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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:
From 2009-03-09 to 2009-03-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 201 with GLP certificate. All validity criteria were fulfilled and no deviation was observed.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
Inspected on: 23 bis 25 August 2006 / Signed on 21 March 2007
Specific details on test material used for the study:
- Water solubility: Low, 840 µg/L in algal medium
- Vapour pressure: 0.04 Pa at 25°C
- Partition coefficient (log Pow): 6.57 (measured)
- Storage condition of test material: Room temperature, dry, well ventilated. Keep container closed and filled, keep away from heat sources, protect against high temperature. Do not re-use empty container.
- Storage stability: stable under normal conditions, avoid oxidizing materials
Analytical monitoring:
yes
Details on sampling:
- Concentrations: the concentrations of test substance were assessed by chemical analysis in the test solution at start and end of the test.
- Sampling method: At test sub-sample from each freshly prepared test solution were taken for chemical analysis. At the end pooled samples were taken from each test concentration. The samples for analysis were filled into 40 mL sample vials. The vials were filled up to the brim to prevent the loss of test substance into the headspace of the vials.
For sample preparation, aliquot samples ranging from 0.1 to 5.0 mL were pipetted into 8 mL test tubes, sample volumes less than 5 mL were filled up to 5 mL with purified water. Then 100 µL of the IS working solution and 400 µL cyclohexane were added. The mixtures were shaken thoroughly by hand and additionally for 10 min on a horizontal shaker (about 200 strokes/min). For a rapid phase separation the tubes are centrifuged at 2000 rpm for 2 minutes. The organic supernatants (250 µL aliquots) were transferred into the autosampler vials using disposal Paster pipettes, aliquots of 1µL were analysed by GC/MS.
- Sample storage conditions before analysis: no data
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 200 mg (214 µL) of test substance were given into 2 L growth medium under sterile conditions in a 2 L brown glass flask with a drain port near the bottom. The preparation was stirred at room temperature with around 300 rpm for 48 h. After 48 hours the preparation was left for around 18 hours (overnight) to enable droplets to move to the surface of the solution. Then, around 200 mL of the aqueous phase were drained off and discarded. The following 600 mL were used for the test preparation without filtration as the highest test concentration. Because adsorption of the test item on filter material cannot be excluded, it is preferred to use test solutions without filtration. The highest test concentration was diluted with growth medium using a spacing factor of 2 to prepare the other 4 test concentrations.
- Eluate: growth medium
- Differential loading: no data
- Controls: yes
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data

The test item is a liquid of poor water solubility. Since the use of a dispersant should be avoided, test solutions were prepared in growth medium as described above to obtain the maximum dissolved concentration of the test substance.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: Pseudokirchneriella subcapitata SAG 61.81, Chlorophycea, Chlorophyta.
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Karspüle 2, 37073 Göttingen, Germany. Catalog No 61.81.
- Age of inoculum (at test initiation): the initial cell concentration in the test cutures was 10 000 cells/mL
- Method of cultivation: Three days prior to testing a pre-culture was established in OECD growth medium to obtain exponentially growing algae for the test.

ACCLIMATION
none
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
none
Post exposure observation period:
no
Hardness:
No data
Test temperature:
temperature was between 21.5°C and 22.5°C
pH:
The pH of the controls increased from 7.54 to 9.63 during the test.
In the test cultures the initial pH ranged between 7.57 and 7.84 and was between 8.11 and 10.1 at the end of the test.
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg/L
Geometric mean measured concentrations: 46.1, 83.1, 163, 371 and 715 µg/L.
The test substance levels decreased to concentrations between 61 and 74% of the initial values during the test. The evaluation of the effect concentrations was based on mean measured concentrations of the test item. See table 6.1.5/1 in "Any other information on results incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: conical glass flasks tightly sealed with glass ground-in stoppers.
- Type (delete if not applicable): tightly sealed
- Material, size, headspace, fill volume: glass, 250 mL
- Aeration: no data
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- Initial cells density: 10 000 cells/mL
- Control end cells density: 42 3400 cells/mL (mean)
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: All stock solutions for the OECD medium were prepared with purified water processed using an ELGA, PURELAB Ultra
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no data
- Photoperiod: no data
- Light intensity and quality: adjusted to approximately 7000 Lux
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell concentrations were determined using an electronic particle counter (CASY 1 Model TT, Schärfe System, Reutlingen, Germany). The correctness of the electronic counts was checked by microscopic counting following internal standard operation procedures.
- Chlorophyll measurement: no
- Other: growth and growth inhibition were quantified from measurements of the algal biomass density (cell counts) as a function of time.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: no
- Test concentrations: control plus 6.25, 12.5, 25, 50 and 100 mg/L
- Results used to determine the conditions for the definitive study: not applicable
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 715 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL not determined
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
596 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL not determined
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
371 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Cell number:
See table 6.1.5/2 in "Any other information on results incl. tables".
Cell number (x 10^4)

- Percent inhibition of growth rate:
See table 6.1.5/3 in "Any other information on results incl. tables"
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 4.25 mg/L
- Other: none
Reported statistics and error estimates:
The test results were statistically analysed to determine EC10 and EC50 values with 95% confidence intervals using Probit-analysis assuming log-normal distribution of the values by using the computer program ToxRat.
The NOEC and LOEC values for growth rate were determined by the Williams Multiple Sequential t-test.

