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Ecotoxicological information

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:
14 September - 17 September 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully documented study run under GLP and according to international guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Temperature in reference study and main study higher as in guideline. pH values of the controls in main study varied more than stated in the guideline. As the controls showed a normal exponential growth, these deviations can be stated as uncritical.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
Temperature in reference study and main study higher as in guideline. pH values of the controls in main study varied more than stated in the guideline. As the controls showed a normal exponential growth, these deviations can be stated as uncritical.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
At the beginning and at the end of the test, the content of test item was determined by DOC-determination in the test solutions.
Vehicle:
no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CHODAT
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)
- Age of inoculum (at test initiation): From September 2010
- Method of cultivation: The algae are kept as stock culture on solid agar at 8 °C. From an aliquot of this stock culture the preculture was prepared.

ACCLIMATION
- Acclimation period: Four days before the start of each test, an aliquot of the stock culture containing a few cells was brought into preculture medium and incubated for 72 hours. The resulting culture is growing exponentially.
Before usage, the culture was checked on the absence of cell aggregates. After the adjustment to a cell concentration of about 6*10 E4/mL through photometric measurement and addition of algal medium, the culture was usable for the test. The adjusted preculture was mixed with the same amount of 10-fold-nutrient solution. This mixture was the test culture.
- Culturing media and conditions (same as test or not): same as test (without substance)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 - 27 °C
pH:
7 - 9.5 (in controls 7 - 9)
Nominal and measured concentrations:
Nominal and measured concentrations: See table in "Any other information on materials and methods incl. tables"
Details on test conditions:
TEST SYSTEM
- Test vessel: closed glass beakers
- Type (delete if not applicable):closed
- Material, size, headspace, fill volume: glass, 130 mL
- Aeration: no (indirectly by shaking)
- Initial cells density: 6*10^4/mL
- Control end cells density:487*10^4/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Intervals of water quality measurement: all 24h

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no: no information
- Adjustment of pH: To avoid a high pH increase, 700 ml deionised water were mixed with 6 mL sodium hydrogencarbonate solution (50 g/L). Then, pH was adjusted to 7.0 (±0.2) with HCl.
- Photoperiod: 72 hours
- Light intensity and quality: 5800 – 5900 Lux
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer
- Chlorophyll measurement: no
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
8.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
dissolved organic carbon
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
8.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
dissolved organic carbon
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
8.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
dissolved organic carbon
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
dissolved organic carbon
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
dissolved organic carbon
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
dissolved organic carbon
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
58 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
dissolved organic carbon
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
23 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
dissolved organic carbon
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
dissolved organic carbon
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC100
Effect conc.:
> 63 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: dissolved organic carbon
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC100
Effect conc.:
> 63 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: dissolved organic carbon
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC100
Effect conc.:
> 63 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: dissolved organic carbon
Basis for effect:
other: yield
Results with reference substance (positive control):
NOEC 72 h 0.4 mg/L
LOEC 72 h 0.63 mg/L
72h ErC50 2.0 mg/L
72h EbC50 0.49 mg/L
72h EyC50 0.34 mg/L
Reported statistics and error estimates:
For the treatments with the nominal test item concentrations 18 and 32 mg/L (measured concentrations 8.6 and 18.7 mg/L), it was tested whether the differences between treatment and control were significant. The values for the area under the curve, yield and the growth rate were used for the assessment of observed effect levels.
In order to select a suitable test for significance, it was checked whether equality of variance was given.
The calculated value F is compared with the F-test table (level of significance 95%). If the calculated value is smaller than the tabular value, equality of variance is given. If equality of variance is given, the t-test is used; else, the WEIR test is used. With the t-Test it was checked whether the differences are significant. Significance is given if the calculated t-value is bigger than the limit of significance (t-value taken from the table with grade of freedom: n1 + n2 – 2, level of significance 95%).

