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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:
2012-05-04 to 2012-05-18
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The saturated solution, further dilution levels and the control were verified at the beginning and the end of the test via LC-MS/MS analysis.
- Sampling method: At test start samples were taken out of additional replicates provided without algae. At the end of the test samples were taken from pooled replicates.
- Sample storage conditions before analysis: All stabilized samples were stored at 6 ± 2 °C until sample preparation.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution with a nominal loading of 3.9 mg test item/L was prepared with dilution water three days prior to application. The test item dispersion was stirred for three days with a magnetic stirrer at 1100 rpm at room temperature. After a separation of the phases after 1 min of standing, the saturated solution was taken from the homogeneous phase.

- Eluate: Dilutioon water
- Differential loadings: 226 - 399 - 638 - 1268 - 2088 µg/L (geom mean measured)
- Controls: Six replictes without test item were tested under the same test conditions as the test replicates.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDAK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Not measured
Test temperature:
Mean: 22.8 °C, Min: 22.5 °C, Max: 23.0 °C
pH:
Geometric mean measured
test item concentration pH-value
[µg/L] Start; 0 h End; 72 h
2088 7.83 9.32
1268 7.90 9.29
638 7.92 9.41
399 7.93 9.40
226 7.94 9.43
Control 7.92 9.36
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to information on materials and methods
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 5466 cells/mL
- Control end cells density: Mean 2139925 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: Yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
According to the guideline (OECD)

Composition of Dilution water (OECD)

Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH-value 8.1  0.2
This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0 – 72 h)


Saturated solution (nominal)
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 9 38
10 -1 -4











Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2 088 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2 088 µg/L
Nominal / measured:
meas. (geom. mean)
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):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.718 (CI 0.699 - 0.737) mg/L
Yield Inhibition: 0.422 (CI 0.386 - 0.466) mg/L
Reported statistics and error estimates:
NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

NOEC, LOEC and EC-Values (0-72 hours)

based on geometric mean measured test item concentration [mg/L] 

Rate-related inhibition

NOEC

2088

LOEC

> 2088

ErC10

> 2088

ErC20

> 2088

ErC50

> 2088

Inhibition of yield

NOEC

2088

LOEC

> 2088

EyC10

1335 (544 - > 2088)

EyC20

> 2088

EyC50

> 2088

Cell Densities

Geometric mean measured test item concentration

Replicate

Cell density [cells/mL]

[µg/L]

No.

0 hours

24 hours

48 hours

72 hours

2088

1

5466

18511

175631

1465644

2

5466

21022

206579

1979843

3

5466

19785

222119

2062240

Mean

5466

19773

201443

1835909

1268

1

5466

19945

209261

1818885

2

5466

18757

194212

1781933

3

5466

17937

235671

2141768

Mean

5466

18880

213048

1914195

638

1

5466

22303

234419

2219360

2

5466

21346

240730

1993700

3

5466

21975

251741

2131189

Mean

5466

21875

242297

2114750

399

1

5466

20429

220592

2055348

2

5466

21148

268168

2221483

3

5466

20940

270798

2010648

Mean

5466

20839

253186

2095826

226

1

5466

23115

255026

2024990

2

5466

25238

297171

2485586

3

5466

23160

305768

2182222

Mean

5466

23838

285988

2230933

Control

1

5466

20955

233451

1839261

2

5466

19871

277317

2174362

3

5466

23041

335561

2017502

4

5466

25838

314648

2163150

5

5466

21990

304099

2363555

6

5466

23756

288983

2281717

Mean

5466

22575

292343

2139925

Evaluation after 72 hours

                                Statistically significant differences of growth rates and yield compared to

                                control values are marked (+), not significant differences are marked (-).

Geometric mean measured test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[µg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

2088

1

1.86

  6.29

1460178

 31.6

2

1.96

  1.25

1974377

     7.50

3

1.98

  0.56

2056774

     3.64

Mean

(-)

1.94

  2.70

(-)

1830443

  14.2

1268

1

1.94

  2.67

1813419

  15.0

2

1.93

  3.01

1776467

  16.8

3

1.99

-0.07

2136302

    -0.09

Mean

(-)

1.95

  1.87

(-)

1908729

  10.6

638

1

2.00

-0.67

2213894

    -3.72

2

1.97

  1.13

1988234

     6.85

3

1.99

  0.01

2125723

     0.41

Mean

(-)

1.99

  0.16

(-)

2109284

     1.18

399

1

1.98

  0.62

2049882

     3.96

2

2.00

-0.68

2216017

    -3.82

3

1.97

  0.99

2005182

    6.06

Mean

(-)

