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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:
March 31, 2006 to July 03, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: OECD Guideline for Testing of Chemicals, No. 201: Alga, Growth Inhibition Test, 1984. EU Commission Directive 92/69/EEC, C.3: Algal Inhibition Test, 1992.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl 2,3-O-(1-methylethylidene)-5-O-[(4-methylphenyl)sulfonyl]-ß-D-ribofuranoside
EC Number:
609-920-2
Cas Number:
4137-56-8
Molecular formula:
C16H22O7S
IUPAC Name:
Methyl 2,3-O-(1-methylethylidene)-5-O-[(4-methylphenyl)sulfonyl]-ß-D-ribofuranoside
Details on test material:
Identity: Tosylfuranosid
Expiration date: May 08, 2006
Solubility in water: 25 mg/L
Aggregate state/physical form
at room temperature: Solid
Color: White
Storage conditions: Refrigerator (approx. 4 °C)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
COUNTING AND EXAMINATION OF THE ALGAL CELLS
Small volumes of the test media and the control (1.0 mL) were taken out of all test flasks after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter®, Model ZM), with at least two measurements per sample.
In addition, after 72 hours exposure, a sample was taken from the control and from a test concentration with reduced algal growth (undiluted filtrate). The shape of the algal cells was examined microscopically in these samples.

ANALYSIS OF THE TEST CONCENTRATIONS
For the analysis of the actual test item concentrations, the following samples were taken:
Just before the start of the test: - duplicate samples from each test medium (without algae)
- duplicate samples from the control (without algae)
After 72 hours: - duplicate samples from each test medium (without algae)
(stability samples) - duplicate samples from the control (without algae)
For the 72-hour stability samples, additional flasks with adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test but without algae.
All samples were deep-frozen (at about -20 °C) immediately after sampling. The test item is sufficiently stable in the test water under these storage conditions.
The concentrations of the test item Tosylfuranosid were analyzed in the test medium samples of the dilution 1:3.2 and the undiluted filtrate from both sampling times (0 and 72 hours). The samples from the lowest test concentrations (dilutions 1:10, 1:32 and 1:10) were not analyzed, since these concentrations were below the 72-hour NOEC determined in this test. From the control samples only one of the duplicate samples was analyzed from each of the sampling times.

