2-Thiophenecarboxylic acid, 5-chloro-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 May - 25 May 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC-UV/VIS

Details on sampling:

- Sampling method: Aliquots of the samples from the biological test were directly analyzed by HPLC and UV/VIS-detection (range of the injection volume: 1-100 µL, depending on the expected concentration).
- Sample storage conditions before analysis: Routinely, the samples are analyzed immediately. Only in exceptional cases, they are stored overnight deeply frozen and protected from light.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: To produce the test concentration, 125.0 mg of the test substance were added to 1 L of dilution water, treated for 1 hour in an ultrasonic bath and afterwards stirred for 24 hour on a magnetic stirrer.
- Controls: 1 control, six replicates
- Test concentration separation factor: 2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None reported

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Non-axenic strain
- Source: obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Age of inoculum (at test initiation): 72 h
- Method of cultivation: Pre-cultures are set up three days before the start of a test. They are grown under identical exposure conditions a the stock culture, except from the use of a different nutrient medium.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): different medium, other conditions are identical
- Any deformed or abnormal cells observed: None

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23 +/- 2 °C

pH:

8.3 to 9.0

Nominal and measured concentrations:

100 mg/L (nominal concentration)

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks with cotton stoppers
- Type: closed
- Initial cells density: 5000 cells/mL
- Control end cells density: 596111 cells/mL
- No. of vessels per concentration (replicates): 3 replicates per concentration
- No. of vessels per control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: yes

OTHER TEST CONDITIONS
- Adjustment of pH: The pH was adjusted o pH 7.9.
- Photoperiod: continuous uniform illumination is provided in the spectral range 400 to 700 nm
- Light intensity and quality: At the average of the test solutions, a light intensity in the range 60 to 120 µE. x m^-2, or an equivalent range of 4000 to 8000 lx, throughout test.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Cell densities are measured in a microcell counter or, alternatively, are determined by means of a microscopic counting chamber. Cell densities are recorded at 24-hour intervals.

TEST CONCENTRATIONS
- Test concentrations: 100 mg/L (nominal concentration)

CULTURING APPARATUS
-Details on culturing apparatus used: Light chamber

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 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
- Any stimulation of growth found in any treatment: No

Analysis test substance

Analysis	0 h	72 h
pH values	8.3	9.0
HPLC replicates [mg/L]	97.905/ 96.699	97.640/ 96.960
HPLC mean value [mg/L]	97.202	97.300

Analysis control

Analysis	0 h	72 h
pH values	8.3	8.9
HPLC replicates [mg/L]	<0.013/ <0.013	<0.013/ <0.013
HPLC mean value [mg/L]	<0.013	<0.013

Validity criteria for the measurement of the algae toxicity

Target condition according to guideline:	Actual condition according to the study:	Validity criteria met:
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period. This corresponds to a specific growth rate of 0.92 day^-1. This criterion may not be met when species that grow slower than those listed in Annex 2 of OECD TG 201 are used. In this case, the test period should be extended to obtain at least a 16-fold growth in control cultures, while the growth has to be exponential throughout the test period. The test period may be shortened to at least 48 hours to maintain unlimited, exponential growth during the test as long as the minimum multiplication factor of 16 is reached.	During the test, the biomass in the control increased exponentially by a factor >16  and the specific growth rate was in the range of 1.58 to 1.60 day^-1 within the 72-hour test period.	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%. This criterion applies to the mean value of coefficients of variation calculated for replicate control cultures.	The mean coefficent of variation for section-by-section specific growth rates in the control was <3%	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	The coefficent of variation of average specific growth rates (0-72h) in the control was <1%	Yes
The concentration of the test item shall be maintained to within 80% of the initial concentration throughout the study. The maintenance of concentration is proved by analytical measurements	Recovery rates correspond to 97.2% of nominal values at 0 hours, and correspond to 97.3% of nominal values at 72 hours, respectively.	Yes
The cultures should be maintained at a temperature in the range of 21 to 24°C, controlled at ± 2°C.	The cultures were maintained at a temperature of 23 +/- 2 °C	Yes

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on results incl. tables'.

Conclusions:

No toxic effects of the test substance against algae at a test concentration of 100 mg/L (nominal concentration) were observed. 

Executive summary:

The study was performed to assess adverse effects of the test substance on the growth (=increase in cell density) and the growth rate (=rate of increase in cell density with time) of the planktonic freshwater algal species Desmodesmus subspicatus over several generations. The study was conducted in accordance with 92/69/EEC Part C, Method 3 'Algal inhibition test' which is in most parts equivalent to the OECD Guideline for Testing of Chemicals No 201 'Alga, Growth Inhibition Test'. Exponentially growing algal cells were exposed for a period of 72 hours to a limit test concentration of nominally 100 mg/L of the test substance dissolved in water. Auxiliaries used to prepare the test media were an ultrasonic bath and a magnetic stirrer. The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth (index b) and growth rate (index r), relative to control cultures grown under identical conditions. Recovery rates correspond to 97.2% of nominal values at 0 hours, and correspond to 97.3% of nominal values at 72 hours, respectively. The following values were determined: E_rC50 (0-72h)>100 mg/L (nominal concentration), and E_bC50 (0-72h)>100 mg/L (nominal concentration). No toxic effects of the test substance against algae at a test concentration of 100 mg/L (nominal concentration) were observed. 

Description of key information

A study was performed to assess adverse effects of the test substance on the growth (=increase in cell density) and the growth rate (=rate of increase in cell density with time) of the planktonic freshwater algal species Desmodesmus subspicatus over several generations. The study was conducted in accordance with 92/69/EEC Part C, Method 3 'Algal inhibition test' which is in most parts equivalent to the OECD Guideline for Testing of Chemicals No 201 'Alga, Growth Inhibition Test'. Exponentially growing algal cells were exposed for a period of 72 hours to a limit test concentration of nominally 100 mg/L of the test substance dissolved in water. Auxiliaries used to prepare the test media were an ultrasonic bath and a magnetic stirrer. The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth (index b) and growth rate (index r), relative to control cultures grown under identical conditions. Recovery rates correspond to 97.2% of nominal values at 0 hours, and correspond to 97.3% of nominal values at 72 hours, respectively. The following values were determined: E_rC50(0-72h)>100 mg/L (nominal concentration), and E_bC50(0-72h)>100 mg/L (nominal concentration). No toxic effects of the test substance against algae at a test concentration of 100 mg/L (nominal concentration) were observed.