Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Apr 2007 - 06 Sept 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP, test substance could not be maintained in solution, all reasonable steps taken to maintain test substance concentration

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MTDID 7145
- Substance type: Multiconstituent substance
- Physical state: Clear, colourless liquid
- Analytical purity: 94.5%
- Lot/batch no.: 142072:43
- Expiration date of the lot/batch: 30 Dec 2007
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: WAFs prepared at loading rates of 1.0, 10 and 100 mg/L for the rangefinding test, and 100 mg/L only for the limit test.

- Sampling method: Aliquots (10 mL) were taken from test bottles at times 0, 24 hours and 48 hours. Parallel samples were taken of two uninoculated test bottles having growth medium at 100 mg/L loading rate. At the end of the test, replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were analyzed day of sampling

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accomodated fraction. A loading rate of 100 mg/L was stirred for approximately 1 hour in air-tight vessels. After a stabilisation period of 5-20 minutes, the Water Accommodated Fraction (WAF) was collected by siphoning and used for the test. The final test solutions were all clear and colourless.
- Controls: Blank
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

Test organisms

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): In-house laboratory culture
- Age of inoculum (at test initiation): 4 days prior to test initiation, cells from algal stock culture were inoculated in culture medium at a cell density of 10,000 cells/mL. Cells counts were obtained using a microscope and counting chamber.
- Method of cultivation: In M1 growth medium, continuously aerated and exposed to 60- 120 uE/m2/s in the wavelength range of 400 to 700 nm at 21 - 24 deg. C.

ACCLIMATION
- Acclimation period: 4 days in M2 pre-culture medium
- Culturing media and conditions (same as test or not): Same during acclimation period
- Any deformed or abnormal cells observed: None noted

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
24 mg CaCO3 / L
Test temperature:
21.7 °C - 22.7 °C
pH:
7.1 - 9.3
Dissolved oxygen:
NA
Salinity:
NA
Nominal and measured concentrations:
WAF based on 100 mg/L loading rate
Mean measured concentration, 0.15 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100-mL septum-sealed all-glass bottles. Fill volume was 75 mL.
- Aeration: Continuous shaking to keep cells in suspension.
- No. of organisms per vessel: initial cell density was 1E+04 cells/mL (fresh dilution of 4 day old culture).
- No. of vessels per concentration (replicates): six, with two additional uninoculated bottles
- No. of vessels per control (replicates): six
- Extra replicates for sampling: yes, inoculated bottles of each test group for sampling at 24 hours.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water used to make growth medium was purified by reverse osmosis.
- Culture medium different from test medium: yes, propagation in M1, test in adjusted M2 (NaHCO3 increased to 100 mg/L to compensate for sealed bottle test).

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: continuous illumination using 30 Watt 'Cool-white' TLD-Iamps. Light intensity within the range of 108 to 112 µE/(m²∙s)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Cell counts at 24 and 48 hours, determined by spectrophotometric measurement at 720 nm using a Varian Cary 50 single beam spectrophotometer with cuvettes. Aliquots were taken by syringe through septum (test bottles left closed). Microscopic observation of cells at end of test only.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: One concentration tested
- Justification for using less concentrations than requested by guideline: Limit test
- Range finding study: Yes
- Test concentrations: WAFs based on 1.0 mg/L, 10 mg/L and 100 mg/L loading rates
- Results used to determine the conditions for the definitive study: Yes. No significant growth inhibition at any concentration tested.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate, reference test done 12 weeks prior to FC-770 study

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: Corresponds to an initial loading rate 100 mg/L
Details on results:
Based on results of range-finding tests, the final test was conducted as a limit test in sealed bottles with a 48-hour duration.

Measured concentrations in the limit test are shown in Table 1. FC-770 concentration passed below the Limit of Quantitation by 24 hours. The average concentration during the test was calculated using:

Concentration = ½*sqrt(C0 x C48)

where C0 and C48 are the concentrations at 0 and 48 hours, and the concentration was set to ½*LOQ if FC-770 was below the Limit of Quantitation.

In the limit test, the control group had >16-fold growth by 48 hours. The mean CV for section-by-section specific growth rate was 20% over this period, with variation in average specific growth rates of 1% (Tables 2, 3).

The test group showed 29% inhibition in growth from 0-24 hours, with recovery by 48 hours (-37% inhibition at 24-48 hrs, 0.2% inhibition overall, Tables 2,3). Recovery of growth from 24 to 48 hours, while not unexpected, is coincident with FC-770 concentrations which were
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No. Test solutions were clear and free of surface film.