Test solutions

Vehicle:
yes
Details on test solutions:
Dosage and Concentrations
The test item was not soluble at a concentration of 100 mg/L in test water and no homogeneous dispersion could be prepared. Therefore, a filtrate of a supersaturated dispersion of the test item and dilutions of the filtrate were prepared. The undiluted filtrate and the dilutions 1:3.2, 1:10, 1:32 and 1:100 were tested. A control was tested in parallel, as well (test water without test item).
A supersaturated dispersion with a loading rate of nominal 100 mg/L was prepared by dispersing 101.76 mg of the test item into 1000 mL of test water using ultrasonic treatment for 15 minutes. No auxiliary solvent or emulsifier was used. This supersaturated stock dispersion was stirred by a magnetic stirrer at room temperature in the dark over 24 hours to dissolve or disperse a maximum amount of the test item. The duration of the stirring period was chosen based on the results of the daphnia study with the test item. The solution equilibrium was reached after this time.
The supersaturated stock dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm) after the 24 hours stirring period just before the preparation of the test media.
The undiluted filtrate of the supersaturated stock dispersion with the maximum concentration of dissolved test item was used as the highest concentrated test medium. In addition, adequate volumes of the filtrate were diluted with test water for the preparation of test media with lower test item concentrations. The test media were prepared just before the start of the test (= addition of algae).
The actual concentrations of the test item in the test media were analytically determined.
The test concentrations were based on the results of a range-finding test. However, concentrations beyond the water solubility limit of the test item were not tested in compliance with EU Commission Directive 92/69/EEC.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
The test organism used for the study was Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, D-37073 Göttingen, Germany). The algae are cultivated in RCC’s laboratories under standardized conditions according to the test guidelines.
Due to the division of the genus Scenedesmus into the genera Scenedesmus and Desmodesmus, Scenedesmus subspicatus was transferred to the new genus Desmodesmus. Thus Scenedesmus subspicatus was renamed as Desmodesmus subspicatus. However, the former name Scenedesmus subspicatus as specified in the guidelines is used in this report.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
0.24 mmol/L (= 24 mg/L as CaCO3).
Test temperature:
22–23 °C
pH:
At the start of the test, the pH values in the test media and the control ranged from 8.1 to 8.3.
At the end of the test, pH values of 7.9 to 8.0 were measured.
Dissolved oxygen:
not applicable
Salinity:
not applicable
Nominal and measured concentrations:
At the start of the test, the analytically determined test item concentrations in the analyzed test media samples (dilution 1:3.2 and the undiluted filtrate) amounted to 8.08 and 25.5 mg/L, respectively.
The values found at the end of the test were 7.98 and 25.4 mg/L.
Details on test conditions:
EXPERIMENTAL CONDITIONS
The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations:
Macro-nutrients:
NaHCO3 50.0 mg/L CaCl2 × 2 H2O 18.0 mg/L NH4Cl 15.0 mg/L MgSO4 × 7 H2O 15.0 mg/L MgCl2 × 6 H2O 12.0 mg/L KH2PO4 1.6 mg/L
Trace elements:
Na2EDTA × 2 H2O 100.0 µg/L FeCl3 × 6 H2O 80.0 µg/L MnCl2 × 4 H2O 415.0 µg/L H3BO3 185.0 µg/L Na2MoO4 × 2 H2O 7.0 µg/L ZnCl2 3.0 µg/L CoCl2 × 6 H2O 1.5 µg/L CuCl2 × 2 H2O 0.01 µg/L
Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L as CaCO3).
The test was started (0 hours) by inoculation of 10,000 algal cells per mL of test medium. These cells were taken from an exponentially growing pre-culture, which was set up four days prior to the test under the same conditions as in the test. One day before the start of the test, the pre-culture was diluted threefold to keep the algae in exponential growth.
The test design included three replicates per test concentration and six replicates of the control. Volumes of 15 mL algal suspension for each replicate were continuously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks.
The flasks were covered with glass dishes. They were incubated in a temperature controlled water bath at a temperature of 22–23 °C, and continuously irradiated at a measured light intensity of about 7890 Lux (mean value), range: 7120 to 8380 Lux (minimum and maximum value of measurements at nine places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed above the test flasks. The test vessels were labeled with the RCC study number and all necessary additional information to ensure unmistakable identification.
The test duration was 72 hours.
Reference substance (positive control):
yes
Remarks:
For evaluation of the algal quality and the experimental conditions, potassium dichromate is tested as a positive control at least once a year to demonstrate satisfactory conditions of the test. The latest result of the positive control test in 2005 (72-h

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
12.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
18.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
8 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
8 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
25.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
25.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
At the start of the test, the analytically determined test item concentrations in the analyzed test media samples (dilution 1:3.2 and the undiluted filtrate) amounted to 8.08 and 25.5 mg/L, respectively. The values found at the end of the test were 7.98 and 25.4, respectively. The lower test concentrations (dilutions 1:10, 1:32 and 1:100) were not analyzed since they were below the 72-hour NOEC. Under the conditions of the test the test item was stable during the test period of 72 hours. The reported biological results are based on the mean measured test item concentrations calculated as an arithmetic mean of all measurements per test concentration. The tabulated values represent rounded results obtained by calculation using the exact raw data.

Dilution Mean measured test item concentration (mg/L)
1:100 not analyzed, since these concentrations were below the 72-hour NOEC determined in this test.
1:32 not analyzed, since these concentrations were below the 72-hour NOEC determined in this test.
1:10 not analyzed, since these concentrations were below the 72-hour NOEC determined in this test.
1:3.2 8.0
undiluted filtrate 25.5

The influence of the test item Tosylfuranosid on the growth of Scenedesmus subspicatus was evaluated. The test item had a statistically significant inhibitory effect on the growth (biomass and growth rate) of Scenedesmus subspicatus after the test period of 72 hours at the highest concentration of 25.5 mg/L corresponding to a loading rate of 100 mg/L (results of Dunnett-tests, one-sided, a = 0.05). Thus, this test concentration was determined as the 72-hour LOEC (lowest concentration tested with toxic effects).
In the undiluted filtrate at a concentration of 25.5 mg/L the percentage inhibition of AUC was 38% and the percentage inhibition of r was 15%. This was the highest test item concentration which could be dissolved in the test water at a loading rate of 100 mg/L. Thus, the EC50 is clearly higher than the loading rate of 100 mg/L and higher than the water solubility limit under test conditions (25.5 mg/L).
The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was 8.0 mg/L, since up to and including this test concentration both the mean biomass and the mean growth rate of the algae were not statistically significantly lower than in the control.

