Benzyl 4-[(1-butyl-5-cyano-1,6-dihydro-2-hydroxy-4-methyl-6-oxopyridin-3-yl)azo]benzoate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

25 October to 3 December, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for the use of a read-across approach is provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: at the start of the test, duplicate samples from both the test medium with algae and also from the medium without algae. After 72 hours, the duplicate samples were taken as like the start of the test. The samples withut algae were performed for a stability check. From the control samples only one ofthe duplicate samples was analyzed from each of both sampling times.
- Sample storage conditions before analysis: samples were analysed immediately after sampling without prior storage

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
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 × 2H2O,18.0 mg/l
- NH4CI, 15.0 mg/l
- MgSO4 × 7H2O, 15.0 mg/l
- MgCl2 × 6H2O, 12.0 mg/l
- KH2PO4, 1.6 mg/l
Trace elements:
- Na2EDTA × 2H2O, 100.0 µg/l
- FeCl3 × 6H2O, 80.0 µg/l
- MnCl2 × 4H2O, 415.0 µg/l
- H3BO3, 185.0 µg/l
- Na2MoO4 × 2H2O, 7.0 µg/l
- ZnCl2, 3.0 µg/l
- CoCl2 × 6H2O, 1.5 µg/l
- CuCl2 × 2H2O, 0.01 µg/l

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Name: Scenedesmus subspicatus Chodat
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Sammlung von Algenkulturen Goettingen (SAG, Experimentelle PHykologie und Sammlung von Algenkulturen, Albrecht-von-Haller-Institut fur Pflanzenwissenschaften, Universitaet Goettingen, 37073 Goettingen, DE)
- Method of cultivation: cells were taken from an exponentially growing pre-culture, grown in laboratory under standardised conditions according to the guidelines
- Acclimation period: 4 days prior to the test
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period

Hardness:

0.24 mmol/l (= 24 mg/l) as CaCO3

Test temperature:

22-23 °C

pH:

7.9 - 8.5

Nominal and measured concentrations:

- 100 mg/l (nominal) corresponding to a mean measured test item concentration of 1.8 µg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml Erlenmeyer flasks
- Volumes of 15 ml algal suspension for each replicate were continuously stirred by magnetic stirrers
- Type: closed: covered with glass dishes
- Initial cells density: 10,000 algal cells per ml test medium
- No. of vessels per concentration: 3 replicates
- No. of vessels per control: 6 replicates
- Adjustment of pH: yes
- Photoperiod: continuously illuminated by fluorescent tubes (Philips TLD 36W840), installed above the test flasks
- Light intensity and quality: measured light intensity of about 8463 Lux (mean value), range: 7770 to 9080 Lux (minimum and maximum value of measurements at 9 places distributed over the experimental area at the surface of the test media)

OTHER TEST CONDITIONS
The test was started (0 hours) by inoculation of 10,000 algal cells per ml test medium. These cells were taken from an exponentially growing pre-culture, which was set up 4 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.

EFFECT PARAMETERS MEASURED
- Counting and examination of 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 exposure, and were not replaced. The algal cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter (R), Model ZM), with at least two measurements per sample ln addition, after 72 hours exposure, a sample was taken from the control and from the single test concentration. The shape of the algal cells was microscopically examined.
- Determination of the algal growth inhibition and the EG values: the inhibition of algal growth was determined from the mean values of counted algal cell densities. The test concentrations corresponding to 10 and 50% inhibition of the algal biomass b (EbC10, EbC50) or of the growth rate r (ErC10, ErC50) could not be calculated due to the absence of a toxic effect of the test item. The LOEC and the NOEC were determined directly from the counted algal cell densities. A statistical evaluation of the algal cell densities was not necessary since the mean algal cell density in the test medium was at all counting dates equal or even slightly higher than in the control.
- Water quality criteria: the pH was measured and recorded in the single test concentration and the control at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was recorded daily.

TEST CONCENTRATIONS
- Range finding study: range-fìnding test and the pre-experiments to the solubility of the test item in test water and stirring period
- Test concentrations: 100 mg/ml and negative control
- Results used to determine the conditions for the definitive study: based on the results of a range-finding test and on results of a pre-experiment to the solubility of the test item (without GLP)
- Additional information: the test item was not soluble at a concentration of 100 mg/l in test water and no homogeneous dispersion could be prepared. The undiluted filtrate of the supersaturated dispersion with the maximum concentration dissolved test item was used as the only test medium. Additionally, a control was tested in parallel (test water without addition of the test item). Thus, a limit test was performed to demonstrate that the test item has no toxic effect on the algae up to this concentration.