Determination of the NOEC
For the endpoints µ, AUC and yield, the differences between the treatment 18 mg/L (measured concentration 8.6 mg/L) and the control can be considered as not significant (level of significance: 97.5%) as the calculated t-values were smaller than the limit of significance. Therefore, the measured concentration 8.6 mg/L is stated as NOEC for these endpoints.

Determination of the LOEC
For the endpoints µ, AUC and yield, the differences between the treatment 32 mg/L (measured concentration 18.7 mg/L) and the control must be considered as significant (level of significance: 97.5%), the measured concentration 18.7 mg/L can be stated as LOEC for these endpoints.

The estimation of the EC50s of the test item was accomplished using the software OriginTM.

Main Study

Cell Numbers

The cell numbers were determined by photometric measurement of optical density. The means and standard deviations of the cell numbers of the control and the treatments are presented in the following table:

Cell Number/mL Main Study

Nominal Concentration in mg/L

Parameter

Cell Number/mL

 

 

0 h

24 h

48 h

72 h

0

Mean

7354

24513

106633

486590

0

SD

0

6005

12084

48873

10

Mean

7354

22062

115213

563807

10

SD

0

0

22467

88350

18

Mean

7354

24513

151983

720692

18

SD

0

4246

4246

36770

32

Mean

7354

25739

73540

290483

32

SD

0

5200

10400

98801

56

Mean

7354

17159

26965

88248

56

SD

0

4246

15309

44733

100

Mean

7354

9805

12257

75991

100

SD

0

4246

8492

29721

SD = Standard Deviation

Temperature, Light Intensity, pH

The pH values in the control ranged from 7.0 to 9.0. The light intensity was in a range of 5800 – 5900 lux. Temperature range was 24.0 – 27.0 °C. The details are given in the following table:

Temperature, Light Intensity, pH Main Study

 

0 h

24 h

48 h

72 h

Temperature [°C]

24

25

24

27

Light intensity [lux]

5800

5800

5900

5900

pH

Conc. in mg/L

 

-

-

 

 

0

7.0

-

-

9.0

 

10

7.0

-

-

9.5

 

18

7.0

 

 

9.5

 

32

7.0

 

 

9.1

 

56

7.0

-

-

7.3

 

100

7.0

-

-

7.1


Microscopical Observations

In the following table, the appearance of the alga at the end of the test is stated:

Microscopical Observation Main Study

Nom. Conc. in mg/L

Observation

0

normal growth

10

normal growth

18

normal growth

32

smaller cells

56

no cells

100

no cells

 

Analytical Determination

The measured and calculated concentrations for treatment and control are given in the following table. The concentration of test item was calculated through measured DOC divided by the content of organic carbon (76%) of the test item.

DOC Values and Recovery

Nominal Conc. mg/L

DOC

mg/L

Test item mg/L

% of Nominal

DOC

mg/L

Test item mg/L

% of Nominal

% Recovery

Geom. Mean of Test Item mg/L

0h

72h

0

2.34

-

-

2.33

-

-

-

-

10

6.82

8.97

90%

4.37

5.75

58%

64%

7.18

18

6.82

8.97

50%

6.31

8.30

46%

93%

8.63

32

15.25

20.07

63%

13.24

17.42

54%

87%

18.70

56

32.46

42.71

76%

29.03

38.20

68%

89%

40.39

100

54.05

71.12

71%

42.85

56.38

56%

79%

63.32

Area under the Curve, Growth Rate, Yield

From the cell numbers, the Growth Rate µ, the Area under the Curve AUC, and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:

Growth Rate µ, Area under the Curve AUC, Yield Main Study

Nom. Concentration in mg/L

Meas. Concentration in mg/L

 

Growth Rate [day-1]

AUC
[Cell Concentration/mL*day]

Yield
[Cell Concentration/mL]

0

 