Table 6.1.5/1: Measured concentrations

Nominal concentrations

Measured concentrations

Test item loading (mg/L)

Test start (µg/L)

Test end (µg/L)

Test end (% of initial concentrations)

Mean measured concentration (µg/L)

Control

6.25

12.5

25

50

100

< LOQ

56.3

102

210

433

840

< LOQ

37.9

67.7

127

318

609

-

67.3

66.4

60.6

73.5

72.5

-

46.1

83.1

163

371

715

Table 6.1.5/2: Cell number (x 10^4)

Test item (µg/L)

Control

46.1

83.1

163

371

715

24 h

1.430

1.490

2.090

1.440

2.580

1.720

2.050

2.230

2.280

2.660

2.540

2.640

2.570

2.060

2.630

2.210

2.330

2.130

1.510

2.120

1.830

Mean :

Std. Dev :

n :

CV :

1.790

0.460

6

25.7

2.190

0.120

3

5.5

2.620

0.064

3

2.4

2.420

0.315

3

13.0

2.220

0.100

3

4.5

1.820

0.304

3

16.7

48 h

11.820

10.640

14.320

9.280

17.910

12.960

13.850

13.830

14.940

14.100

14.400

12.770

12.390

12.950

13.440

10.4600

12.070

10.500

6.860

6.680

6.760

Mean :

Std. Dev :

n :

CV :

12.820

3.048

6

23.8

14.210

0.635

3

4.5

13.760

0.868

3

6.3

12.930

0.525

3

4.1

11.010

0.918

3

8.3

6.770

0.089

3

1.3

72 h

49.100

40.160

40.860

38.910

45.270

39.760

47.950

39.210

36.820

45.870

45.060

51.730

51.870

43.050

39.190

40.860

43.010

38.000

22.740

24.260

23.060

Mean :

Std. Dev :

n :

CV :

42.340

3.991

6

9.4

41.330

5.859

3

14.2

47.550

3.640

3

7.7

44.700

6.500

3

14.5

40.620

2.513

3

6.2

23.350

0.801

3

3.4

Table 6.1.5/3: Percent inhibition of growth rate by the test item during the 72 hours test period

Test item, mean measured (µg/L)

% inhibition of growth rate

46.1

83.1

163

371

715

0.7

-3.1

-1.3

1.0

15.2

Validity criteria fulfilled:
yes
Conclusions:
The 72h- growth rate ErC50 and ErC10 in Pseudokirchneriella subcapitata is above 0.715 mg test item/L and is equal to 0.596 mg test item/L (geometric mean measured concentration), respectively.
Executive summary:

This study was performed to determine the toxicity of the test substance on the growth of the green alga Pseudokirchneriella subcapitata according to the OECD guideline 201, with GLP certificate.

Since the test item is a liquid of poor water solubility, the highest test solution was prepared with growth medium to obtain the maximum dissolved concentration of the test item.

Nominal loadings were: 6.25, 12.5, 25, 50 and 100 mg test item/L. The test item concentrations were chemically analysed at start and end of the test. At the start of the test, the measured concentrations were 0.0563, 0.102, 0.210, 0.433 and 0.840 mg test item/L. At end of the test, the measured concentrations were 0.0379, 0.0677, 0.127, 0.318 and 0.609 mg test item/L. Based on this, the test substance concentrations decreased from 61 to 74% of the initial values during the test. Therefore, the evaluation of the effect concentrations was based on geometric mean measured concentrations, corresponding to 0.0461, 0.0831, 0.163, 0.371 and 0.715 mg test item/L.

Effects on algal growth could be observed only at the highest test concentration where growth rate was significantly reduced by 15.2% compared to controls. Therefore, based on geometric mean measured concentrations, the 72h-growth rate ErC50 is above 0.715 mg test item/L and the 72h-growth rate NOEC is equal to 0.371 mg test item/L. However, the preferred long-term endpoint value is the 72h-growth rate EC10, determined at 0.596 mg test item/L.

Description of key information

OECD Guideline 201, GLP, key study, validity 1:

72h-ErC50 > the highest attainable test substance concentration in the test conditions (geometric mean measured concentration: 0.715 mg/L)

72h-ErC10 = 0.596 mg/L (geometric mean measured concentration)

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
0.596 mg/L

Additional information

One key study is available to assess the toxicity of the registered substance on the growth of the green alga Pseudokirchneriella subcapitata.

This study (Fraunhofer, 2009) was performed on the registered substance, according to the OECD guideline 201 with GLP certificate.

Since the test item is a liquid of poor water solubility, the highest test solution was prepared with growth medium to obtain the maximum dissolved concentration of the test item. Nominal loadings were: 6.25, 12.5, 25, 50 and 100 mg test item/L. The test item concentrations were chemically analysed at start and end of the test. At the start of the test, the measured concentrations were 0.0563, 0.102, 0.210, 0.433 and 0.840 mg test item/L. At end of the test, the measured concentrations were 0.0379, 0.0677, 0.127, 0.318 and 0.609 mg test item/L. Based on this, the test substance concentrations decreased from 61 to 74% of the initial values during the test. Therefore, the evaluation of the effect concentrations was based on geometric mean measured concentrations, corresponding to 0.0461, 0.0831, 0.163, 0.371 and 0.715 mg test item/L.

Effects on algal growth could be observed only at the highest test concentration where growth rate was significantly reduced by 15.2% compared to controls. Therefore, based on geometric mean measured concentrations, the 72h-growth rate ErC50 is above 0.715 mg test item/L and the 72h-growth rate NOEC is equal to 0.371 mg test item/L. However, the preferred long-term endpoint value is the 72h-growth rate EC10, determined at 0.596 mg test item/L.