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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:
13 - 16 Feb 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
March 2006
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
July 2009
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Version / remarks:
October 2004
GLP compliance:
yes (incl. QA statement)
Remarks:
Food and Consumer Product Safety Authority (VWA), Utrecht, The Netherlands
Analytical monitoring:
no
Remarks:
No suitable analytical method could be established to quantify the test substance in test medium.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with individually prepared loading rates of 1.0, 10 and 100 mg/L applying a 1 h period of magnetic stirring. The obtained mixtures containing undissolved material were allowed to settle for a period of 1 h after which the Water Accommodated Fractions were siphoned off and used as test solutions. The lowest test concentration was prepared as a 10-fold dilution of the WAF prepared at 1.0 mg/L. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10E+04 cells/mL.
- Eluate: no
- Differential loading: yes, except for the lowest test concentration
- Controls: yes, test medium control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The final test solutions were all clear and colourless.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24 °C. The stock culture medium was M1 medium according to NPR 6505 formulated in Milli-RO water. 3 d before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use. The medium for the pre-culture was M2; according to the OECD 201 Guideline, formulated using Milli-Q water (tap water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
21.5 - 22.2 °C
pH:
8.0 - 8.2
Nominal and measured concentrations:
nominal: control, 0.1, 1.0, 10, 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: all-glass test vessels
- Material, size, headspace, fill volume: all-glass, 100 mL, headspace: 50 mL, fill volume: 50 mL
- Aeration: continuous shaken
- Initial cells density: 1.00E+04 cells/mL
- Control end cells density: 79.5 - 134E+04 cells/mL
- No. of vessels per concentration (replicates): 3 (0.1, 1.0 and 10 mg/L), (100 mg/L)
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-Q water (tap water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges preventing precipitation.
- Culture medium different from test medium: M1 medium was used for stock culture, M2 medium was used for pre-culture
- Intervals of water quality measurement: The pH was measured at the beginning and end of the test in the control and the highest test concentration. Temperature was continuously monitored in a temperature control vessel.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: TLD-lamps of the type ‘Cool-white’ of 30 W, with a light intensity within the range of 75 to 87 µE*m-2*s-1.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Algal density was determined by use of a microscope and a counting chamber throughout the test.
- Appearance: At the end of the test microscopic observations were performed on the limit concentration to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
- Any stimulation of growth found in any treatment: yes, stimulation of growth compared to the control was recorded in each tested loading rate.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: In the three highest test groups a floating layer was observed at 24 and 48 h. At the end of the test small particles of the test substance were observed in the highest concentration.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: ErC50 (72 h) 1.8 mg/L (95% CI: 1.4 - 2.4 mg/L), EyC50 (72 h) 0.69 mg/L (95% CI 0.50 - 0.95 mg/L)
- Other: The historical ranges for growth rate reduction lies between 0.82 and 2.3 mg/L. The historical ranges of the EC50 (72 h) for yield inhibition lie between 0.43 and 1.1 mg/L.
Reported statistics and error estimates:
Statistical analysis of the data was not needed as the effects recorded were not significant (<10%). Stimulation of growth was recorded.

Table 1: Growth rate and reduction of growth rate during exposure.

Loading rate [mg/L]

Test vessel

Growth rate [µ]

Growth rate reduction [%]

0-24 h

24-48 h

48-72 h

0-24 h

24-48 h

48-72 h

control

1

0.07103

0.06512

0.04617

 

 

 

2

0.08108

0.05701

0.05870

3

0.07956

0.04778

0.06060

4

0.07466

0.06636

0.06306

5

0.07636

0.04407

0.06541

6

0.08395

0.05099

0.05458

Mean

0.07777

0.05522

0.05809

CV

6%

17%

12%

10% of 1.0

1

0.06706

0.06622

0.06568

14

-20

-13

2

0.06909

0.06862

0.05479

11

-24

6

3

0.06029

0.07036

0.06558

22

-27

-13

1.0

1

0.05776

0.08108

0.06064

26

-47

-4

2

0.06029

0.07818

0.06136

22

-42

-6

3

0.07636

0.06395

0.05517

2

-16

5

10

1

0.07103

0.06927

0.05281

9

-25

9

2

0.07288

0.06596

0.06267

6

-19

-8

3

0.07103

0.06855

0.06059

9

-24

-4

100

1

0.06909

0.07049

0.06373

11

-28

-10

2

0.06267

0.07388

0.06594

19

-34

-14

3

0.06267

0.07103

0.06863

19

-29

-18

4

0.07288

0.06952

0.05725

6

-26

1

5

0.07103

0.06391

0.06383

9

-16

-10

6

0.07636

0.04949

0.07131

2

10

-23

Analytical measurements

The test substance is an organic compound with the element tin (Sn) in its molecular structure. An ICP-MS method was developed for the determination of the test substance based on the element Sn. Validation of the analytical assay was carried out, however, it was found that the accuracies on test samples prepared with the test substance were inadequate. The ICP-MS method developed was considered to be the best possible for the determination of the test substance:

- Oxygen and helium gas were used to remove the organic and tetrahydrofuran (THF) content of the test substance and accuracy samples.

- The analytical sequence had a normal number of samples and the instrument was cleaned prior to use.

- The substance must be dissolved in THF since it was not directly soluble in (aqueous) strong acids such as HNO3.

According to the results obtained it was concluded that it is not possible to use the ICP-MS method for quantification of test samples to support physico-chemical- and (eco)toxicologal studies. Other analytical techniques such as GC, HPLC and UV-Vis were not suitable for the determination of the test substance due to its hydrophobic molecular structure.

Validity criteria fulfilled:
yes

Description of key information

ErL50 (72 h) > 100 mg/L (nominal, OECD 201)

NOErLR (72 h) ≥ 100 mg/L (nominal, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

One experimental study is available investigating the effects of the substance to freshwater algae.The study was performed according to OECD 201 (GLP) with Pseudokirchneriella subcapitata under static conditions. Test solutions were prepared as loading rates of 0.1, 1.0, 10.0 and 100 mg/L by adding appropriate amounts of the substance to test medium followed by 1 h stirring 1 h settling and siphoning off the soluble fraction to be used for testing. Only the lowest loading rate of 0.1 mg/L was prepared by serial dilution (10x) of 1 mg/L. All test solutions were clear and colorless throughout the test. Analytical measurements were not possible to be conducted since no suitable analytical method could be established (see also IUCLID section 5.1.2). No inhibition of growth was recorded after 72 h resulting in an ErL50 (72 h) of > 100 mg/L and a NOErLR (72 h) ≥ 100 mg/L.