Mean

1.40

356056

479236

SD

0.04

41236

48873

10

7.2

Mean

1.44

400793

556453

SD

0.05

66492

88350

18

8.6

Mean

1.53

518457

713338

SD

0.02

22963

36770

32

18.7

Mean

1.22

226136

283129

SD

0.12

65001

98801

56

40.4

Mean

0.80

69863

80894

SD

0.16

41763

44733

100

63.3

Mean

0.76

41673

68637

SD

0.12

27598

29721

SD = Standard Deviation

Reference Study

Cell Numbers

The cell numbers were determined by photometric measurement of optical density. The means and standard deviations of the cell numbers of the control and the treatments are presented in the following table:

Cell Number/mL Reference Study

Nominal Concentration in mg/L

Parameter

Cell Number/mL

 

 

0 h

24 h

48 h

72 h

0

Mean

7354

60303

238270

760404

0

SD

0

9588

69882

187966

0.04

Mean

7354

63735

230425

823648

0.04

SD

0

8492

4246

19457

0.063

Mean

7354

66186

227974

728046

0.063

SD

0

0

7354

74635

0.1

Mean

7354

58832

166691

539293

0.1

SD

0

7354

48965

176700

0.16

Mean

7354

51478

134823

485364

0.16

SD

0

19457

73662

247099

0.25

Mean

7354

56381

149531

436337

0.25

SD

0

4246

74029

276077

0.4

Mean

7354

71089

174045

509877

0.4

SD

0

4246

4246

29721

0.63

Mean

7354

61283

115213

296611

0.63

SD

0

4246

8492

15309

1.0

Mean

7354

53929

83345

98053

1.0

SD

0

4246

18507

16983

1.6

Mean

7354

58832

56381

83345

1.6

SD

0

0

8492

8492

SD = Standard Deviation

 Area under the Curve, Growth Rate, Yield

From the cell numbers, the Growth Rate µ, the Area under the Curve AUC, and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:

Growth Rate µ, Area under the Curve AUC, Yield Reference Study

Nominal Concentration in mg/L

 

Growth Rate [day-1]

AUC
[Cell Concentration/mL*day]

Yield
[Cell Concentration/mL]

0

Mean

1.54

660389

753050

SD

0.09

166860

187966

0.04

Mean

1.57

687599

816294

SD

0.01

18385

19457

0.063

Mean

1.53

639798

720692

SD

0.03

42405

74635

0.1

Mean

1.42

476784

531939

SD

0.11

138039

176700

0.16

Mean

1.36

410598

478010

SD

0.18

215107

247099

0.25

Mean

1.31

405696

428983

SD

0.22

215327

276077

0.4

Mean

1.41

481687

502523

SD

0.02

13258

29721

0.63

Mean

1.23

306417

289257

SD

0.02

18869

15309

1.0

Mean

0.86

167916

90699

SD

0.06

25826

16983

1.6

Mean

0.81

138500

75991

SD

0.04

11233

8492

 

 Inhibition

The following mean inhibition values were calculated for the Growth Rate µ, the Area under the Curve AUC, and the yield. Individual inhibition values are given in the annex.

Inhibition Values Reference Study

Nominal Concentration in mg/L

% Inhibition

Growth Rate µ

Area under the Curve AUC

Yield

0

0!

0!

0!

0.04

-2.36

-4.12

-8.40

0.063

0.39

3.12

4.30

0.1

7.63

27.80

29.36

0.16

11.16

37.82

36.52

0.25

14.56

38.57

43.03

0.4

8.06

27.06

33.27

0.63

19.81

53.60

61.59

1.0

44.01

74.57

87.96

1.6

47.41

79.03

89.91

Validity criteria fulfilled:
yes
Remarks:
except for the pH in the reference study
Conclusions:
The following results for the test item "2-propanol and 2-butanol production, distn. residues" were determined:
The NOECs of the growth rate, AUC and yield were 8.6 mg/L and the LOECs 19 mg/L. The EC50 value for the growth rate was 58 mg/L, for the AUC 23 mg/L and for the yield also 23 mg/L.
Based on these data in combination with the fact that the substance is not readily biodegradable, a classification into aquatic chronic category 3 (H412) (corresponding to R52/53) is necessary (EC50 band 10 - 100 mg/L).
Executive summary:

