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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:
4-17.1.2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION OF TEST SOLUTION (especially for difficult test substances)
Since the test substance is poorly soluble in test medium at concentrations needed for the test, saturated solutions of the test substance in test medium were prepared by adding measured amounts of the test substance to the test medium. The solutions were stirred for period of 72 hours on a shaft stirrer and consequently filtered through 0.45 μm filter. These solutions were used for testing.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: Brinkmann 1953/SAG 86.81
- Source (laboratory, culture collection): the collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The strain culture was pre-inoculated from the stock solution and cultivated in flasks with the test medium in indirect daylight at laboratory temperature.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): Yes
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.5 to 23.5 °C
pH:
7.7 to 8.0
Conductivity:
0.188 mS/m
Nominal and measured concentrations:
- Nominal concentrations: 100, 53, 28, 15, 8, 4 and 2 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): Closed with stoppers venting air
- Material, size, headspace, fill volume: 50 mL in each test flask
- Aeration: No
- Initial cell density: 5 000 cells/mL
- Control end cell density: 237 500 in 1 mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: Yes
The test medium was prepared by admixing 10 mL of basic stock solution A and 1 mL each of basic stock solutions B, C, D in 1000 mL deionised water. The solutions were kept in the dark at 4 ± 1 °C in fridge. The 4 basic solutions contained the following components and each was made up to 1000 mL with deionised water.
Solution A: NH4Cl 1.5 g, MgC12.6H2O 1.2 g, CaCl2.2H2O 1.8 g, MgSO4.7H2O 1.5 g and KH2PO4 0.16 g
Solution B: FeCl3.6H2O 64 mg and Na2EDTA.2H2O 100 mg
Solution C: H3BO3 185 mg, MnCl2.4H2O 415 mg, ZnCl2 3.0 mg, CoCl2.6H2O 1.5 mg, CuCl2.2H2O 0.01 mg and Na2MoO4.H2O 7.0 mg
Solution D: NaHCO3 50 g

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water with conductivity smaller than 5 μS/cm was used for the preparation of solutions.
- Culture medium different from test medium: No
- Intervals of water quality measurement: At the beginning and the end of the test the pH of the test mixtures was measured. The temperature was measured every 24 hours.

OTHER TEST CONDITIONS
- Sterile test conditions: No data
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: white light, 6800 to 6900 lux (measured every 24 hours)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: The density of algae culture was evaluated microscopically at 24, 48 and 72 hours. The cell density was measured by direct counting of living cells in Burker's counting chamber.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.9
- Range finding study: Yes
- Test concentrations: 100, 60, 40, 20 and 10 mg/L (nominal concentrations)
- Results used to determine the conditions for the definitive study: Yes. Due to toxicity observed in the preliminary test the full definitive test was performed. The percentage reduction in growth rate observed in the preliminary test was 15.9, 20.1, 22.7, 27.8 and 28.9 % at nominal concentrations of 10, 20, 40, 60 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
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
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence limit: 1.0 to 2.8 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % confidence limit: 4.1 to 4.9 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % confidence limit: 0.0 to 0.6 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
Reduction in the growth rate and inhibition of the yield was seen in every test concentration.
In the main test, the biomass in the control cultures increased more than 45 times during the 72-hour test period.
The mean coefficient of variation for section-by-section specific growth rates in the control cultures is 13.1 % (CV0-24 h = 2.4 %, CV24-48 h = 12.6 % and CV48-72 h = 24.1 %).
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures is 2.7 %.

TEST QUALITY CRITERIA
- The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.
- The mean coefficient of variation for section-by-section specific growth rates in the control cultures must not exceed 35 %.
- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7 % in tests.
All three criteria were fulfilled.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 72 hour ErC50 = 0.94 mg/L (95 % confidence limit: 0.084 to 1.04 mg/L)
- Other: The inhibition of growth rate by the reference substance obtained from the most recent testing facility reference was in the appropriate range. The sensitivity of test species and proficiency of the laboratory in test performance was therefore verified.
Reported statistics and error estimates:
For the determination EC and NOEC values the nominal concentrations were used.
The values of ErC50 and EyC50 were calculated using the software “Toxicita” (Water Research Institute, Ostrava, Czech Republic).
The values and confidence intervals of ErC10 and EyC10 were calculated using the software QC.Expert 2.5 © 1998-2000 (product of TriloByte Ltd., Czech Republic).
The model fits the regression line to the data (EC x inhibition) and calculates the EC value and 95 % confidence interval for desired % of inhibition.
The determination of NOEC values was done using comparisons in ANOVA (Analysis of Variance) analysis. The ANOVA method used is the part of statistical software QC.Expert 2.5 © 1998-2000.

