Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1999-07-27 to 1999-11-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline compliant GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
as at 1984
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
as at 1992
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Version / remarks:
as at 15 November 1989
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): dl-Lactone
- Physical state: white crystalline mass
- Analytical purity: 100.0 %
- Lot/batch No.: 805046
- Expiration date of the lot/batch: 01 May 2002
- Stability under test conditions: Stable under storage conditions
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L
- Sampling method: 10 mL samples taken at t=0 h, t=24 h and t=72 h
- check for compliance with the Quality criteria regarding maintenance of actual concentrations: running a test vessel at the highest substance
concentration but without algae + sampling at the start and the end of the test period.
- taking reserve samples of 10 mL from all test solutions (stored in a deepfreeze until possible analysis for a maximum of three months or until
delivery of the final report)
- Sample storage conditions before analysis: all samples analysed on sampling day (except for the reserves, see above)

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: preparation of a stock solution of 100 mg/L in the ISO medium
- Controls: medium control (ISO medium)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
- Medium:
M2-medium according to the ISO-Standard "Algal growth inhibition test" Nov. 1989; formulated using Milli-Q water preventing precipitation and with the following composition:
NH4CI 15 mg/L
MgC12 .6H2O 12 mg/L
CaC12.2H2O 18 mg/L
MgSO4 .7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeC13.6H2O 80 µg/L
Na2 EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnC12.4H2O 415 µg/L
ZnC12 3 µg/L
CoC12 .6H2O 1.5 µg/L
CuC12.2H2O 0.01 µg/L
Na2Mo04.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L(24 mg CaCO3/L )
prepared with Milli-Q water: Tap water purified by reverse osmosis and then passed over activated carbon and ion-exchange cartridges (Millipore Corp., Bedford, Mass., USA).

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: Selenastrum capricornutum, strain: CCAP 278/4.
- Age of inoculum (at test initiation): 4 days before the start of the test
- Method of cultivation: continuously aerated and exposed to light (4000-9000 lux) in a climate room at a temperature of 23 ± 2°C, starting from a
pure culture on agar

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): same

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
no

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
21.5 - 22.5 °C
pH:
7.9 - 8.7 (see table 5)
Nominal and measured concentrations:
nominal: 100 mg/L
measured: 78 mg/L
see table 1
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all-glass
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 100 mL, headspace not described, 50 mL
- Aeration: passive via shacking
- Renewal rate of test solution (frequency/flow rate): not applicable, static cultures
- Initial cells density: 10^4 cells/mL
- Control end cells density: 107.6 * 10^4 cell/mL
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates):
- range finding test: triplicate
- main test: 6 replicates of the test concentration
- No. of vessels per control (replicates):
- range finding test: triplicate
- main test:
6 replicates of the blank-control
2 replicate of the test concentration without algae
1 extra replicate of both test concentrations necessary for sampling
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes, M2-medium according to the ISO-Standard "Algal growth inhibition test" Nov. 1989 (see above under test solutions)

TEST MEDIUM / WATER PARAMETERS
- Medium parameters: see above under test solutions
- Culture medium different from test medium: no
- Intervals of water quality measurement:
- pH: At the beginning and at the end of the test, pH of the solutions should preferably not deviate by more than 1.5 units during the test
- temperature of medium: every day in a temperature-control vessel

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continous
- Light intensity and quality: TLD-lamps of the type `GroLux' of 30 Watt (Sylvania), with a light intensity within the range of 60 to 120 .t.E.m .2 .s-1, yielding 3500 — 4000 lux.
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer, based on a calibration curve
- Chlorophyll measurement: no


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- pre-test: factor = 10
- main test: limit test at 100 mg/L only
- Justification for using less concentrations than requested by guideline: limit test
- Range finding study
- Test concentrations:
- pre-test: 0.1, 1.0, 10, 100 mg/L
- main test: limit test at 100 mg/L only
- Results used to determine the conditions for the definitive study: 100 mg/L only (from the main test)
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.:
> 78 mg/L
Nominal / measured:
meas. (arithm. mean)
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:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
78 mg/L
Nominal / measured:
meas. (arithm. 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): no
- Any stimulation of growth found in any treatment: slight stimulation in the main test (limit test at 100 mg/L)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.75 mg/L
- see table 6 for details
Reported statistics and error estimates:
not necessary as the main test was a limit test

Any other information on results incl. tables

- Table 1: Concentrations of DL-LACTONE in test medium (final test).

Time of
sampling
[hours]

Date of
sampling
[dd-mm-yy]

Date of
analysis
[dd-mm-yy]

Concentration

Nominal
[mg/L]

Analysed (1)
[mg/L]

Relative to
nominal [%]

0

24-08-99

24-08-99

0

n.d.

n.a.

0

 

 

100

106

106

0

 

 

100 (2)

105

105

24

25-08-99

25-08-99

0

n.d.

n.a.

 

 

 

100

98.6

99

 

 

 

100 (2)

96.3

96

72

27-08-99

27-08-99

0

n.d.

n.a.

