2

E

0.0917

0.0789

0.0670

15.6

F

0.1059

0.0815

0.0602

27.7

 

 

 

Mean:

22.4

1Coefficient of Variation = S.D./mean *100

Results were generated using Excel 2010 in the full precision mode. Manual calculations may differ slightly.

 

Average Specific Growth Rate for the Negative Control

 

 

Control Replicate

0-72 Hour

Growth Rate

A

0.0782

B

0.0810

C

0.0784

D

0.0821

E

0.0792

F

0.0826

Mean=
Standard Deviation=
% COV1=

0.0803
0.0019
2.3

1Coefficient of Variation = S.D./mean *100

Results were generated using Excel 2010 in the full precision mode. Manual calculations may differ slightly.


 


Validity criteria fulfilled:
yes
Conclusions:
CONCLUSIONS
The freshwater alga, Raphidocelis subcapitata, was exposed to a series of six nominal WAF loading rates of 1,12-Dodecanediol Bismethacrylate (CAS #72829-09-5) ranging from 1.0 to 100 µg a.i./L. The toxicity of 1,12-Dodecanediol Bismethacrylate to Raphidocelis subcapitata was assessed based on effects on cell density, growth rate, and yield relative to the negative control. The 72-hour NOEC was determined to be 2.6 µg a.i./L based on statistically significant reductions in cell density and yield. The 72 hour EC50, ErC50 and EyC50 values for cell density, growth rate, and yield were determined to be 8.1, 17, and 8.3 µg a.i./L, respectively.
Executive summary:

The freshwater alga, Raphidocelis subcapitata, was exposed to a series of six nominal WAF loading rates of 1,12-Dodecanediol Bismethacrylate (CAS #72829-09-5) ranging from 1.0 to 100 µg a.i./L.  The toxicity of 1,12-Dodecanediol Bismethacrylate to Raphidocelis subcapitata was assessed based on effects on cell density, growth rate, and yield relative to the negative control.  The 72-hour NOEC was determined to be 2.6 µg a.i./L based on statistically significant reductions in cell density and yield.  The 72 hour EC50, ErC50 and EyC50 values for cell density, growth rate, and yield were determined to be 8.1, 17, and 8.3 µg a.i./L, respectively. The 72 hour EC10, ErC10 and EyC10 values for cell density, growth rate, and yield were determined to be 3.8, 6.4, and 4.3 µg a.i./L, respectively.

Description of key information

A study assessing the toxicity of the test item to Raphidocelis subcapitata (Pseudokirchneriella subcapitata)  was conducted in accordance with the OECD 201 Test Guideline and GLP requirements.

Following exposure to a series of six nominal WAF loading rates of the test item ranging from 1.0 to 100 µg a.i./L, the 72 hour ErC50 (growth rate) was found to be 17 µg a.i./L and the ErC10 (growth rate) was equal to 6.4 µg a.i./L.

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
17 µg/L
EC10, LC10 or NOEC for freshwater algae:
6.4 µg/L

Additional information