Black HM 2482

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:

From December 5th, 2003 to January 8th, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

EU Commission Directive 92/69/EEC

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

400-920-6

EC Name:

-

Cas Number:

89857-06-7

Molecular formula:

C50 H53 N11 O14 S

IUPAC Name:

5'-[2-(7-{2-[4-(2-{1'-[3-(dimethylazaniumyl)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-8-hydroxy-6-sulfonatonaphthalen-2-yl)diazen-1-yl]-6'-hydroxy-3,4'-dimethyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium bis(2-hydroxypropanoate)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

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.
In addition, after 72 hours exposure, a sample was taken from the control and from a test concentration with reduced algal growth in experimental part A (nominal 32 mg/L).

Test solutions

Vehicle:

no

Details on test solutions:

The test medium of the highest test item concentration of nominal 100 mg/L was prepared by dissolving 50.1 mg of the test item completely in 500 mL of test water by intense stirring (15 minutes at room temperature). Adequate volumes of this intensively mixed test medium were diluted with test water to prepare the test media with the lower test item concentrations. The test media were prepared just before addition of algae (= start of the test).

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus
- Strain: No. 86.81 SAG
- Source: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, D-37073 Göttingen, Germany).
- Age of inoculum (at test initiation):
- Method of cultivation: all flasks were incubated in a temperature controleld water bath at 23 to 24 °C and continuosly illuminated.

ACCLIMATION
- Culturing media and conditions: the algae were cultivated and tested in synthetic test water (water hardness = 0.24 mmol/L = 24 mg/L as CaCO3).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg/l as CaCO3

Nominal and measured concentrations:

Nominal: 0.10, 0.32, 1.0, 3.2, 10, 32, and 100 mg/L
Measured: 0.95 (nominal 1.0 mg/L), 1.7 (nominal 3.2 mg/L), 6.7 (nominal 10 mg/l), 29 (nominal 32 mg/L) and 96 mg/L (nominal 100 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks, each filled with 15mL algal suspension
- Type: closed
- Material, size, headspace, fill volume: glass
- Initial cells density: 10.000 algal cells per mL of test medium
- No. of vessels per concentration (replicates): 7. 3 replicates
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 1


TEST MEDIUM / WATER PARAMETERS
Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations.
Macronutrients:
NaHCO3 50 mg/l
CaCl2 x 2H2O 18 mg/l
NH4Cl 15 mg/l
MgSO4 x 7 H2O 15 mg/l
KH2PO4 1.6 mg/l
Trace elements:
Na2EDTA x 2H2O 100 µg/l
FeCl3 x 6H2O 80 µg/l
MnCl2 x 4 H2O 415 µg/l
H3BO3 185 µg/l
Na2MoO4 x 2 H2O 7 µg/l
ZnCl2 3 µg/l
CoCl2 x 6H2O 1.5 µg/l
CuCl2 x 2H2O 0.01 µg/l


OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: ca. 8940 Lux (mean value); range 8460 - 9580 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%-confidence limits 6 - n.d (not determined)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.3 mg/L

95% CI:

0 - 8.3

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%-confidence limits n.d. (not determined)

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Any other information on results incl. tables

Experimental part A

Parameter (0-72h)	Biomass b (mg/L)	Growth rate r (mg/L)
EC50 95%-confidence limits	6.0 1.4 - 28	25 6 – n.d.
EC10 95%-confidence limits	0.8 0.0 – 2.7	2.3 0.0 – 8.3
EC90 95%-confidence limits	44 13 – n.d.	>100 n.d.
NOEC	0.95	0.95
LOEC	1.7	1.7

n.d.: could not be determined

 

Experimental part B

Parameter (0-72h)	Biomass b (mg/L)	Growth rate r (mg/L)
EC50 95%-confidence limits	26 16 - 43	65 56 – 78
EC10 95%-confidence limits	6.2 2.1 – 11	19 14 – 24
EC90 95%-confidence limits	113 63 – n.d.	>100 n.d.

n.d.: could not be determined

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The influence of the test item on the growth of the green algal species Scenedesmus subspicatus was investigated in a 7Z-hour static test according to the EU Commission Directive 92/69/EEC, Annex Part C.3, 1992, and the OECD Guideline No. 201, 1984. However, the test method was modified to quantify the algicidal effect of the test item, but also the growth inhibition effect caused by reduced light intensities in the colored test solutions.
The nominal test concentrations were 0.10, 0.32, 1.0,3.2, 10,32, and 100 mg/L in parallel with a control. All test media down to the nominal test concentration of 1.0 mg/L were slightly to strongly colored by the test item.
The analytically determined test item concentrations in the test media of the nominal concentrations of 3.2 to 100mg/L were in the range of 87 to 102o/o of the nominal values at the start of the test demonstrating the correct preparation of the test media. At the end of the test, the measured test item concentrations were 21,35,78, and 94% of nominal at the
nominal test concentrations of 3.2, 10,32, and 100 mg/L, respectively. At the nominal test concentration of 1.0 mg/L, the measured concentration at the start of the test was 145% of nominal. At the end of the test, the test item concentration was below the LOQ (limit of quantification) of 0.88 mg/L at this test concentration. Thus, the test item concentration was approximately constant over the test period at the highest nominal test concentration of 100 mg/L. A decrease of the test concentration was observed at all lower test concentrations.
The reported biological results are related to the mean measured test item concentrations of 0.95 mg/L (nominal 1.0 mg/L), 1.7 mglL (nominal 3.2 mg/L), 6.7 mg/L (nominal 10 mg/L), 29 mglL (nominal 32 mg/L), and 96 mg/L (nominal 100 mg/L).
At the mean measured test concentration of 1.7 mg/L (nominal 3.2 mg/L) and above, the growth of Scenedesmus subspicatus was statistically significantly smaller than in the control. However, the study demonstrates that only at the two highest test concentrations of 29 and 96 mg/L (nominal 32 and 100 mg/L), a significant growth inhibition was caused by the indirect effect, the light absorption by the colored test item. At all lower test concentrations, the light absorption of the test item was too small to result in a growth inhibition.
Thus, the growth inhibition was caused, at least in part, by a real toxic effect of the test item.
Therefore, the results of this experimental part, where the algae grew in the test media with dissolved test item as in a usual algal growth inhibition test should be taken into consideration for the determination of the toxic etfect of the test item on the growth of Scenedesmus subspicatus.

ln this experimental part, test item had a statistically significant inhibition effect on the growth of Scenedesmus subspicatus after the exposure period of 72 hours first at the mean measured concentration of 1.7 mglL (nominal 3.2 mg/L). This test concentration was determined to be the 72-hour LOEC (lowest concentration tested with toxic effects). The 72-hour NOEC (highest concentration tested without toxic effects) was 0.95 mg/L (nominal 1.0 mg/L) since at this test concentration, the biomass b and growth rate r of the algae was statistically not significant lower than in the control.