Disodium 4,4'-methylenebis[3-hydroxy-2-naphthoate]

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:

27 NOV 2017 - 19 MAR 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted on March 23, 2006; Annex 5 corrected: July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Version / remarks:

January 1993

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft and Verbraucherschutz, Mainzer Straße 80, 65189 Wiesbaden (1 Feb 2013)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: For analysis, samples of control and test media were taken daily. The algae were separated from the medium by centrifugation at a low g-force, sufficient to settle the algae. The samples were diluted 1:4 with methanol:buffer pH 9.00 (1:1, v:v) and filtered. The concentrations of the test material in the samples were quantified using the calibration curve.

Vehicle:

yes

Remarks:

reconstituted water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium (reconstituted water and test item) was prepared freshly. For this purpose, the test item was weighed into freshly reconstituted water and then shortly stirred with a mini shaker.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus (unicellular green algae)
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, Germany; this strain was further cultivated in the internal laboratories
- Method of cultivation: For reproduction, outside of studies, the algae were agitated during the entire study, and lit between 4440 and 8880 Lux. Fluorescent tubes installed above the flasks serve for lighting. The maximum variation of the light intensity should be ± 15% from the light intensity average.

ACCLIMATION
- Acclimation period: Prior to start the study, the algae were grown in reconstituted water as preculture and the growth rate was determined.
- Culturing media and conditions (same as test or not): same (reconstituted water)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

for details see composition of reconstituted water "details on test conditions"

Test temperature:

21 - 24 °C

pH:

7.41 - 7.56

Nominal and measured concentrations:

nominal: 0, 10, 17.8, 31.6, 56.2, 100 mg/L
analytical (0 h): < 0.094 mg/L (LOD), 8.275, 13.906, 26.174, 47.899, 92.686 mg/L
analytical (24 h): < 0.094 mg/L (LOD), 3.009*, 7.396, 15.841, 29.471, 55.998 mg/L
analytical (48 h): < 0.094 mg/L (LOD), 1.880*, 3.856*, 9.913, 16.488, 33.309 mg/L
analytical (72 h): < 0.094 mg/L (LOD), 1.407*, 2.863*, 5.452, 12.893, 29.093 mg/L

*Value outside (below) calibration range of 5 - 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask (300 mL)
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 50 mL/300 mLflask (Due to the fact, that the test item preparations were slightly brownish colored, the light path was reduced by reducing the volume to 50 mL per flask instead of 100 mL normally used.)
- Aeration: continuously shaken
- Initial cells density: 10,000 cells/mL
- Control end cells density: 45,700 cells/mL (mean)
- No. of organisms per vessel: 500,000 cells
- No. of vessels per concentration (replicates): 3
- No. of vessels per vehicle control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
Details on composition of the reconstituted water:

Nutrient mg/L
NH4CI 15.0
MgCl2 * 6H2O 12.0
CaCl2 * 2H2O 18.0
MgSO4 * 7H2O 15.0
KH2PO4 1.60
FeCl3 * 6H2O 0.0640
Na2EDTA * 2H2O 0.100
H3BO3 0.185
MnCl2 * 4H2O 0.415
ZnCl2 0.00300
CoCl2*6H2O 0.00150
CuCl2*2H2O 0.00001
Na2Mo04 * 2 H2O 0.00700
NaHCO3 50.0
The pH of this medium after equilibration with air is approximately 8.

OTHER TEST CONDITIONS
- Photoperiod: continuos illumination
- Light intensity and quality: Due to the fact, that the test item preparations were slightly brownish colored, the study was performed at a higher light intensity of about 13000 - 14000 Lux instead of 4440 - 8880 Lux, since the growth rate above light saturation is almost independent from light intensity. The light intensity measured immediately before start of the study was 13954 Lux ± 2.8% and 13648 Lux ± 6.3% at the end of the study.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Small samples with algae were taken from all concentrations after 24, 48, or 72 hours of exposure and were not replaced.
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.78
- Test concentrations: nominal 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 45.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 45.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 45.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 45.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 45.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 45.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No morphological changes of the treated algal cells in comparison to the control algal cells were observed.
- Unusual cell shape: Not observed
- Colour differences: Not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The stability of the test item in reconstituted water was determined at a concentration of 100 mg/L in an open system. It was shown, that the test item concentrations could not be maintained ≥ 80% of the fresh prepared test item concentrations throughout the study, due to instability of the test item. Therefore, analysis was performed daily to calculate the geometric mean exposure concentration. The analytically determined test item concentration immediately after preparation of the medium was 92.7-78.1% and 72 hours thereafter 29.1-14.1% of the nominal concentration. Therefore, the EC values were calculated with the geometric mean exposure concentration.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.64 mg/L based on growth rate

Validity of the Test

In the control group the biomass increased by a factor of 45.7 over 72 hours. Therefore, the validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled.