The 72h-EC50 of reaction mass of "2-propanol and 2-butanol production, distn. residues" (main constituent 2,2'-oxybisbutane (DSBE), DIPE, SBA and 2 -methylpropan-2 -ol (TBA)) against Desmodesmus subspicatus was determined according to OECD 201 resp. EU C.3.

One valid main study was performed.

The study was performed using five concentrations ranging from 10 to 100 mg/L. The treatments were used to incubate the unicellular freshwater green alga Desmodesmus subspicatus for a period of 72 hours. The cell concentration of each replicate was determined by measuring the absorption of the cuvettes at 440 nm every 24 hours with a spectral photometer. With these measured values, the number of cells was calculated (linear correlation between cell concentration and absorption given). Then the growth rate µ, the area under the growth curve (AUC [1]) and the Yield [2] were determined.

At the beginning and at the end of the test, the content of test item was determined by DOC-determination in the test solutions. The content of the test item was calculated from content of organic carbon of the test item (76 %) The recovery after 72 hours ranged from 64 to 93 % of the start concentration. The correlation between nominal and measured concentration was poor. Therefore, the geometric mean of the measured concentration was used for the determination of the biological results and the determination of the biological results was based on the mean of the measured concentrations.

The EC50s of potassium dichromate were tested in a current reference test. The values lay within the normal range of the laboratory.

The following results for the test item "2-propanol and 2-butanol production, distn. residues" were determined:

Growth Rate: NOEC 8.6 mg/L, LOEC 19 mg/L, EC50 58 mg/L

AUC: NOEC 8.6mg/L, LOEC 19 mg/L, EC50 23 mg/L

Yield: NOEC 8.6mg/L, LOEC 19 mg/L, EC50 25 mg/L

 

[1] AUC1 (Area Under Curve according to EU Method C.3) means the integral of the biomass.

[2] Yield (according to OECD Guideline 201) is defined as the biomass at the end of the exposure period minus the biomass at the start of the exposure period.

Description of key information

The following results were determined for the test item "2-propanol and 2-butanol production, distn. residues" (species: Desmodesmus subspicatus): 72h-ErC50 (growth rate) = 58 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
58 mg/L
EC10 or NOEC for freshwater algae:
8.6 mg/L

Additional information

The EC50 value and the NOEC for growth inhibition, AUC and yield of "2 -propanol and 2 -butanol, distn residues" was measured according to OECD 201, respectively EU C.3 guideline under GLP. Following results were obtained:

Endpoint

NOEC

LOEC

EC50

Growth Rate

8.6 mg/L

19 mg/L

58 mg/L

AUC

8.6 mg/L

19 mg/L

23 mg/L

Yield

8.6 mg/L

19 mg/L

25 mg/L

The analytical determinations of the test item showed poor correlation between nominal and measured concentrations and acceptable recovery rates. The poor correlation is probably caused by the high volatility of the test item. Therefore, the determination of the results of the test item "2 -propanol and 2 -butanol, distn. residues" was based on the geometric means of the measured concentrations.

Based on these data in combination with the fact that the substance is not readily biodegradable, a classification into aquatic chronic category 3 (H412) according to the CLP regulation (corresponding to R52/53) is necessary (EC50 10 - 100 mg/L).

Based on the lowest EC50 value of three species daphnia, algae and fish (available studies), an aquatic PNEC value was calculated for the risk assessment. As values from algae test based on growth rate, not on biomass or yield, should be employed for classification and risk assessment, the relevant EC50 value is 58 mg/L. Therefore, the value for algae does not represent the lowest value of three species daphnia, algae and fish.