Propanoic acid, 2-[(ethoxythioxomethyl)thio]-, methyl ester

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 25 september 2000 to 19 july 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

see below / These deviations are assumed not to have affected the validity of this study.

Principles of method if other than guideline:

Deviations from the protocol:
The test substance was added using tert. butanol as a carrier, and not added directly in medium, as the test substance did not dissolve at the appropriate concentrations.
The algal density at the range-finding test was only measured after 51 hours, not 72 hours.
The Department of Environmental Toxicology, part of TNO Nutrition and Food Research, has decided to issue reports and protocols under the name of 'TNO Chemistry'.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the start of the growth inhibition test, single 30 ml samples were taken from the background series without algae containing nominal test substance concentrations of 0, 0.33, 1.0 and 10.3 mg/l
At the end of the test, samples were taken from the pooled (algae containing) media with the same test substance concentrations. The samples were placed in 40 ml glass vials with a teflon coated closure (EPA vials), and they were immediately transferred to the Residue Analysis Department at Zeist for analysis.

Vehicle:

yes

Remarks:

Tertiary Butyl Alcohol (TBA)

Details on test solutions:

For the range-finding test, an amount of 1002.1 mg, was dissolved in 10 ml of tertiar butanol (TBA). Dilutions were then prepared in TBA so as to yield a test substance concentration series of (0.32, 1.0, 3.2 and 10) x 10000 mg/l.
For the growth inhibition test, an amount of 1027.8 mg, was dissolved in 10 ml of TBA. Dilutions were then prepared in TBA so as to yield a test substance concentration series of (0.10, 0.33, 1.0, 3.3 and 10.3) x 10000 mg/l.

Test organisms (species):

Scenedesmus capricornutum

Details on test organisms:

TEST ORGANISM
- Common name: The fresh-water green alga Selenastrum capricornutum (CCAP 278/4), which belongs to the order of Chlorococcales (class Chlorophyceae), was used as the test organism.
- Strain: CCAP 278/4
- Source (laboratory, culture collection): The culture was supplied by the CCAP, The Freshwater Biological Association, the Ferry House, Far Sawrey, Ambleside, Cumbria LA22 OLP, England.
- Age of inoculum (at test initiation): no data
- Method of cultivation: A pre-culture of algae in the exponential growth phase was prepared as detailed in OECD Guideline no. 201.
A suspension of algae in the algal medium containing 10000 cells/ml was prepared by dilution of a pre-culture containing 4.0 x 1000000 cells/ml.


ACCLIMATION
- Acclimation period: no data
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no data

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24.2 mg equivalent CaCO3/L

Test temperature:

23 +/- 2°C

pH:

7.6 - 9.4

Dissolved oxygen:

No data

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

For the range-finding test
Nominal concentrations: 0, 0.32, 1.0, 3.2 and 10 mg/L.

For the definitive test
Nominal concentrations: 0, 0.10, 0.33, 1.0, 3.3 and 10.3 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical test flask
- Material, size, headspace, fill volume: 200 mL filled with 100 ml of this algal suspension and 10 μI of the appropriate solutions of the test substance
- Aeration: No
- Initial cells density: 1 x 10e4 cells/mL
- Control end cells density: Yes
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3 . The test was carried out in triplicate with three controls containing algae only and three controls containing algae and 10 μI TBA. Furthermore a single background
series containing test substance without algae was added.

GROWTH MEDIUM
- Standard medium used: yes
The medium was prepared from concentrated stock solutions in ultra pure water . It was sterilized by micropore filtration and contained 150 mg/l NaHCO3 (not 50 mg/l as specified in the OECD Guideline, this in order to improve the buffer capacity of the medium). Furthermore, the medium contained Fe-citrate, because the growth of the algae would be erratic in the absence of complexed iron.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ultra pure water
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: constant lighting
- Light intensity and quality: The light intensity radiated by the fluorescent lamps was within the standard range of 60-120 μ,mot.s· 1 .m-2 (measured with a Bottemanne Weather Instruments Photosynthetic Radiometer RA200 Q).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: After two days of incubation, algal densities (cells/ml) and algal biovolume (μm 3/ml) were determined after 0,23, 48.5 and 68.5 h with the electronic particle counter Coulter
Multisizer Ile. Measured values were corrected for the background values in the appropriate blanks. The mean values were used for further calculations, without the values of the control without carrier. There was a
small difference between the control with and without TBA addition.
The effect values were calculated using a statistic parametric model. The following parameters were calculated:
NOEC: Estimated no-observed-effect concentration
ErC-values: Effect concentration with regard to the growth rate


TEST CONCENTRATIONS
- Spacing factor for test concentrations: no data
- Justification for using less concentrations than requested by guideline: no data
- Range finding study: yes
- Test concentrations: 0.32, 1.0, 3.2 and 10 mg/L
- Results used to determine the conditions for the definitive study: The range-finding test revealed that inhibiting effects could be expected at nominal test substance concentrations > = 0.32 mg/l