 

 

 

100

44.9

45

 

 

 

100 (2)

75.7

76

(1) Mean of duplicate analysis. The maximum deviation between the responses was calculated for each sample and was < 10%.

(2) Without algea.

n.d.: not detected.

n.a.: not applicable.

In the chromatograms recorded for the nominal 100 mg/L samples taken at t=24 hours and t=72 hours, an extra peak was observed at a retention time smaller than the retention time of RS-Pantolactone. The extra peak increased in time. The presence of an extra peak in the chromatograms indicated that the decrease of the test item was probably related to degradation. Based on the measured concentrations the algal suspensions had been exposed to an average measured concentration of 78 mg/L.

- Table 2: Extinction values measured during the range-finding test:

Concentration
DL-LACTONE
(mg/L)

Hours of exposure

24       48       72

Control

40

239

1005

 

36

243

1007

 

39

285

1058

0.1

40

246

1000

 

39

259

999

 

39

298

1055

1.0

37

219

947

 

39

275

1055

 

37

246

1056

10

40

240

1062

 

37

245

1037

 

41

284

1001

100

39

251

1007

 

36

275

957

 

34

247

984

Based on these results the EC50 was expected to be above 100 mg/I and the project was continued with a final limit study.

- Table 3: Mean cell densities during the limit test

Concentration
(mg/L)
DL-LACTONE
Nominal / Actual *

Exposure time (hours)

0

24

48

72

Blank-control

1.0

1.0

26.8

107.6

100 / 78

1.0

1.0

30.5

111.1

* Average measured concentration.

- Table 4: Percentage inhibition of cell growth and reduction of growth rate during the limit test.

Concentration (mg/L)
DL-LACTONE
Nominal / Actual *

Cell growth (0-72 hrs)

Mean growth rate p: (cells/mL/h)

Mean area (A)

Inhibition (%)

µ (0-72 hrs)

Reduction (%)

Blank-control

1898.09

 

0.06491

 

100 / 78

2029.91

-6.9

0.06534

-0.7

* Average measured concentration.

- Table 5: pH levels recorded during the limit study.

Concentration (mg/L)

DL-LACTONE

Nominal / Actual *

Exposure time (hours)

 

0

72

Blank-control

8.1

8.7

100 / 78

7.9

8.5

* Average measured concentration. - Table 6: Calculation of % inhibition of growth and % reduction in growth rate in the reference test:

Concentration
K2Cr2O7
(mg/L)

Total cell growth:

Growth rate:

Mean Area for 0-72h

Inhibition in %

Interval 0-72h

Reduction in %

0.00

2519.09

 

0.06691

 

0.18

2637.32

-4.7

0.06744

-0.8

0.32

2618.97

-4.0

0.06793

-1.5

0.56

1677.65

33.4

0.06132

8.4

1.00

503.84

80.0

0.03914

41.5

1.80

224.26

91.1

0.02416

63.9

3.20

145.45

94.2

0.01806

73.0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Selenastrum capricomutum, RS-Pantolactone induced no effect on algal growth at a concentration of 100 mg/L during 72 hours of incubation.
The exposure included degradation of RS-Pantolactone, which was at least partly related with algal growth.
The EC50 for both algal growth and growth rate exceeded a concentration of 100 mg/L (NOEC), corresponding with an average measured concentration of 78 mg/L.
Executive summary:

RS-Pantolactone (named DL-LACTONE in the report) was tested for it's toxicity to aquatic algae and cyanobacteria with Selenastrum capricormutum in the Fresh Water Algal Growth Inhibition Test. The study procedures described in this report were based on the EEC Directive 92/69, Publication No. L383 Part C-3 adopted December, 1992; OECD guideline No. 201, Adopted June 7, 1984; and ISO Standard 8692, First edition, 15 November 1989.

The test item was completely soluble at the concentrations tested. After a range-finding test, a limit test was performed exposing exponentially growing algal cultures to a RS-Pantolactone concentration of 100 mg/L and to a blank-control. The initial cell density was 10^4 cells/mL. The total test period was 72 hours. Samples for analysis of actual exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test period. Sampling included a test solution prepared at 100 mg/L incubated without algal cells present.

Analysis of the samples showed that the concentrations measured at the start of the test were in agreement with nominal. Further, the measured concentration remained above 80 % relative to the initial concentration during the first 24-hour exposure period. At the end of the 72-hour exposure period, the concentration measured in the sample taken from the solution with algae had decreased to 45 mg/L, whereas the concentration measured in the solution without algae remained at 76 mg/L. The presence of an extra peak in the chromatograms indicated that this decrease was probably related to degradation.

RS-Pantolactone induced no effect on algal growth at a concentration of 100 mg/L during 72 hours of incubation. The exposure included degradation of the test item, which was at least partly related with algal growth.

The EC50 for both algal growth and growth rate exceeded a concentration of 100 mg/L (NOEC), corresponding with an average measured concentration of 78 mg/L.

By expert judgement it is concluded that L-Pantolactone will get the same results.

L-Pantolactone: Algea, 72h EC50: 100 mg/L