Reference substance (positive control):

no

Remarks:

potassium dichromate is tested as a positive control at least once per year to demonstrate satisfactory test conditions but was not used in this test

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 1.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 1.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

MAIN TEST
- Growth inhibition: the mean algal cell densities in the test medium were at all counting dates even slightly higher than those in the parallel control cultures. Thus, the test item had clearly no inhibitory effect on the growth of Scenedesmus subspicatus during the exposure period of 72 hours at a loading rate of 100 mg/l (mean measured test item concentration of 1.8 µg/l). This test concentration was therefore determined as the 72-hour NOEC. The NOEC might even be higher but concentrations in excess of the loading rate of 100 mg/l have not been tested, due to the low water solubility of the test item in the used test water.
- Microscopic examination: microscopic examination of the algal cells after 72 hours test period showed no difference between the algae growing in the test concentration at a loading rate of 100 mg/l and the algal cells in the control. The shape and size of the algal cells growing up to this test concentration were obviously not affected.
- Fulfillment of validity criteria: ln the control the cell density increased from nominal N = 1 × 10^4 cells/ml at the start of the test (0 hours) to N = 60 × 10^4 cells/ml (mean value) after 72 hours. Thus, the algal growth in the control was sufficiently high under the test conditions and the validity criterion of increase by at least a factor of 16 over the duration of the study was fulfilled.
- pH: control and treatment at start of test pH = 7.9; at the end of the test control and treatment pH ranged from 8.4-8.5
- Appearance of test medium: no remarkable observations were made concerning the appearance of test medium; the test medium remained a clear yellow colored solution throughout the entire test period

ANALYTICAL DETERMINATION OF CONCENTRATIONS
The analytically determined test item concentration in the analyzed test medium samples of the undiluted filtrate amounted to 2.1 µg/l at the start of the test and 1.5 µg/l at the end of the test. The biological results are based on the mean measured test item concentration of 1.8 µg/l (calculated as the average over all measurements in the undiluted filtrate) as well as on the loading rate of 100 mg/l.

Validity criteria fulfilled:

yes

Remarks:

Control cell density of nominal: N = 1 × 10^4 cells/ml (nominal) at start of test; and N = 60 × 10^4 cells/ml (mean value) at end. Therefore, control algal growth increased by more than factor 16 during the study period.

Conclusions:

The 72-hour EC50 for both mean algal biomass and mean growth rate are greater than 100 mg/l (nominal).

Executive summary:

The influence of the test item on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, C.3, 1992, and the OECD guideline 201. Due to the low water solubility of the test item, a supersaturated dispersion of the test item with a loading rate of nominal 100 mg/l was continuously stirred at room temperature in the dark over 3 hours. Then the dispersion was filtered. The undiluted filtrate with the maximum concentration of dissolved test item was used as the only test medium. Additionally, a control was tested in parallel. A limit test was performed at 100 mg/l.

The analytically determined test item concentrations in the analysed test medium samples of the undiluted filtrate amounted to 2.1 µg/l at the start of the test and 1.5 µg/l at the end of the test. The biological results are based on the mean measured test item concentration of 1.8 µg/l (calculated as the average over all measurements in the undiluted filtrate) as well as on the loading rate of 100 mg/l. The mean algal cell densities in the test medium were even slightly higher than those in the parallel control cultures at all counting dates. Thus, the test item had clearly no inhibitory effect on the growth of Scenedesmus subspicatus during the exposure period of 72 hours at a loading rate of 100 mg/l (mean measured test item concentration of 1.8 µg/l).

The 72 h LOEC and the 72 h EC₅₀ for both mean algal biomass and mean growth rate are greater than the loading rate of 100 mg/l. The NOEC might even be higher but concentrations in excess of the loading rate of 100 mg/l have not been tested, due to the low water solubility of the test item in the used test water. In conclusion, the substance had no toxic effect on the algae up to its solubility limit in test water.

Description of key information

E_rC₅₀ and E_yC₅₀ (72 h, Scenedesmus subspicatus) > 100 mg/l (nominal)

Additional information

No data on Disperse Yellow 231 was available, thus 2 experimental studies on a structural analogue, i.e. Similar Substance 01, were used for the assessment. Details on the read-across are available in section 13.

Both studies were 72 hours of exposure of Scenedusmus subspicatus in a static, freshwater test, performed according to the OECD guideline 201 (1984) and the EU method C.3 (1992). Both studies demonstrated that the concentration which leads to 50 % reduction in growth rate (E_rC₅₀) of S. subspicatus after 72 hours of exposure is greater than 100 mg/l, reported as nominal concentration due to poor water solubility.