The ECX values as well as the NOEC and LOEC were calculated for both parameters, the algal biomass (b) and the growth rate (r), after 72 hours test duration:

Parameter Biomass b Growth rate r
(0-72 h) (mg/L) (mg/L)
EC50 > 25.5 > 25.5
EC10 12.9 18.2
NOEC 8.0 8.0
LOEC 25.5 25.5

The microscopic examination of the algal cells after 72 hours test period showed no difference between the algae growing in the undiluted filtrate and the algal cells in the control. The shape and size of the algal cells growing in test media containing the test item at up to this test concentration were not affected.
In the control the cell density has increased from nominal N = 1 × 104 cells/mL at the start of the test (0 hours) to N = 59 × 104 cells/mL (mean value) after 72 hours. Thus, the algal growth in the control was sufficiently high under the conditions of the test. The validity criterion of increase of cell density by at least a factor of 16 over 72 hours was fulfilled.
No remarkable observations were made concerning the appearance of test media. The test media of all test concentrations remained clear throughout the entire test period.
At the start of the test, the pH values in the test media and the control ranged from 8.1 to 8.3. At the end of the test, pH values of 7.9 to 8.0 were measured. The water temperature was 22–23 °C.
Results with reference substance (positive control):
For evaluation of the algal quality and the experimental conditions, potassium dichromate is tested as a positive control at least once a year to demonstrate satisfactory conditions of the test. The latest result of the positive control test in 2005 (72-h EC50 for the growth rate: 0.78 mg/L, RCC Study No. 858774) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 1996 to 2005: 72-h EC50: 0.44–1.16 mg/L).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Executive summary:

The influence of the test item Tosylfuranosid on the growth of the green algal species Scenedesmus subspicatus was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, C.3, 1992, and the OECD Guideline No. 201, 1984. Due to the low water solubility of the test item, a supersaturated stock dispersion of the test item with a loading rate of 100 mg/L was stirred continuously at room temperature in the dark over 24 hours. Then, the stock dispersion was filtered. The undiluted filtrate of the stock dispersion and the dilutions 1:3.2, 1:10, 1:32 and 1:100 were used as test media. A control was tested in parallel. The test item was stable over the test period of 72 hours. At the start of the test, the analytically determined test item concentrations in the analyzed test media samples (dilution 1:3.2 and the undiluted filtrate) amounted to 8.08 and 25.5 mg/L, respectively. The values found at the end of the test were 7.98 and 25.4 mg/L. The lower test concentrations (dilutions 1:10, 1:32 and 1:100) were not analyzed since they were below the 72-hour NOEC. Under the conditions of the test the test item was stable during the test period of 72 hours. All biological results are based on the mean measured test item concentrations of 8.0 mg/L (dilution 1:3.2) and 25.5 mg/L (undiluted filtrate), calculated as the average over all measurements per test concentration during the test period. The test item had a statistically significant inhibitory effect on the growth (biomass and growth rate) of Scenedesmus subspicatus after the test period of 72 hours at the highest concentration of 25.5 mg/L corresponding to a loading rate of 100 mg/L (= 72-hour LOEC: lowest concentration tested with toxic effects after a test period of 72 hours). In the undiluted filtrate at a concentration of 25.5 mg/L the percentage inhibition of AUC was 38% and the percentage inhibition of r was 15%. This was the highest test item concentration which could be dissolved in the test water at a loading rate of 100 Thus, the EC50 is clearly higher than the loading rate of 100 mg/L and higher than the water solubility limit under test conditions (25.5 mg/L). mg/L. The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was 8.0 mg/L, since up to and including this test concentration both the mean biomass and the mean growth rate of the algae were not statistically significantly lower than in the control. The ECX values as well as the NOEC and LOEC were calculated for both parameters, the algal biomass (b) and the growth rate (r), after 72 hours test duration:

Parameter Biomass b Growth rate r

(0-72 h) (mg/L) (mg/L)

EC50 > 25.5 > 25.5

EC10 12.9 18.2

NOEC 8.0 8.0

LOEC 25.5 25.5