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 and/or 3,5- dichlorophenol

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes
- Unusual cell shape: No
- Colour differences: No
- Flocculation:No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Other: Microscopic inspection of the morphology of algal cells in the pre-culture at the start of the test revealed normal cells. At the end of the test no abnormal cells were observed in the cultures containing different concentrations of Rhodixan A-1.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: yes, It is important to note that the test substance loss was highest for treatments with high algal biomass levels, and this loss can likely be attributed to biotransformation by algae and possibly by bacteria.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? yes
- The test system used is checked with reference substances (K2Cr2O7 and/or 3,5-dichlorophenol) on an approximately yearly basis. The results of these reference tests demonstrate a constant quality of the test
system during an extended period of at least 15 years. The resulting EC50 values are also comparable to the mean values found in international ring tests

Reported statistics and error estimates:

no data

Test substance concentrations appeared to be rather unstable during the test. The effect estimates will therefore also have to be calculated from measured concentrations.

It is important to note that the test substance loss was highest for treatments with high algal biomass levels, and this loss can likely be attributed to biotransformation by algae and possibly by bacteria. These high biomass levels are first reached during the last day of the test, suggesting that the highest loss took place at the end of the test. The average of the initial and the final test substance concentration then represents a relatively low estimate of the overall exposure concentration during the test, and this leads in turn to a conservative estimation of the test substance toxicity. Nominal concentrations in the range of 10 mg/l (i.e. at the level of the ErC 50-value) can be translated to measured concentrations by multiplication with the conversion factor of 0.496 ( = average of 74.3% and 25.0%). The translation of nominal concentrations to measured concentrations at the lower dosing levels is compromised by the substantial concentration decrease during the test. The algal cultures followed balanced growth, which can be seen by the Iinear

growth curves in the semi-logarithmic plot . The growth inhibition is then best described as reduction of the algal growth rate and therefore by the ErC-50 value. Based on the average measured concentration, the ErC50-value was calculated to be 5.1 mg/l with a 95% confidence interval of 4.1 - 6.4 mg/l. No growth inhibition was observed at a nominal concentration of 0.1 mg/l. The algal cultures were generally exposed to more then 80% of the nominal concentration at the start of the experiment. The NOEC based on nominal and on measured concentrations is thus assessed to be 0.1 mg/l.

Validity criteria:

The OECD Guideline No. 201 recognises two validity criteria, i.e. a sufficient control growth rate, and a limited increase of the test medium pH value during the test (one pH unit). The control growth rate (0.076 h-1 ) is higher than the minimum cell multiplication factor of 16 during a three day test given in the Guideline (corresponding to a growth rate of 0.038 h-1). The mean pH in the control cultures was 1.0 pH units higher than the pH in the medium without algae, which is just the limit given in the Guideline. The test therefore met the validity criteria of the Guideline.

Validity criteria fulfilled:

yes

Remarks:

a sufficient control growth rate, and a limited increase of the test medium pH value during the test (one pH unit).

Conclusions:

Based on the average percentage of the test substance present during the test, the 72h-NOEC was estimated to be 0.1 mg/l and the 72h-EC50 is calculated to be 5.1 mg/l for the growth rate and 1.9 mg/l for the biomass expresses as measured concentration. The Rhodixan A-1 is considered as toxic to the aquatic organisms tested.

Executive summary:

The toxicity of Rhodixan A-1 to the fresh water green algae Selenastrum capricornutum was determined in a 72 hours growth inhibition test. The test was carried out according to OECD guideline No.201 and Directive 67/548/EEC, Annexe V, test method C.3 and in compliance with Good Laboratory Practices.

 

Stock solutions in Tertiary Butyl Alcohol (TBA) containing the appropriate amounts of the test substance were used to obtain the following nominal concentrations : 0.1 – 0.33 – 1 - 3.3 – 10.3 mg/l. The inoculum cell density was 10000 cells/ml. The algal growth was measured with an electronic particle counter.

    

The actual concentration of Rhodixan A-1 in the exposure media was determined by HPLC with UV-detection. Samples of the exposure media were taken at T=0h just after dosing, and also in the spent media at the end of the test. The average percentage of the test substance present during the test was 49%.

The validity criteria related to the control algal growth and the pH variation were fulfilled. Based on the average percentage of the test substance present during the test, the 72h-NOEC was estimated to be 0.1 mg/l and the 72h-EC50 is calculated to be 5.1 mg/l for the growth rate and 1.9 mg/l for the biomass expresses as measured concentration. The Rhodixan A-1 is considered as toxic to the aquatic organisms tested.

Description of key information

Based on the average percentage of the test substance present during the test, the 72h-NOEC was estimated to be 0.1 mg/l and the 72h-EC50 is calculated to be 5.1 mg/l for the growth rate and 1.9 mg/l for the biomass expresses as measured concentration. The Rhodixan A-1 is considered as toxic to the aquatic organisms tested.

Key value for chemical safety assessment

EC50 for freshwater algae:

5.1 mg/L

Additional information

A GLP-compliant study, scored as Klimisch 1 and flagged as a key study, is available on the Rhodixan A-1; giving a 72h-EC50: 5.1 mg/L (growth rate) (measured concentration) and revealing that the Rhodixan A-1 is toxic to algae (TNO, 2001).