Table 1: Mean cell Density in Definitive Test

Nominal Concentration (mg/L)

Mean Cell Density in 1 mL

0 hours

24 hours

48 hours

72 hours

100

5000

18 750

27 083

39 583

53

5000

20 833

29 167

50 000

28

5000

22 917

35 417

54 167

15

5000

27 083

37 500

77 083

8

5000

31 250

43 750

97 917

4

5000

33 333

50 000

133 333

2

5000

43 750

62 500

147 917

Control

5000

60 417

106 250

237 500

 

Table 2: Reduction of Growth and Inhibition of Yield in Definitive Test

Nominal Concentration (mg/L)

Growth rate

 

Mean Growth (µ)

Reduction (%)

Mean Yield (Y)

Inhibition (%)

100

0.69

46.4

34 583

85.1

53

0.77

40.4

45 000

80.6

28

0.79

38.3

49 167

78.9

15

0.91

29.1

72 083

69.0

8

0.99

23.0

92 917

60.0

4

1.09

15.0

128 333

44.8

2

1.13

12.3

142 917

38.5

Control

1.29

0.0

232 500

0.0

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the 72 hour ErC50 was determined to be >100 mg/L and the 72 hour NOEC was <2 mg/L.
Executive summary:

The test substance, Solvent Red 19E, was tested for growth inhibition on algae Desmodesmus subspicatus under GLP conditions.

The test was performed according to method C.3. - Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EC) No. 761/2009, Annex IV and OECD Test Guideline No.201, Freshwater Alga and Cyanobacteria, Growth Inhibition Test, Adopted March 23, 2006.

In a preliminary study, algae were exposed the test material at nominal concentrations of 10, 20, 40, 60 and 100 mg/L under static conditions. Following this, a definitive study was carried out at nominal concentrations of 2, 4, 8, 15, 28, 53 and 100 mg/L under static conditions.

Since the test substance is poorly soluble in test medium at the concentrations needed for the test, saturated solutions of the test substance in test medium were prepared by adding measured amounts of the test substance to the test medium. The saturated solutions were stirred for a period of 72 hours on a shaft stirrer and consequently filtered through 0.45 μm filter.

The nominal concentrations were used for all evaluations and results. The study was performed without analytical determination of test substance concentrations because no suitable analytical method was found.

Reduction in the growth rate and inhibition of the yield was seen in every test concentration.

Under the conditions of this study, the 72 hour ErC50 was determined to be >100 mg/L and the 72 hour NOEC for growth was <2 mg/L. The 72 h EyC50 was determined to be 4.5 mg/L (95 % condidence limit 4.1 to 4.9 mg/L) and the 72 h NOEC for yield was <2 mg/L.

Description of key information

The 72 hour ErC50 was determined to be >100 mg/L and the 72 hour NOEC for growth was <2 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
1.8 mg/L

Additional information

The test substance, Solvent Red 19E, was tested for growth inhibition on algae Desmodesmus subspicatus under GLP conditions.

The test was performed according to method C.3. - Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EC) No. 761/2009, Annex IV and OECD Test Guideline No.201, Freshwater Alga and Cyanobacteria, Growth Inhibition Test, Adopted March 23, 2006.

In a preliminary study, algae were exposed the test material at nominal concentrations of 10, 20, 40, 60 and 100 mg/L under static conditions. Following this, a definitive study was carried out at nominal concentrations of 2, 4, 8, 15, 28, 53 and 100 mg/L under static conditions.

Since the test substance is poorly soluble in test medium at the concentrations needed for the test, saturated solutions of the test substance in test medium were prepared by adding measured amounts of the test substance to the test medium. The saturated solutions were stirred for a period of 72 hours on a shaft stirrer and consequently filtered through 0.45 μm filter.

The nominal concentrations were used for all evaluations and results. The study was performed without analytical determination of test substance concentrations because no suitable analytical method was found.

Reduction in the growth rate and inhibition of the yield was seen in every test concentration.

Under the conditions of this study, the 72 hour ErC50 was determined to be >100 mg/L and the 72 hour NOEC for growth was <2 mg/L. The 72 h EyC50 was determined to be 4.5 mg/L (95 % condidence limit 4.1 to 4.9 mg/L) and the 72 h NOEC for yield was <2 mg/L.