The mean coefficient of variation of the daily growth rates in the control group (section-by-section growth rates) during 72 hours was 37.0%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was slightly exceeded. It can be assumed that the slightly decreased growth rate throughout the study was related to the high light intensity used in this study due to the coloration of the test item preparation. At higher light intensities the photosynthesis apparatus of the algae can be affected, resulting in lower growth rates (Shuter, 1979; Han et al, 2000). It is considered that this minor deviation does not affect the integrity of the study.

The coefficient of variation of the average specific growth rates in the replicates of the control group after 72 hours was 1.9%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

Han et al.: Effect of photoinhibition on algal photosynthesis: a dynamic model. Journal of Plankton Research, Volume 22 (5), 865-885, 2000

Shuter, A: Model of Physiological Adaption in Unicellular Algae. J. theor. Biol., Volume 78, 519-552, 1979

Validity criteria fulfilled:

yes

Remarks:

Please refer to 'Any other information on results incl. tales'

Conclusions:

The test item solved in reconstituted water was tested for toxicity in algae in an open static test system. Under the conditions of the present study, an aqueous solution of nominal 100 mg/L revealed no aquatic toxicity in the test system. The EC50 (0-72h) for growth rate and yield was > 45.9 mg/L based on analytical concentration.

Executive summary:

In this study the influence of the test item on the growth and growth rate of the green algae species Desmodesmus subspicatus was tested.The GLP study was performed according to OECD TG  201. The study design included one control group with six replicates and five test item groups with three replicates, each containing 50 mL reconstituted water or test medium and about 10000 cells/mL at the start of the experimental phase. The study was performed with nominal concentrations of 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L in reconstituted water in a static test system in open vessels. The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test item and control group. Due to the results of a stability determination before start of study, analytical controls of the test item preparations were performed at the start of the study and then daily.

The analytically determined test item concentration immediately after preparation of the medium was 92.7-78.1% and 72 hours thereafter 29.1-14.1% of the nominal concentration. The concentrations of the test item in test media without algae were slightly higher after 24 and 48 hours, respectively compared to the preparations with algae. However, no difference has been observed between analytical concentrations of samples with and without algae after 72 hours. During the experimental phase of the study, the test item concentration could not be maintained within ± 20% of the initial analytical concentration, due to instability of the test item. Therefore, the EC values were calculated with the geometric mean exposure concentration. Due to the fact that an aqueous preparation of a nominal concentration of 100 mg/L of the test item revealed no toxic effect in Desmodesmus subspicatus, the geometric mean exposure concentration was calculated only for the highest test item concentration. The EC₅₀ (0-72h) for growth rate and yield were determined to be > 45.9 mg/L.

Description of key information

Key, growth inhibition, Desmodesmus subspicatus, static, open, OECD 201, GLP: E_rC₅₀(72h) > 45.9 mg/L and E_rC₁₀(72h) > 45.9 mg/L

Additional information

In this study the influence of the test item on the growth and growth rate of the green algae species Desmodesmus subspicatus was tested.The GLP study was performed according to OECD TG  201. The study design included one control group with six replicates and five test item groups with three replicates, each containing 50 mL reconstituted water or test medium and about 10000 cells/mL at the start of the experimental phase. The study was performed with nominal concentrations of 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L in reconstituted water in a static test system in open vessels. The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test item and control group. Due to the results of a stability determination before start of study, analytical controls of the test item preparations were performed at the start of the study and then daily.

The analytically determined test item concentration immediately after preparation of the medium was 92.7-78.1% and 72 hours thereafter 29.1-14.1% of the nominal concentration. The concentrations of the test item in test media without algae were slightly higher after 24 and 48 hours, respectively compared to the preparations with algae. However, no difference has been observed between analytical concentrations of samples with and without algae after 72 hours. During the experimental phase of the study, the test item concentration could not be maintained within ± 20% of the initial analytical concentration, due to instability of the test item. Therefore, the EC values were calculated with the geometric mean exposure concentration. Due to the fact that an aqueous preparation of a nominal concentration of 100 mg/L of the test item revealed no toxic effect in Desmodesmus subspicatus, the geometric mean exposure concentration was calculated only for the highest test item concentration. The EC₅₀ (0-72h) for growth rate and yield were determined to be > 45.9 mg/L.