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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Regulation (EU) No.2016/266
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
PRELIMINARY TESTThe analytical determination of the test substance concentrations was performed at the beginning and at the end of the test. The samples for analysis were taken from the highest (100 mg/L), the lowest (1 mg/L) test concentration and from the highest (100 mg/L) concentration without algae. The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory. The samples were analyzed on the day of delivery. The samples for analysis at the end of the test (72 hours) were delivered to analytical laboratory immediately after the end of testing. The samples were analyzed on the day of delivery. All samples were stored at laboratory temperature.The analytical results showed that the test substance was stable in the test medium under the conditions of testing.LIMIT TEST The analytical determination of the test substance concentration was performed at the beginning and at the end of the test. The samples for analysis were taken from the stock solution (100 mg/L) with and without algae. The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory. The samples were analyzed on the day of delivery. The samples for analysis at the end of the test (72 hours) were delivered to analytical laboratory immediately after the end of testing. The samples were analyzed on the day of delivery. All samples were stored at laboratory temperature.
Vehicle:
no
Details on test solutions:
BASIC STOCK SOLUTIONSThe four basic stock solutions were prepared according to following data. The solutions were kept in the dark at temperature of 4 ± 1 °C in fridge. The temperature was checked every day.Solution A: NH4Cl: 1.5 g MgCl2 · 6H2O: 1.2 g CaCl2 · 2H2O: 1.8 g MgSO4 · 7H2O: 1.5 g KH2PO4: 0.16 g water up to 1000 mL Solution B: FeCl3 · 6H2O: 64 mg Na2EDTA · 2H2O: 100 mg water up to 1000 mLSolution C: H3BO3: 185 mg MnCl2 · 4H2O: 415 mg ZnCl2: 3.0 mg CoCl2 · 6H2O: 1.5 mg CuCl2 · 2H2O: 0.01 mg Na2MoO4 · 2H2O: 7.0 mg water up to 1000 mL Solution D: NaHCO3: 50 g water up to 1000 mLPREPARATION OF THE TEST MEDIUMThe test medium was prepared by admixing 10 mL of basic stock solution A and 1 mL of basic stock solutions B, C, D in 1 000 mL deionized water.PREPARATION OF SOLUTIONS OF THE TEST SUBSTANCEThe stock solution of the test substance was prepared in the test medium. 50 mg of the test substance was weighed into 500 mL of the test medium for the preliminary and the limit test. The stock solution was ultrasonicated for 10 minutes. The concentrations of test solutions used in the preliminary test were obtained by dilution of the stock solution with test medium.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM - Common name: Desmodesmus subspicatus - Strain: Brinkmann 1953/SAG 86.81 - Source (laboratory, culture collection): collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň on date 27.5.2016. - Method of cultivation: The strain culture was preinoculated from the stock solution and cultivated in flasks with the test medium on indirect daylight at laboratory temperature. Algae inoculum for the test was sampled from exponentially growing inoculum culture. The strain culture was always set to pre-culturing of cells for 3-4 days before the start of the test. Inoculum culture was kept 3-4 days under conditions at which the test was performed. After this period the culture was in the state of the exponential growth and had the cell density suitable for the performance of the test. The cell density of the pre-culture was measured just before the start of the test and the needed inoculum volume was calculated.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
21 - 24°C controlled at ± 2°C
pH:
7.6 - 7.8
Nominal and measured concentrations:
PRELIMINARY TESTNominal concentrations: 1 mg/L 0 h: 0.96 mg/L 72 h: 1.00 mg/LNominal concentrations: 100 mg/L 0 h: 99.4 mg/L 72 h: 101.7 mg/LNominal concentrations: 100 mg/L without algae 0 h: 100.2 mg/L 72 h: 104.0 mg/LLIMIT TESTNominal concentrations: 100 mg/L 0 h: 98.7 mg/L 72 h: 100.9 mg/LNominal concentrations: 100 mg/L without algae 0 h: 99.5 mg/L 72 h: 101.0 mg/L
Details on test conditions:
TEST SYSTEM - Test vessel: Erlenmeyers’ flasks - Aeration: without aeration - Initial cells density: 5000 cells per mL - Control end cells density: Burker´s counting chamber - No. of vessels per concentration (replicates): 3 in preliminary test, 6 in limit test - No. of vessels per control (replicates): 3 in preliminary test, 6 in limit test GROWTH MEDIUM - Standard medium used: yes OTHER TEST CONDITIONS - Adjustment of pH: no - Photoperiod: continuous white light - Light intensity and quality: 4 440 lux to 8 880 lux EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : - Determination of cell concentrations: Burker´s counting chamber - Observation intervals: 0, 24, 48 and 72 hours TEST CONCENTRATIONS - Range finding study - Test concentrations: 1, 5, 10 50 and 100 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate (CAS: 7778-50-9)
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
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
see Table 1 and 2
Results with reference substance (positive control):
RESULTS OF REFERENCE TESTErC50 = 1.05 mg/L (potassium dichromate) (72 h)INTERLABORATORY TEST DATAFrom the results of the last interlaboratory test arranged by CSlab spol. s r.o. in 2016 we have obtained data for potassium dichromate 72h – ErC50 generated at 13 laboratories. The following reference range of values could be calculated from those data: ErC50 = 0.54 – 1.26 mg/L (72 h)