- Effect concentrations exceeding solubility of substance in test medium: Yes
Results with reference substance (positive control):
In a test of potassium dichromate conducted 12 weeks prior to the FC-770 test, a 48-h EC50 of 1.9 mg/L was observed. This EC50 is within the range of values for reference tests of P. subcapitata performed by the testing lab, but the reference did not follow the sealed bottle method.

Any other information on results incl. tables

Table 1, Concentrations of FC-770 in the test medium

Time of sampling (hour)

Concentration

Loading rate (mg/L)

Analysed(mg/L)

0

0

<LOQ²

100

0.479

100¹

0.217

 

24

0

<LOQ

100

<LOQ

100¹

<LOQ

 

48

0

<LOQ

100

<LOQ

100¹

<LOQ

1, Test bottle prepared without algae

2, < LOQ, less than Limit of Quantitation (0.1 mg/L)

 

Table 2, Individual cell densities in test bottles during the algae tests

Loading

rate

Vessel

number

Cell density (x 1E+04) at exposure time:

0 hours

24 hours

48 hours

Control

1

1.00

8.71

39.42

 

2

1.00

8.39

33.98

 

3

1.00

7.76

36.78

 

4

1.00

7.36

37.61

 

5

1.00

7.62

38.93

 

6

1.00

7.29

37.28

 

 

100 mg/L

1

1.00

4.36

38.16

 

2

1.00

3.93

36.81

 

3

1.00

3.94

36.52

 

4

1.00

5.06

38.34

 

5

1.00

4.64

39.17

 

6

1.00

4.22

33.13

Table 3, Specific growth rates and Percent growth inhibition during the algae tests

Loading rate

Vessel number

Growth rate (/hour) during section:

Percent growth inhibition, section:

0-24 h

24-48 h

0-48 h

0-24 h

24-48 h

0-48 h

Control

1

0.09019

0.06291

0.07655

 

2

0.08863

0.05828

0.07345

 

3

0.08537

0.06483

0.07510

 

4

0.08317

0.06797

0.07557

 

5

0.08462

0.06796

0.07629

 

6

0.08277

0.06800

0.07538

 

mean

0.08579

0.06499

0.07539

 

CV

3%

6%

1%

Mean CV for section-by-section specific growth rate: 20%

100 mg/L

1

0.06135

0.09039

0.07587

28

-39

-1

 

2

0.05703

0.09321

0.07512

34

-43

0

 

3

0.05713

0.09278

0.07496

33

-43

1

 

4

0.06756

0.08438

0.07597

21

-30

-1

 

5

0.06395

0.08888

0.07641

25

-37

-1

 

6

0.05999

0.08586

0.07293

30

-32

3

 

mean

0.06117

0.08925

0.07521

28.7

-37.3

0.2

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
16-fold growth in controls, <1% Coefficient of Variation (COV) for average specific growth rate, 20% COV for section specific growth rates
Conclusions:
The 48-hour EC50 for FC-770 to the green algae Pseudokirchneriella subcapitata is >0.15 mg/L
Executive summary:

The toxicity of FC-770 to the green algae Pseudokirchneriella subcapitata was assessed in a 48-hour limit test conducted according to OECD TG201. The test was performed as a limit test after a rangefinding test indicated no toxicity in water accomodated fractions (WAFs) made using loading rates of 1.0, 10, and 100 mg/L. Other rangefinding tests indicated that the final test would necessarily be conducted in sealed bottles with a 48-hour test duration, due to volatility of test substance and limitation on growth in sealed test vessels. The limit test used a WAF made with a loading rate of 100 mg/L. FC-770 concentrations after 24 hours were below the limit of quantitation. Growth during the 0-24 hour section was 29% inhibited v. controls, but recovered by 48 hours. The 48-hour EC50 of FC-770 to Pseudokirchneriella subcapitata magna was >0.15 mg/L based on measured concentration, which corresponds to an initial loading rate >100 mg/L. Because recovery of growth coincided with concentrations of FC-770 below LOQ in the 24-48 hour section, a no-effect concentration cannot be determined.

The study was conducted under accepted guidelines and according to GLP criteria. The concentration of FC-770 could not be maintained above the limit of quantitation. However, the testing lab took all reasonable steps to assure that FC-770 remained in solution. Therefore, the study is classified as reliable with restrictions.

Results Synopsis

Test Type: Static

EC50:  >0.15 mg/L                        95% C.I.:  NA

NOEC: NA                  Probit Slope:  NA