1.98

  0.31

(-)

2090360

     2.07

226

1

1.97

  0.87

2019524

     5.38

2

2.04

-2.57

2480120

-16.2

3

2.00

-0.38

2176756

    -1.98

Mean

(-)

2.00

-0.69

(-)

2225467

    -4.26

Control

1

1.94

1833795

2

2.00

2168896

3

1.97

2012036

4

1.99

2157684

5

2.02

2358089

6

2.01

2276251

Mean

1.99

2134459

Section-by-Section and Average Specific Growth Rates of the Control Group
(0 – 72 hours)

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 hours)

SD

±

CV
[%]

Mean CV [%]

section-by-section                           

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.34

2.41

2.06

1.94

0.544

28.1

29.1

2

1.29

2.64

2.06

2.00

0.675

33.8

3

1.44

2.68

1.79

1.97

0.639

32.4

4

1.55

2.50

1.93

1.99

0.477

23.9

5

1.39

2.63

2.05

2.02

0.618

30.5

6

1.47

2.50

2.07

2.01

0.517

25.7

Mean

1.99

 

SD ±

0.03

 

CV [%]

1.51

 

Validity criteria fulfilled:
yes
Conclusions:
In this study VESTANAT B 1370 (solvent free) was not found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata significantly, when tested with the saturated solution and further dilution levels. The effect values after 72 hours (geometric mean measured test item concentrations) were: The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 hours were > 2088 µg/L for both endpoints. The NOEC-values for inhibition of specific growth rate and yield after 72 hours were 2088 µg/L (highest test concentration) for both endpoints. All effect values are given based on geometric mean measured concentrations of VESTANAT B 1370 (solvent free).
Executive summary:

The toxicity of Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked  to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 from 2012-05-04 to with the definitive exposure phase from to at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany. The aim of the study was the determination of NOEC, LOEC, EC10 - , EC20- and EC50 - values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5466 cells/mL. A saturated solution* (i.e. 100 %) and further four dilution levels of 9.96 - 17.7 - 31.6 - 56.2 % of the saturated solution were tested, corresponding to geometric mean measured test item concentrations of 226 - 399 – 638 – 1268 – 2088 µg/L. Three replicates for each dilution level and six replicates for the control were tested. Environmental conditions were determined to be within the acceptable limits.

The concentrations of VESTANAT B 1370 (solvent free) were analytically verified by LC-MS/MS analysis at test start and test end of all dilution levels and the control. The measured concentrations of VESTANAT B 1370 (solvent free) at test start were in the range of 180 – 2154 µg/L. The measured concentrations at the end of the test were in the range of 283 – 2024 µg/L. All effect values given are based on the geometric mean measured concentrations of the test item VESTANAT B 1370 (solvent free).

Table 1:    NOEC, LOEC and EC-Values (0-72 hours)

based on geometric mean measured test item concentration [µg/L] 

Rate-related inhibition

NOEC

2088

LOEC

> 2088

ErC10

> 2088

ErC20

> 2088

ErC50

> 2088

Inhibition of yield

NOEC

2088

LOEC

> 2088

EyC10

1335 (544 - > 2088)

EyC20

> 2088

EyC50

> 2088

Description of key information

The toxicity of Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201. The aim of the study was the determination of NOEC, LOEC, EC10 - , EC20- and EC50 - values of growth rate and yield over a period of 72 hours.
The study was conducted under static conditions with a saturated solution* (i.e. 100 %) and further four dilution levels of 9.96 - 17.7 - 31.6 - 56.2 % of the saturated solution, corresponding to geometric mean measured test item concentrations of 226 - 399 – 638 – 1268 – 2088 µg/L.
The concentrations of the test item were analytically verified by LC-MS/MS analysis at test start and test end of all dilution levels and the control. The measured concentrations at test start were in the range of 180 – 2154 µg/L. The measured concentrations at the end of the test were in the range of 283 – 2024 µg/L. All effect values given are based on the geometric mean measured concentrations of the test item.

Key value for chemical safety assessment

EC50 for freshwater algae:
2 088 µg/L
EC10 or NOEC for freshwater algae:
2 088 µg/L

Additional information

In this study the test item was not found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata significantly, when tested with the saturated solution and further dilution levels. The effect values after 72 hours (geometric mean measured test item concentrations) were: The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 hours were > 2088 µg/L for both endpoints. The NOEC-values for inhibition of specific growth rate and yield after 72 hours were 2088 µg/L (highest test concentration) for both endpoints.