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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-04-23 - 2002-05-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well reported guideline study with plausible results and GLP compliance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
equivalent or similar to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes
Remarks:
OECD-Principles of Good Laboratory Practice

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
Analytical monitoring had not been feasible, as substance solubility (below 10 µg/l) was beyond analytical sensitivity.

Test solutions

Vehicle:
no
Details on test solutions:
The batch tested was a yellow powder with a purity of 94.5%. The test substance was insoluble in water based on information supplied by the sponsor. Analytical confirmation of the actually dissolved concentration in test medium was not possible as no analytical method was available or could be developed with the necessary sensitivity to measure the soluble fraction of the test item.
This was based on previous work on a similar compound, which showed that the water solubility was less than 10 µg/I (NOTOX Project 289979). The soluble fraction present in saturated solutions prepared during that project were not distinct from the background responses. All possible efforts were taken to improve the detection limit of the analytical method. However, these were not successful.
Preparation of a test solution at maximum saturation of the test item in test medium started with a nominal concentration of 100 mg/I. This solution was treated with ultrasonic waves and subsequently magnetically stirred for 4 days. The resulting dispersion was then pre-filtered through a paper filter (Schleicher and Schuell 604) to remove the larger undissolved test substance particles (ca. > 5µm), followed by filtration through a 0.45 µm membrane filter. The final test solution was a clear and colourless solution. The blank-control received similar treatment to correct for possible treatment related effects on algal growth.
Note that the use of centrifugation to obtain the water-soluble fraction proved not to be successful as a consequence of the behaviour of the test substance in water, i.e. large amounts remained floating an the water surface. After preparation, volumes of 50 ml were added to each replicate of the respective test concentration.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Test organism: Selenastrum capricornutum, strain: NIVA CHL 1
- Stock culture: 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 (4000-9000 lux) in a climate room at a temperature of 23 ± 2°C.
- Preculture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2x10^4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
22.5°C at the start of the test. Temperature was maintained during the test between 21.5 nd 23°C.
pH:
The pH was determined at the beginning and the end of the incubation period:
Control: 8.4 and 8.2
Test sample: 8.3 and 8.4
Dissolved oxygen:
not applicable
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentration (limit test): 100 mg/L;
Due to the poor water solubility (less than 10 µg/L), no analytical determination of the concentration was possible as analytical sensitivity had been too low.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- 100 ml, all glass
- 50 ml
- The algal cells were kept in suspension by continous shaking
- Initial cells density: 10E4 Cells/mL
- Control end cells density: 103.1x10E4 cells/mL
- No. of vessels per concentration (replicates): 6 replicates for the control and the limit test (100 mg/L nominal), each

GROWTH MEDIUM
- Standard medium used: yes, M2-medium according to ISO-Standard Standard "Algal growth inhibition test" Nov. 1989

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water - Tap water purified by reverse osmosis and then passed over activated carbon and ion-exchange cartridges (Millipore Corp., Bedford, Mass., USA).



OTHER TEST CONDITIONS

- Photoperiod: continuously
- Light intensity and quality: TLD-lamps of the type 'Cool-white' of 30 Watt, with a light intensity within the range of 60 to 120 µE/mE2/s, not varying by more than 20%.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted by microscope, using a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength =20 mm). Varian Nederland BV., Houten, The Netherlands. Algal medium was used as blank.

TEST CONCENTRATIONS
Limit test, nominal concentration of 100 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no

Exposure of the fresh water algal species Selenastrum capricornutum to the maximum soluble fraction of the test item in test medium resulted in 4% reduction in growth rate and 13% inhibition of total cell growth (biomass). Both, the inhibition of Algal cell growth and the reduction of growth rate were not statistically different from the control values.
Hence, the NOEC equalled the maximum solubility and the EC50 for both algal growth inhibition and growth rate reduction exceeded the maximum solubility of the test item in test medium at a 0.45 pm filtered test solution prepared at 100 mg/I, the regulatory limit concentration.

Results with reference substance (positive control):
Selenastrum capricornutum, fresh water algal growth inhibition test with potassium dichromate (NOTOX Project 352417).
The positive control experiment was performed analogous to the actual study.
Start of first exposure: May 21, 2002 Completion last exposure: May 24, 2002
The EC50 for cell growth inhibition (EBC50: 0-72h) was 0.74 mg/L with a 95 % confidence interval ranging from 0.46 to 1.2 mg/I. The historical ranges of the 72h EC50 for growth inhibition lie between 0.49 and 1.4 mg/L. Hence, the EBC50: 0-72h for the present batch corresponds with this range.
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.5 mg/L with a 95 % confidence interval ranging from 1.2 to 1.9 mg/L. The historical ranges for growth rate reduction are in the range of 0.82 and 2.3 mg/I. Hence, the ERC50: 0-72h for the present batch is within this range.

Any other information on results incl. tables

Table 1 shows mean cell densities measured at 24-hour intervals at the different concentrationsof the test item. The respective growth curves are shown in Figure 1 (see the Appendix I for the calibration curve and extinctions and cell densities per replicate).

Table 1: Mean cell densities (x 104cells/ml) during the limit test

Nominal conc.*
test item
(mg/1)

Exposure time (hours)

0

24

48

72

Blank-control

1.0

5.2

30.0

103.1

100

1.0

6.2

27.8

84.1

 * 0.45 µm filtrate

Table 2: Percentage inhibition of cell growth during the final test.

Nominal conc.*
test item
(mg/1)

Cell growth (0-72 hrs)

Mean area (A)

Inhibition(%)

Blank-control

2022.74

 

100

1765.66

12.7

* 0.45 µm filtrate

Table 3: Percentage reduction of growth rate at different time intervals during the final test.

Nominal conc.*
test item

(mg/L)

Mean growth rate

µ (0-24 hrs)

Reduction (%)

µ (0-48 hrs)

Reduction (%)

µ (0-72 hrs)

Reduction(%)

Blank-control

0.06869

 

0.07073

 

0.06426

 

100

0.07542

-9.8

0.06915

2.2

0.06141

4.4

*0.45 µm filtrate

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
1.      In the controls, cell density increased by an average factor of > 16 within three days. 2.      Further, all test conditions (pH and temperature) remained within the ranges prescribed by the protocol.
Conclusions:
Limit study with nominal test substance concentration of 100 mg/L. Because of the poor substance solubility in water (below 10 µg/L) analytical detection or monitoring was not feasible.
EC50: > 100 mg/L (nominal)
NOEC: = 100 mg/L (nominal)
Executive summary:

Exposure of the fresh water algal species Selenastrum capricornutum to the maximum soluble fraction of the test item in the test medium resulted in 4% reduction in growth rate and 13% inhibition of total cell growth (biomass) which were not statistically different from the control values.

The NOEC equalled the maximum solubility. The EC50for both algal growth inhibition and growth rate reduction exceeded the maximum solubility of the test item in test medium prepared with a 0.45 µm filtered test solution (nominal concentration 100 mg/L).