Table 1: Preliminary test results

Nominal

concentration

(mg/L)

Replicates

Cell density in 1 mL

Growth rate

Yield

0 h

24 h

48 h

72 h

Mean

Reduction

(%)

Mean

Inhibition

(%)

100

1

5000

18750

37500

125000

1.07

13.4

117917

40.2

2

5000

31250

50000

112500

3

5000

25000

43750

131250

50

1

5000

25000

50000

131250

1.10

10.8

130417

33.8

2

5000

25000

43750

143750

3

5000

18750

56250

131250

10

1

5000

31250

50000

143750

1.11

9.6

136667

30.7

2

5000

31250

50000

137500

3

5000

25000

56250

143750

5

1

5000

31250

62500

150000

1.14

7.3

149167

24.3

2

5000

25000

56250

143750

3

5000

31250

62500

168750

1

1

5000

31250

62500

162500

1.16

5.5

159583

19.0

2

5000

25000

62500

168750

3

5000

31250

68750

162500

Control

1

5000

25000

87500

187500

1.23

-

197083

-

2

5000

31250

75000

225000

3

5000

25000

62500

193750

Table 2: Limit test results

Nominal

concentration

(mg/L)

Replicates

Cell density in 1 mL

Growth rate

Yield

0 h

24 h

48 h

72 h

Mean

Reduction

(%)

Mean

Inhibition

(%)

100

1

5000

25000

43750

137500

1.10

7.2

130417

23.3

2

5000

25000

50000

150000

3

5000

18750

37500

118750

4

5000

18750

56250

143750

5

5000

31250

50000

125000

6

5000

25000

56250

137500

Control

1

5000

18750

62500

137500

1.19

-

170000

-

2

5000

25000

75000

175000

3

5000

25000

56250

162500

4

5000

25000

68750

162500

5

5000

31250

87500

212500

6

5000

25000

87500

200000

Validity criteria fulfilled:
yes
Conclusions:
The inhibition of growth rate was 7.2 % and the inhibition of yield was 23.3 % in the limit test. Therefore exact value of ErC50 and EyC50 could not be calculated and the values of EC are given in the form of a range.The determination of NOEC value was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 1998-2000 (product of TriloByte Ltd., Czech Republic). There is stated in the guideline that, if evidence is available to demonstrate that the concentration of the test substance has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the test, the results can be based on nominal or measured initial values. So the nominal concentrations were used for all evaluations and results.ErC50 > 100 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)EyC50 > 100 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)NOECr < 1 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)NOECy < 1 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)
Executive summary:

The test substance, Direct Yellow 28, was tested for growth inhibition on algae Desmodesmus subspicatus. The test was performed according to method C.3. - Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EU) No.2016/266.

The preliminary test was performed in a range of the test substance nominal concentrations 1 – 100 mg/L. The preliminary test was performed twice. The first preliminary test was stopped due to the test would not meet the validity criteria.

The analytical results showed, that the test substance Direct Yellow 28 was sufficiently stable in the test medium at conditions of the test in the concentration range 1 – 100 mg/L. The highest inhibition of growth rate was 13.4 % and the highest inhibition of yield was 40.2 % in the preliminary test. Based on no toxicity of the test substance found in the preliminary test, the limit test was performed subsequently. The concentration of 100 mg/L was tested in the limit test. The inhibition of growth rate was 7.2 % and the inhibition of yield was 23.3 %.

The guideline specify that if evidence is available to demonstrate that the concentration of the test substance in the limit test has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the limit test, then the results can be based on nominal or measured initial values, which is the case of this study. The nominal concentrations were used for all evaluations and results.

ErC50 > 100 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)

EyC50 > 100 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)

NOECr < 1 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)      

NOECy < 1 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)

Description of key information

The effective concentration EC50 > 100 mg/L was determined based on inhibition of growth rate of aquatic algae.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information